Re: [ccp4bb] Molecular replacement problem

[Nukri Sanishvili]

Hi Robert,
In addition to the great suggestions you already have received, maybe you
should also consider SIMBAD or similar programs? The behavior you are
describing is typical of, albeit not exclusive to, having crystallized a
contaminant protein.
Good luck!
Nukri


On Thu, Jun 18, 2020, 08:01 Robert S Phillips  wrote:

> I've been pulling out my hair with this for a few months now.  I have data
> sets to 2.6 A for a new enzyme in the aminotransferase superfamily.
> Unfortunately, the closest structure is only 25% identity.  MR with PHASER
> using the monomer was a complete failure.  Since the minimum structure of
> enzymes in the family is a dimer (the active site is formed at the
> monomer-monomer interface), I used dimers for MR with PHASER.  Most of the
> results were marginal, but one looks good.  However, it will not refine.
> Everything I have done with this solution has failed, simulated annealing,
> morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not
> give any useable models, since they have poor statistics and low
> completeness.  The output from PHASER is below.
>
> ** SINGLE solution
>
> ** Solution written to PDB file:  DGL_phaser.1.pdb
> ** Solution written to MTZ file:  DGL_phaser.1.mtz
>Solution annotation (history):
>SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1
> PAK=0 LLG=994 TFZ==20.1
>SOLU SPAC P 2 2 21
>SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
> BFAC -0.12 MULT 2 #TFZ==20.1
>SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89
>
> With LLG = 994 and TFZ = 20.1, isn't this a real solution?
>
> Robert S. Phillips
> Professor of Chemistry and of Biochemistry and Molecular Biology
> University of Georgia
> Athens, GA 30602
> Phone: (706) 542-1996
> Fax: (706) 542-9454
> E-mail: rsphill...@chem.uga.edu
> Web:  http://tryptophan.net
> 
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Molecular replacement problem

[Fischmann, Thierry]

It may be worth trying rigid body-refinement of the solution, using two rigid 
bodies, one per monomer, if you haven’t tried already. Then perform positional 
refinement.

When refining with the original sequence, first remember that R/Rfree are bound 
to be and stay high with at most 25% homology. Does the map look reasonable? 
Can you see density matching the true sequence, even if refining with the 
molecular replacement model, in places where the side-chains are ordered (not 
facing the solvent) but there is an unmistakable side-chain difference (i.e. 
from a small side chain such as Ala or Val etc. to Phe Tyr Trp, or vice-versa)

If so you can either use an automated approach to fit the new sequence or do it 
“manually” in Coot, then refine again. R’s should drop quickly.

I’ve had good success using the above approach, all refinements performed with 
autoBUSTER.

Thierry

From: CCP4 bulletin board  On Behalf Of Robert S Phillips
Sent: Thursday, June 18, 2020 9:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Molecular replacement problem

EXTERNAL EMAIL – Use caution with any links or file attachments.
I've been pulling out my hair with this for a few months now.  I have data sets 
to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, 
the closest structure is only 25% identity.  MR with PHASER using the monomer 
was a complete failure.  Since the minimum structure of enzymes in the family 
is a dimer (the active site is formed at the monomer-monomer interface), I used 
dimers for MR with PHASER.  Most of the results were marginal, but one looks 
good.  However, it will not refine.  Everything I have done with this solution 
has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD 
and BUCCANEER with it do not give any useable models, since they have poor 
statistics and low completeness.  The output from PHASER is below.

** SINGLE solution

** Solution written to PDB file:  DGL_phaser.1.pdb
** Solution written to MTZ file:  DGL_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 
LLG=994 TFZ==20.1
   SOLU SPAC P 2 2 21
   SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00 BFAC 
-0.12 MULT 2 #TFZ==20.1
   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

With LLG = 994 and TFZ = 20.1, isn't this a real solution?

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu<mailto:rsphill...@chem.uga.edu>
Web:  
http://tryptophan.net<https://pod51004.outlook.com/owa/redir.aspx?C=ccbf42ffea5f48b1bf8e9bb950454bab=http%3a%2f%2ftryptophan.net>



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Re: [ccp4bb] Molecular replacement problem

[Eleanor Dodson]

Or try the other orthorhombic SGs..?
Some general ideas for any such problem...

Is there evidence for a dimer in the asymmetric unit, or could your dimer
be generated by the crystal 2-folds?
Checks on data - all easily accessed from CCP4I2 ..
First is the data OK - Wilson plot? twinning? r factors v batch etc..
Second - likely contents of asymmetric unit?
Third , if there is likelty to be more than one molecule per asymm unit
a)Is there non-crystallographic translation
b) is the self rotation any help?

But here your solution is  the identity - ie the model fits your data
without either rotation or translation!

 SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
BFAC -0.12 MULT 2 #TFZ==20.
Could the model structure be isomorphous with your new one?
Eleanor

On Thu, 18 Jun 2020 at 14:06, David Briggs  wrote:

> Hi Robert,
>
> Have you tried lower symmetry spacegroups? Maybe your crystal is
> 'almost-but-not-quite' orthorhombic and is in fact monoclinic, pretending
> to be orthorhombic.
>
> Zanuda can do this for you.
>
> https://www.ccp4.ac.uk/newsletters/newsletter48/articles/Zanuda/zanuda.html
>
> Good luck,
>
> Dave
>
> --
>
> Dr David C. Briggs
>
> Senior Laboratory Research Scientist
>
> Signalling and Structural Biology Lab
>
> The Francis Crick Institute
>
> London, UK
>
> ==
>
> Diamond User Committee MX representative
>
> ==
>
> about.me/david_briggs
> --
> *From:* CCP4 bulletin board  on behalf of Robert S
> Phillips 
> *Sent:* 18 June 2020 14:00
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] Molecular replacement problem
>
> I've been pulling out my hair with this for a few months now.  I have data
> sets to 2.6 A for a new enzyme in the aminotransferase superfamily.
> Unfortunately, the closest structure is only 25% identity.  MR with PHASER
> using the monomer was a complete failure.  Since the minimum structure of
> enzymes in the family is a dimer (the active site is formed at the
> monomer-monomer interface), I used dimers for MR with PHASER.  Most of the
> results were marginal, but one looks good.  However, it will not refine.
> Everything I have done with this solution has failed, simulated annealing,
> morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not
> give any useable models, since they have poor statistics and low
> completeness.  The output from PHASER is below.
>
> ** SINGLE solution
>
> ** Solution written to PDB file:  DGL_phaser.1.pdb
> ** Solution written to MTZ file:  DGL_phaser.1.mtz
>Solution annotation (history):
>SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1
> PAK=0 LLG=994 TFZ==20.1
>SOLU SPAC P 2 2 21
>SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
> BFAC -0.12 MULT 2 #TFZ==20.1
>SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89
>
> With LLG = 994 and TFZ = 20.1, isn't this a real solution?
>
> Robert S. Phillips
> Professor of Chemistry and of Biochemistry and Molecular Biology
> University of Georgia
> Athens, GA 30602
> Phone: (706) 542-1996
> Fax: (706) 542-9454
> E-mail: rsphill...@chem.uga.edu
> Web:  http://tryptophan.net
> <https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpod51004.outlook.com%2Fowa%2Fredir.aspx%3FC%3Dccbf42ffea5f48b1bf8e9bb950454bab%26URL%3Dhttp%253a%252f%252ftryptophan.net=02%7C01%7C%7Cd804f2e8aae94225588808d81387ab6d%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637280820702952452=DR11jkYMX2Y0wTZjbuY362yf%2FGNaIN5htG6HQaJ0XcE%3D=0>
>
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>
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>
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Re: [ccp4bb] Molecular replacement problem

[Roger Rowlett]

I managed to solve a structure by MR at 2.4 A with a 27% identity model.
Like you, I had to use a dimer search model to make any headway. To get
usable maps and an initial model, I used Chainsaw to truncate the search
model, Phaser (MR), Parrot (DM) with NCS averaging, then auto building with
Buccaneer. I had the advantage of 8 chains in the ASU, which greatly
improved the NCS averaging. NCS helps as the square root of the chains
averaged.

Before going down this road, all potential MR solutions were carefully
checked by examining crystal lattice packing to ensure the MR solution was
reasonable and space group was likely correct, and that I had the correct
number of chains per ASU. My initial MR attempts, based on Matthew's number
estimates, were 2 chains short, which was obvious from crystal packing.

Cheers, and good luck!

Roger Rowlett
Dorothy & Gordon Kline Professor, Emeritus
Colgate University

On Thu, Jun 18, 2020, 9:01 AM Robert S Phillips  wrote:

> I've been pulling out my hair with this for a few months now.  I have data
> sets to 2.6 A for a new enzyme in the aminotransferase superfamily.
> Unfortunately, the closest structure is only 25% identity.  MR with PHASER
> using the monomer was a complete failure.  Since the minimum structure of
> enzymes in the family is a dimer (the active site is formed at the
> monomer-monomer interface), I used dimers for MR with PHASER.  Most of the
> results were marginal, but one looks good.  However, it will not refine.
> Everything I have done with this solution has failed, simulated annealing,
> morphing, etc., it will not refine; AUTOBUILD and BUCCANEER with it do not
> give any useable models, since they have poor statistics and low
> completeness.  The output from PHASER is below.
>
> ** SINGLE solution
>
> ** Solution written to PDB file:  DGL_phaser.1.pdb
> ** Solution written to MTZ file:  DGL_phaser.1.mtz
>Solution annotation (history):
>SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1
> PAK=0 LLG=994 TFZ==20.1
>SOLU SPAC P 2 2 21
>SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00
> BFAC -0.12 MULT 2 #TFZ==20.1
>SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89
>
> With LLG = 994 and TFZ = 20.1, isn't this a real solution?
>
> Robert S. Phillips
> Professor of Chemistry and of Biochemistry and Molecular Biology
> University of Georgia
> Athens, GA 30602
> Phone: (706) 542-1996
> Fax: (706) 542-9454
> E-mail: rsphill...@chem.uga.edu
> Web:  http://tryptophan.net
> 
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Molecular replacement problem

[David Briggs]

Hi Robert,

Have you tried lower symmetry spacegroups? Maybe your crystal is 
'almost-but-not-quite' orthorhombic and is in fact monoclinic, pretending to be 
orthorhombic.

Zanuda can do this for you.

https://www.ccp4.ac.uk/newsletters/newsletter48/articles/Zanuda/zanuda.html

Good luck,

Dave


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee MX representative

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Robert S 
Phillips 
Sent: 18 June 2020 14:00
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Molecular replacement problem

I've been pulling out my hair with this for a few months now.  I have data sets 
to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, 
the closest structure is only 25% identity.  MR with PHASER using the monomer 
was a complete failure.  Since the minimum structure of enzymes in the family 
is a dimer (the active site is formed at the monomer-monomer interface), I used 
dimers for MR with PHASER.  Most of the results were marginal, but one looks 
good.  However, it will not refine.  Everything I have done with this solution 
has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD 
and BUCCANEER with it do not give any useable models, since they have poor 
statistics and low completeness.  The output from PHASER is below.

** SINGLE solution

** Solution written to PDB file:  DGL_phaser.1.pdb
** Solution written to MTZ file:  DGL_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 
LLG=994 TFZ==20.1
   SOLU SPAC P 2 2 21
   SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00 BFAC 
-0.12 MULT 2 #TFZ==20.1
   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

With LLG = 994 and TFZ = 20.1, isn't this a real solution?

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fpod51004.outlook.com%2Fowa%2Fredir.aspx%3FC%3Dccbf42ffea5f48b1bf8e9bb950454bab%26URL%3Dhttp%253a%252f%252ftryptophan.net=02%7C01%7C%7Cd804f2e8aae94225588808d81387ab6d%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637280820702952452=DR11jkYMX2Y0wTZjbuY362yf%2FGNaIN5htG6HQaJ0XcE%3D=0>



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[ccp4bb] Molecular replacement problem

[Robert S Phillips]

I've been pulling out my hair with this for a few months now.  I have data sets 
to 2.6 A for a new enzyme in the aminotransferase superfamily.  Unfortunately, 
the closest structure is only 25% identity.  MR with PHASER using the monomer 
was a complete failure.  Since the minimum structure of enzymes in the family 
is a dimer (the active site is formed at the monomer-monomer interface), I used 
dimers for MR with PHASER.  Most of the results were marginal, but one looks 
good.  However, it will not refine.  Everything I have done with this solution 
has failed, simulated annealing, morphing, etc., it will not refine; AUTOBUILD 
and BUCCANEER with it do not give any useable models, since they have poor 
statistics and low completeness.  The output from PHASER is below.

** SINGLE solution

** Solution written to PDB file:  DGL_phaser.1.pdb
** Solution written to MTZ file:  DGL_phaser.1.mtz
   Solution annotation (history):
   SOLU SET  RFZ=18.9 TFZ=23.0 PAK=0 LLG=193 TFZ==8.3 LLG=994 TFZ==20.1 PAK=0 
LLG=994 TFZ==20.1
   SOLU SPAC P 2 2 21
   SOLU 6DIM ENSE ense_1 EULER  360.00.00.0 FRAC -0.00 -0.00 -0.00 BFAC 
-0.12 MULT 2 #TFZ==20.1
   SOLU ENSEMBLE ense_1 VRMS DELTA -3.5403 #RMSD  2.08 #VRMS  0.89

With LLG = 994 and TFZ = 20.1, isn't this a real solution?

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net



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Re: [ccp4bb] AW: [ccp4bb] Molecular replacement with template having low sequence identity

[Eleanor Dodson]

Please tell us what worked.
Eleanor

On 6 June 2017 at 04:52, Shankar Prasad Kanaujia <spkanau...@gmail.com>
wrote:

> Dear All,
>
> I am very happy to inform you that I have finally solved the structure.
> Thanks to all for your kind suggestions.
>
> With best regards,
> Shankar
>
> On Wed, Aug 3, 2016 at 8:35 PM, Sudipta Bhattacharyya <
> sudiptabhattacharyya.iit...@gmail.com> wrote:
>
>> Dear Shankar,
>>
>> The high TFZ and LLG you mentioned indeed indicate a possible solution.
>> However, how certain are you about the number of ensembles placed in the
>> ASU? Also this high Rfree value sometime indicates the presence of
>> t-NCS/twining, Did you check that (the presence of large off origin t-NCS
>> peak)? Although phaser should detect it and correct it accordingly but only
>> if you are searching for even number of ensembles in the ASU.
>>
>> Good luck,
>> Sudipta.
>>
>> On Wed, Aug 3, 2016 at 9:14 AM, Shankar Prasad Kanaujia <
>> spkanau...@gmail.com> wrote:
>>
>>> Dear All,
>>>
>>> As suggested, I tried several things. I am getting MR solutions with TFZ
>>> > 8.0 (e.g. 12, 16) and high LLG (e.g. 150, 160). However, Rw/Rf remains
>>> above 0.50 after first round of refinement. In some cases, after some
>>> manual model building, Rw falls up to 0.46, however, Rf remains above 0.50.
>>>
>>> Any suggestions for further model building. The data is of ~1.9 Angst
>>> processed in P1211.
>>>
>>> With best regards,
>>> Shankar
>>>
>>> On Mon, Aug 1, 2016 at 1:48 PM, <herman.schreu...@sanofi.com> wrote:
>>>
>>>> Dear Schankar,
>>>>
>>>>
>>>>
>>>> you could also try automatic packages like Balbes or Morda. However, it
>>>> might not be a bad idea to try a more rational approach as well. With 27%
>>>> sequence identity, you template may or may not have the same fold. With 27%
>>>> sequence identity AND a similar biological function (e.g. similar reaction
>>>> catalysed, binding to a similar receptor etc.), your bets are much better.
>>>> (Except cases with large conformational changes like antibodies and
>>>> calmodulin).
>>>>
>>>>
>>>>
>>>> If molecular replacement fails, you should also look very carefully in
>>>> the space group assignment and in case of ambiguity try all possible space
>>>> groups for your MR searches.
>>>>
>>>>
>>>>
>>>> Best,
>>>>
>>>> Herman
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>
>>>> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag
>>>> von *Sivakumar N
>>>> *Gesendet:* Montag, 1. August 2016 08:30
>>>> *An:* CCP4BB@JISCMAIL.AC.UK
>>>> *Betreff:* Re: [ccp4bb] Molecular replacement with template having low
>>>> sequence identity
>>>>
>>>>
>>>>
>>>> Dear Shankar,
>>>>
>>>> How do you appreciate this approach of generating an Ensemble of
>>>> superposed homologous structures as a MR search probe? I found the FUGUE
>>>> server helpful in pulling out the distantly related structural homologues
>>>> for a given query sequence.  Also, a manual or an automated method of
>>>> refining or sieving the superposed homologues to cut out the residues that
>>>> fall in the loop regions and possibly other residues at loci that introduce
>>>> any noisy-correspondences between the superposed structures can be useful,
>>>> in order to arrive at a better RMSD values between the equivalent Cα atoms
>>>> of this hybrid MR search probe.
>>>>
>>>> Regards
>>>>
>>>> Sivakumar,N.
>>>>
>>>>
>>>>
>>>> On Sat, Jul 30, 2016 at 6:20 PM, Shankar Prasad Kanaujia <
>>>> spkanau...@gmail.com> wrote:
>>>>
>>>> Dear All,
>>>>
>>>> Is it possible to solve the structure of a protein having template with
>>>> a sequence identity of 27%. If yes, what is the best possible method.
>>>>
>>>> Any program which can automatically give some clue.
>>>>
>>>> With best regards,
>>>>
>>>> Shankar
>>>>
>>>>
>>>>
>>>
>>>
>>>
>>> --
>>> Shankar Prasad Kanaujia, Ph.D.
>>> Assistant Professor
>>> Department of Biosciences and Bioengineering
>>> Indian Institute of Technology Guwahati
>>> Guwahati - 781039 Assam, India
>>> Tele: 0361 258 2228
>>> Fax:  0361 258 2249
>>> Email: spkanau...@iitg.ernet.in
>>> Homepage: http://www.iitg.ernet.in/spkanaujia/
>>> Google Scholar: https://scholar.google.com/cit
>>> ations?user=Zt4JSNYJ=en
>>>
>>
>>
>
>
> --
> Shankar Prasad Kanaujia, Ph.D.
> Associate Professor
> Department of Biosciences and Bioengineering
> Indian Institute of Technology Guwahati
> Guwahati - 781039 Assam, India
> Tele: 0361 258 2228
> Fax:  0361 258 2249
> Email: spkanau...@iitg.ernet.in
> Homepage: http://www.iitg.ernet.in/spkanaujia/
> Google Scholar: https://scholar.google.com/citations?user=Zt4JSNYJ;
> hl=en
>

Re: [ccp4bb] AW: [ccp4bb] Molecular replacement with template having low sequence identity

[Shankar Prasad Kanaujia]

Dear All,

I am very happy to inform you that I have finally solved the structure.
Thanks to all for your kind suggestions.

With best regards,
Shankar

On Wed, Aug 3, 2016 at 8:35 PM, Sudipta Bhattacharyya <
sudiptabhattacharyya.iit...@gmail.com> wrote:

> Dear Shankar,
>
> The high TFZ and LLG you mentioned indeed indicate a possible solution.
> However, how certain are you about the number of ensembles placed in the
> ASU? Also this high Rfree value sometime indicates the presence of
> t-NCS/twining, Did you check that (the presence of large off origin t-NCS
> peak)? Although phaser should detect it and correct it accordingly but only
> if you are searching for even number of ensembles in the ASU.
>
> Good luck,
> Sudipta.
>
> On Wed, Aug 3, 2016 at 9:14 AM, Shankar Prasad Kanaujia <
> spkanau...@gmail.com> wrote:
>
>> Dear All,
>>
>> As suggested, I tried several things. I am getting MR solutions with TFZ
>> > 8.0 (e.g. 12, 16) and high LLG (e.g. 150, 160). However, Rw/Rf remains
>> above 0.50 after first round of refinement. In some cases, after some
>> manual model building, Rw falls up to 0.46, however, Rf remains above 0.50.
>>
>> Any suggestions for further model building. The data is of ~1.9 Angst
>> processed in P1211.
>>
>> With best regards,
>> Shankar
>>
>> On Mon, Aug 1, 2016 at 1:48 PM, <herman.schreu...@sanofi.com> wrote:
>>
>>> Dear Schankar,
>>>
>>>
>>>
>>> you could also try automatic packages like Balbes or Morda. However, it
>>> might not be a bad idea to try a more rational approach as well. With 27%
>>> sequence identity, you template may or may not have the same fold. With 27%
>>> sequence identity AND a similar biological function (e.g. similar reaction
>>> catalysed, binding to a similar receptor etc.), your bets are much better.
>>> (Except cases with large conformational changes like antibodies and
>>> calmodulin).
>>>
>>>
>>>
>>> If molecular replacement fails, you should also look very carefully in
>>> the space group assignment and in case of ambiguity try all possible space
>>> groups for your MR searches.
>>>
>>>
>>>
>>> Best,
>>>
>>> Herman
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag
>>> von *Sivakumar N
>>> *Gesendet:* Montag, 1. August 2016 08:30
>>> *An:* CCP4BB@JISCMAIL.AC.UK
>>> *Betreff:* Re: [ccp4bb] Molecular replacement with template having low
>>> sequence identity
>>>
>>>
>>>
>>> Dear Shankar,
>>>
>>> How do you appreciate this approach of generating an Ensemble of
>>> superposed homologous structures as a MR search probe? I found the FUGUE
>>> server helpful in pulling out the distantly related structural homologues
>>> for a given query sequence.  Also, a manual or an automated method of
>>> refining or sieving the superposed homologues to cut out the residues that
>>> fall in the loop regions and possibly other residues at loci that introduce
>>> any noisy-correspondences between the superposed structures can be useful,
>>> in order to arrive at a better RMSD values between the equivalent Cα atoms
>>> of this hybrid MR search probe.
>>>
>>> Regards
>>>
>>> Sivakumar,N.
>>>
>>>
>>>
>>> On Sat, Jul 30, 2016 at 6:20 PM, Shankar Prasad Kanaujia <
>>> spkanau...@gmail.com> wrote:
>>>
>>> Dear All,
>>>
>>> Is it possible to solve the structure of a protein having template with
>>> a sequence identity of 27%. If yes, what is the best possible method.
>>>
>>> Any program which can automatically give some clue.
>>>
>>> With best regards,
>>>
>>> Shankar
>>>
>>>
>>>
>>
>>
>>
>> --
>> Shankar Prasad Kanaujia, Ph.D.
>> Assistant Professor
>> Department of Biosciences and Bioengineering
>> Indian Institute of Technology Guwahati
>> Guwahati - 781039 Assam, India
>> Tele: 0361 258 2228
>> Fax:  0361 258 2249
>> Email: spkanau...@iitg.ernet.in
>> Homepage: http://www.iitg.ernet.in/spkanaujia/
>> Google Scholar: https://scholar.google.com/citations?user=Zt4JSNYJ;
>> hl=en
>>
>
>


-- 
Shankar Prasad Kanaujia, Ph.D.
Associate Professor
Department of Biosciences and Bioengineering
Indian Institute of Technology Guwahati
Guwahati - 781039 Assam, India
Tele: 0361 258 2228
Fax:  0361 258 2249
Email: spkanau...@iitg.ernet.in
Homepage: http://www.iitg.ernet.in/spkanaujia/
Google Scholar: https://scholar.google.com/citations?user=Zt4JSNYJ=en

[ccp4bb] Molecular Replacement

[Muhammed bashir Khan]

Hi All;

I have a native data set of membrane protein at 3.8A. I nearly use all the
options for Molrep. I would like to ask, is some body has some special
strategy which can work for difficult Molrep

Thanks you in Advance

Bashir


-- 
Muhammad Bashir Khan
**
Department Of Biochemistry
University of Alberta, Edmonton
Canada

Re: [ccp4bb] molecular replacement with poor model

[Ursula Schulze-Gahmen]

Thanks for all the good suggestions.  This gives me a lot more things to
try.

Ursula

On Sat, Dec 13, 2014 at 2:44 AM, Claudia Millán Nebot cmn...@ibmb.csic.es
wrote:

 Dear Ursula,

 If you have a resolution around 2.0 A you can try some of the following:

 - Expand the partial solution with shelxe autotracing feature.

 - Do a search with ARCIMBOLDO_LITE using the partial solution. You can fin
 the program at http://chango.ibmb.csic.es/arcimboldo_lite. Then, instead
 of searching for ideal alpha helices, you can input your solution and
 search for 2 copies.

 Hope it helps. Best,

 Claudia


 

 Claudia Millán (cmn...@ibmb.csic.es)

 Crystallographic Methods Group

 http://chango.ibmb.csic.es

 Institut de Biologia Molecular de Barcelona (IBMB-CSIC)

 Barcelona, Spain

 LinkedIn: es.linkedin.com/in/claudiamillan/
 http://es.linkedin.com/pub/claudia-mill%C3%A1n/60/a76/821/

 ResearchGate:
 https://www.researchgate.net/profile/Claudia_Millan?ev=hdr_xprf



 2014-12-12 22:38 GMT+01:00 Ursula Schulze-Gahmen uschulze-gah...@lbl.gov
 :

 I am trying molecular replacement with a very poor model. The model
 consists mainly of 1 long helix and two slightly bent antiparallel helices.
 After dividing it into 2 fragments, I was able to find a solution for one
 of the fragments ( at least I think so after looking at maps, packing,
 refinement etc). But even if I place the first solution as fixed ensemble
 in phaser, I cannot find a solution for the second fragment ( 18% sequence
 identity). From the structure of the model and the packing it seems clear
 where the fragment should go roughly.

 Are there any other programs other than phaser that might be able to
 solve this problem? I tried already epmr and mr_rosetta without success.
 I also tried to just superimpose the complete model onto the partial
 solution. This results in quite nice packing, but doesn't refine. Is there
 a rigid program refinement program with very large convergence?

 Ursula

 --
 Ursula Schulze-Gahmen, Ph.D.
 Project Scientist
 UC Berkeley, QB3
 360 Stanley Hall #3220
 Berkeley, CA 94720-3220
 (510) 643 9491



-- 
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491

Re: [ccp4bb] molecular replacement with poor model

[Claudia Millán Nebot]

Dear Ursula,

If you have a resolution around 2.0 A you can try some of the following:

- Expand the partial solution with shelxe autotracing feature.

- Do a search with ARCIMBOLDO_LITE using the partial solution. You can fin
the program at http://chango.ibmb.csic.es/arcimboldo_lite. Then, instead of
searching for ideal alpha helices, you can input your solution and search
for 2 copies.

Hope it helps. Best,

Claudia



Claudia Millán (cmn...@ibmb.csic.es)

Crystallographic Methods Group

http://chango.ibmb.csic.es

Institut de Biologia Molecular de Barcelona (IBMB-CSIC)

Barcelona, Spain

LinkedIn: es.linkedin.com/in/claudiamillan/
http://es.linkedin.com/pub/claudia-mill%C3%A1n/60/a76/821/

ResearchGate:
https://www.researchgate.net/profile/Claudia_Millan?ev=hdr_xprf



2014-12-12 22:38 GMT+01:00 Ursula Schulze-Gahmen uschulze-gah...@lbl.gov:

 I am trying molecular replacement with a very poor model. The model
 consists mainly of 1 long helix and two slightly bent antiparallel helices.
 After dividing it into 2 fragments, I was able to find a solution for one
 of the fragments ( at least I think so after looking at maps, packing,
 refinement etc). But even if I place the first solution as fixed ensemble
 in phaser, I cannot find a solution for the second fragment ( 18% sequence
 identity). From the structure of the model and the packing it seems clear
 where the fragment should go roughly.

 Are there any other programs other than phaser that might be able to solve
 this problem? I tried already epmr and mr_rosetta without success.
 I also tried to just superimpose the complete model onto the partial
 solution. This results in quite nice packing, but doesn't refine. Is there
 a rigid program refinement program with very large convergence?

 Ursula

 --
 Ursula Schulze-Gahmen, Ph.D.
 Project Scientist
 UC Berkeley, QB3
 360 Stanley Hall #3220
 Berkeley, CA 94720-3220
 (510) 643 9491

Re: [ccp4bb] molecular replacement with poor model

[Daniel Rigden]

Dear Ursula

AMPLE is also worth trying, especially but not exclusively for smaller 
and predominantly helical targets.  It was originally conceived for 
novel folds but you may well find ab initio modelling methods get closer 
to the real structure of distant homologs than the nearest available 
templates.  AMPLE will produce a range of search models of different 
sizes, through clustering and truncation of a model set, and this 
sampling can be helpful.  You can install Rosetta for the modelling or, 
thanks to Yang Zhang's efforts, take a tar file direct from his Quark 
server (http://zhanglab.ccmb.med.umich.edu/QUARK/) and give it to AMPLE. 
Higher resolution helps generally, but we have had success out to 2.9A.


Good luck

Dan


On 13/12/14 10:44, Claudia Millán Nebot wrote:

Dear Ursula,

If you have a resolution around 2.0 A you can try some of the following:

- Expand the partial solution with shelxe autotracing feature.

- Do a search with ARCIMBOLDO_LITE using the partial solution. You can 
fin the program at http://chango.ibmb.csic.es/arcimboldo_lite. Then, 
instead of searching for ideal alpha helices, you can input your 
solution and search for 2 copies.


Hope it helps. Best,

Claudia



Claudia Millán (cmn...@ibmb.csic.es mailto:cmn...@ibmb.csic.es)

Crystallographic Methods Group

http://chango.ibmb.csic.es

Institut de Biologia Molecular de Barcelona (IBMB-CSIC)

Barcelona, Spain

LinkedIn: es.linkedin.com/in/claudiamillan/ 
http://es.linkedin.com/in/claudiamillan/http://es.linkedin.com/pub/claudia-mill%C3%A1n/60/a76/821/


ResearchGate: 
https://www.researchgate.net/profile/Claudia_Millan?ev=hdr_xprf




2014-12-12 22:38 GMT+01:00 Ursula Schulze-Gahmen 
uschulze-gah...@lbl.gov mailto:uschulze-gah...@lbl.gov:


I am trying molecular replacement with a very poor model. The
model consists mainly of 1 long helix and two slightly bent
antiparallel helices. After dividing it into 2 fragments, I was
able to find a solution for one of the fragments ( at least I
think so after looking at maps, packing, refinement etc). But even
if I place the first solution as fixed ensemble in phaser, I
cannot find a solution for the second fragment ( 18% sequence
identity). From the structure of the model and the packing it
seems clear where the fragment should go roughly.

Are there any other programs other than phaser that might be able
to solve this problem? I tried already epmr and mr_rosetta without
success.
I also tried to just superimpose the complete model onto the
partial solution. This results in quite nice packing, but doesn't
refine. Is there a rigid program refinement program with very
large convergence?

Ursula

-- 
Ursula Schulze-Gahmen, Ph.D.

Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220 tel:94720-3220
(510) 643 9491



--
Dr Daniel John Rigden Tel:(+44) 151 795 4467
Institute of Integrative Biology  FAX:(+44) 151 795 4406
Room 101, Biosciences Building
University of Liverpool   http://pcwww.liv.ac.uk/~drigden/
Crown St.,
Liverpool L69 7ZB, U.K.

[ccp4bb] molecular replacement with poor model

[Ursula Schulze-Gahmen]

I am trying molecular replacement with a very poor model. The model
consists mainly of 1 long helix and two slightly bent antiparallel helices.
After dividing it into 2 fragments, I was able to find a solution for one
of the fragments ( at least I think so after looking at maps, packing,
refinement etc). But even if I place the first solution as fixed ensemble
in phaser, I cannot find a solution for the second fragment ( 18% sequence
identity). From the structure of the model and the packing it seems clear
where the fragment should go roughly.

Are there any other programs other than phaser that might be able to solve
this problem? I tried already epmr and mr_rosetta without success.
I also tried to just superimpose the complete model onto the partial
solution. This results in quite nice packing, but doesn't refine. Is there
a rigid program refinement program with very large convergence?

Ursula

-- 
Ursula Schulze-Gahmen, Ph.D.
Project Scientist
UC Berkeley, QB3
360 Stanley Hall #3220
Berkeley, CA 94720-3220
(510) 643 9491

Re: [ccp4bb] molecular replacement with poor model

[Pavel Afonine]

Hi Ursula,


I also tried to just superimpose the complete model onto the partial
 solution. This results in quite nice packing, but doesn't refine. Is there
 a rigid program refinement program with very large convergence?



depending on what you call very large, this may be helpful:

Automatic multiple-zone rigid-body refinement with a large convergence
radius. J. Appl. Cryst. 42, 607-615 (2009).

Pavel

Re: [ccp4bb] molecular replacement with poor model

[Randy Read]

Dear Ursula,

18% identity is really in the twilight zone for molecular replacement, where it 
may work but there are certainly no guarantees.

However, there’s a feature in Phaser that is useful for this kind of problem, 
i.e. the “rotate around” option, where you take advantage of knowing the 
approximate orientation of a domain.  In the current version of Phaser in 
either CCP4 or Phenix, this can be done as part of an automated search (instead 
of doing separate rotation/translation/packing/refinement steps as in earlier 
versions).  Let’s assume that the model from the first fragment was cut out of 
a larger model, from which you would now like to place the second fragment.  
The rotation to apply to the second one should be similar to what was applied 
to the first.  In ccp4i, you can go to the “Additional Search Parameters” 
folder, and choose “Brute” for the “Rotation function target”.  In the “Search” 
pulldown that appears then, choose “Around an angle”, give the Euler angles 
that were found in the search for the first domain, and choose something like 
20-30 degrees for “Range”, i.e. allow the second domain to differ in 
orientation by up to 20 or 30 degrees from the orientation for the first 
domain.  It’s probably better to do translation searches with all orientations 
generated by this rotation search, so open the “Expert parameters” folder, 
under “Rotation search peak selection” choose “All peaks”, and turn “Rotation 
clustering” “Off”.

We’ve had good luck in a number of cases where the hinge angle changes by more 
than even the best rigid-body refinement would manage.

Good luck!

Randy

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical ResearchTel: +44 1223 336500
Wellcome Trust/MRC Building Fax: +44 1223 336827
Hills RoadE-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   
www-structmed.cimr.cam.ac.uk

On 12 Dec 2014, at 21:38, Ursula Schulze-Gahmen uschulze-gah...@lbl.gov wrote:

 I am trying molecular replacement with a very poor model. The model consists 
 mainly of 1 long helix and two slightly bent antiparallel helices. After 
 dividing it into 2 fragments, I was able to find a solution for one of the 
 fragments ( at least I think so after looking at maps, packing, refinement 
 etc). But even if I place the first solution as fixed ensemble in phaser, I 
 cannot find a solution for the second fragment ( 18% sequence identity). From 
 the structure of the model and the packing it seems clear where the fragment 
 should go roughly.
 
 Are there any other programs other than phaser that might be able to solve 
 this problem? I tried already epmr and mr_rosetta without success. 
 I also tried to just superimpose the complete model onto the partial 
 solution. This results in quite nice packing, but doesn't refine. Is there a 
 rigid program refinement program with very large convergence?
 
 Ursula
 
 -- 
 Ursula Schulze-Gahmen, Ph.D.
 Project Scientist
 UC Berkeley, QB3
 360 Stanley Hall #3220
 Berkeley, CA 94720-3220
 (510) 643 9491

Re: [ccp4bb] molecular replacement with poor model

[xaravich ivan]

What is the resolution of your data? I have been able to get a solution for
my protein with 30% identity but my resolution of data was 1.4 Angs. I
believe to get a solution at 18% identity your search model has to be very
close, like using Robetta to make the 3 mer and 9mer peptides and then work
from a homology model. But your resolution should be around 1 angstrom.

On Fri, Dec 12, 2014 at 2:38 PM, Ursula Schulze-Gahmen 
uschulze-gah...@lbl.gov wrote:

 I am trying molecular replacement with a very poor model. The model
 consists mainly of 1 long helix and two slightly bent antiparallel helices.
 After dividing it into 2 fragments, I was able to find a solution for one
 of the fragments ( at least I think so after looking at maps, packing,
 refinement etc). But even if I place the first solution as fixed ensemble
 in phaser, I cannot find a solution for the second fragment ( 18% sequence
 identity). From the structure of the model and the packing it seems clear
 where the fragment should go roughly.

 Are there any other programs other than phaser that might be able to solve
 this problem? I tried already epmr and mr_rosetta without success.
 I also tried to just superimpose the complete model onto the partial
 solution. This results in quite nice packing, but doesn't refine. Is there
 a rigid program refinement program with very large convergence?

 Ursula

 --
 Ursula Schulze-Gahmen, Ph.D.
 Project Scientist
 UC Berkeley, QB3
 360 Stanley Hall #3220
 Berkeley, CA 94720-3220
 (510) 643 9491

Re: [ccp4bb] Molecular Replacement model preparation

[Sheriff, Steven]

All:



While Phil Jeffrey attributed to me the trick of aligning the hinge axis of 
an Fab along the Z direction, I, in turn, must give credit to Mirek Cygler, who 
explained this to me at the Diffraction Methods in Molecular Biology [now 
Structural Biology] Gordon Research Conference in 1986.



To Scott's query about searching for 1 domain sequentially and then other 
domains OR searching for multiple domains all at once?



Inevitably a program like PHASER searches for domains (ensembles in its 
terminology) sequentially. However, as to the practical question of whether to 
feed PHASER all of the domains in one run, that is certainly how I start and 
it is usually (almost always?) successful. In fact, while I no longer bother to 
align the hinge axis of an Fab along the Z axis, I now break Fabs into three 
parts: CL:CH1, VH, and VL to allow molecular replacement to accommodate the 
tilt angle between VH and VL (tilt angle is a term I learned from Gary 
Gilliland's talk at the Diffraction Methods in Structural Biology GRC in 2014). 
This also allows me to search for the highest identity VL and, separately, VH 
in the PDB to use as probe models.  N.B. since I'm usually studying antigen/Fab 
complex, I'm usually searching for 4 ensembles in one PHASER run: CL:CH1, 
antigen, VH and VL.



Steven



P.S. to Tassos Perrakis: I'm sorry that these plugs for the Diffraction Methods 
GRC are ~4 months too late!

P.P.S. to Eddie Snell: And I'm sorry that these plugs are ~18 months too early 
for 2016's Diffraction Methods GRC!





==

Date:Mon, 6 Oct 2014 18:33:26 +

From:Scott Thomas Walsh swals...@umd.edumailto:swals...@umd.edu

Subject: Re: Molecular Replacement model preparation



Hi Phil,



Thank you for the input.  I would like to get CCP4ers input.  I deal with 
multiple domain cytokine receptors in a manner very similar to antibody 
molecules.



Have people have more correct solutions searching for 1 domain sequentially and 
then other domains OR searching for multiple domains all at once?  I am curious 
to hear peoples' experiences on this topic?



Cheers,



Scott





Scott T. R. Walsh, PhD

Assistant Professor

University of Maryland

IBBR/CBMG

3127E CARB-2

9600 Gudelsky Drive

Rockville, MD  20850  USA

phone: (240) 314-6478

fax: (240) 314-6225

email: swals...@umd.edumailto:swals...@umd.edu





On Oct 6, 2014, at 2:11 PM, Phil Jeffrey 
pjeff...@princeton.edumailto:pjeff...@princeton.edu wrote:



 That document is fairly old and is in dire need of revision to reflect the 
 modern arsenal of programs.



 Nevertheless:

 Putting the hinge axis along Z was a trick told to me by Steven Sheriff back 
 in the days when we worked on Fab structures - which after all are classical 
 examples of hinged molecules.  One would search with separate domain 
 fragments - split either side of the hinge - and the Z-orientation trick 
 makes it easier to spot pairs of peaks from each search model that are 
 related to each other.  In the Fab world we searched with Fv models (VH:VL 
 heterodimer) and CH1:CL constant region heterodimeric models.  Peaks related 
 solely by hinge motion would have similar alpha and beta angles and 
 potentially different gamma (Crowther convention Eulerian angles).  
 Historical note: this was back in the days when it was possible to remember 
 the names of all the Fab fragments that were in PDB and their respective IDs.



 This ploy was more important in the days before Phaser or Molrep, which will 
 now gleefully try a long list of rotation function peaks for you quite 
 quickly, so manually parsing the list of rotation function peaks is rather 
 unnecessary.  And perhaps counter-productive.





 Split your molecule apart at the hinge, giving fragment1 and fragment2.  
 Attempt to find both fragments independently.  Choose the one that gives the 
 best results: TFZ score or LLG score or discrimination between possible space 
 group or whatever you like.  Then, attempt to find the *other* fragment in 
 the context of that first solution.





 Phil Jeffrey

 Princeton





 On 10/5/14 3:34 AM, Luzuokun wrote:

 Dear all,

 I'm doing molecular replacement using Phaser. My protein is predicted

 to have two domain with a hinge linking them. The model sequence

 identity is 0.27. But the MR result is poor. I've tried other

 programme (Molrep, MrBump, Balbes,,,_.) But no improvement was

 observed. I think that this is due to the open or closed

 conformation around the hinge. I was told that I could place the Z

 axis along the hinge

 (http://xray0.princeton.edu/~phil/Facility/Guides/MolecularReplacemen

 t.html),  could anyone tell me more details about how to do next?



 Thanks!

 Lu Zuokun





This message (including any attachments) may contain confidential, proprietary, 
privileged and/or private information. The information is intended to be for 
the use of the 

Re: [ccp4bb] Molecular Replacement model preparation

[Scott Thomas Walsh]

Hi Steven,

Thank you for the information and guidance.  When you search for all the 
ensembles with Phaser do you use “AND” or “OR”
searching?

Cheers,

Scott


While Phil Jeffrey attributed to me the “trick” of aligning the hinge axis of 
an Fab along the Z direction, I, in turn, must give credit to Mirek Cygler, who 
explained this to me at the Diffraction Methods in Molecular Biology [now 
Structural Biology] Gordon Research Conference in 1986.

To Scott’s query about searching for “1 domain sequentially and then other 
domains OR searching for multiple domains all at once?”

Inevitably a program like PHASER searches for domains (“ensembles” in its 
terminology) sequentially. However, as to the practical question of whether to 
“feed” PHASER all of the domains in one run, that is certainly how I start and 
it is usually (almost always?) successful. In fact, while I no longer bother to 
align the hinge axis of an Fab along the Z axis, I now break Fabs into three 
parts: CL:CH1, VH, and VL to allow molecular replacement to accommodate the 
“tilt” angle between VH and VL (tilt angle is a term I learned from Gary 
Gilliland’s talk at the Diffraction Methods in Structural Biology GRC in 2014). 
This also allows me to search for the highest identity VL and, separately, VH 
in the PDB to use as probe models.  N.B. since I’m usually studying antigen/Fab 
complex, I’m usually searching for 4 ensembles in one PHASER run: CL:CH1, 
antigen, VH and VL.




Scott T. R. Walsh, PhD
Assistant Professor
University of Maryland
IBBR/CBMG
3127E CARB-2
9600 Gudelsky Drive
Rockville, MD  20850  USA
phone: (240) 314-6478
fax: (240) 314-6225
email: swals...@umd.edumailto:swals...@umd.edu

Re: [ccp4bb] Molecular Replacement model preparation

[Randy Read]

Dear Scott,

By “AND” searching, presumably you mean adding another SEARCH command? (Or 
clicking “Add another search” in the ccp4i interface.)  For Steven’s strategy, 
you want to specify separate searches for separate ensembles, i.e. give several 
SEARCH commands in a script or add extra searches in ccp4i.

The “OR” option (to specify several ensembles to search for in one SEARCH 
command, available through ccp4i by opening the “Additional Search parameters” 
pane and turning on “Allow search with alternative ensembles...” uses 
alternative models to search for the same component, which can be useful but 
isn’t what you’re interested in for defining a hinge angle.

At some point in the distant past, you had to tell Phaser what order to search 
for the components in, but then we implemented a method to automatically choose 
a good search order and, more recently, implemented an improved method to 
choose the optimal search order.  If the information you give Phaser about the 
quality of the model (RMSD or sequence identity, which Phaser turns into an 
RMSD estimate) is correct, then the strength of the signal for different models 
can be estimated.  Phaser will use this to define an initial search order.  If 
each search gives an unambiguous solution, then everything will proceed in the 
predefined order.  However, whenever there is ambiguity about whether a correct 
solution has been found, then Phaser will automatically try strategies like 
choosing a different search order or changing the resolution of the data used 
for the calculation.  

So, in most cases, it’s best to tell Phaser everything you’re looking for and 
set up one job to find everything, because this will allow the greatest level 
of optimisation and the most flexibility in trying different strategies.  It’s 
only if this fails that you’ll want to choose the strategy and parameters 
manually.

Best wishes,

Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical ResearchTel: +44 1223 336500
Wellcome Trust/MRC Building Fax: +44 1223 336827
Hills RoadE-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   
www-structmed.cimr.cam.ac.uk

On 7 Oct 2014, at 15:16, Scott Thomas Walsh swals...@umd.edu wrote:

 Hi Steven,
 
 Thank you for the information and guidance.  When you search for all the 
 ensembles with Phaser do you use “AND” or “OR”
 searching?
 
 Cheers,
 
 Scott
 
 
 While Phil Jeffrey attributed to me the “trick” of aligning the hinge axis 
 of an Fab along the Z direction, I, in turn, must give credit to Mirek 
 Cygler, who explained this to me at the Diffraction Methods in Molecular 
 Biology [now Structural Biology] Gordon Research Conference in 1986.
  
 To Scott’s query about searching for “1 domain sequentially and then other 
 domains OR searching for multiple domains all at once?”
  
 Inevitably a program like PHASER searches for domains (“ensembles” in its 
 terminology) sequentially. However, as to the practical question of whether 
 to “feed” PHASER all of the domains in one run, that is certainly how I 
 start and it is usually (almost always?) successful. In fact, while I no 
 longer bother to align the hinge axis of an Fab along the Z axis, I now 
 break Fabs into three parts: CL:CH1, VH, and VL to allow molecular 
 replacement to accommodate the “tilt” angle between VH and VL (tilt angle is 
 a term I learned from Gary Gilliland’s talk at the Diffraction Methods in 
 Structural Biology GRC in 2014). This also allows me to search for the 
 highest identity VL and, separately, VH in the PDB to use as probe models.  
 N.B. since I’m usually studying antigen/Fab complex, I’m usually searching 
 for 4 ensembles in one PHASER run: CL:CH1, antigen, VH and VL.
  
 
 
 
 Scott T. R. Walsh, PhD
 Assistant Professor
 University of Maryland
 IBBR/CBMG
 3127E CARB-2
 9600 Gudelsky Drive
 Rockville, MD  20850  USA
 phone: (240) 314-6478
 fax: (240) 314-6225
 email: swals...@umd.edu
 
 

Re: [ccp4bb] Molecular Replacement model preparation

[Phil Jeffrey]

That document is fairly old and is in dire need of revision to reflect 
the modern arsenal of programs.


Nevertheless:
Putting the hinge axis along Z was a trick told to me by Steven Sheriff 
back in the days when we worked on Fab structures - which after all are 
classical examples of hinged molecules.  One would search with separate 
domain fragments - split either side of the hinge - and the 
Z-orientation trick makes it easier to spot pairs of peaks from each 
search model that are related to each other.  In the Fab world we 
searched with Fv models (VH:VL heterodimer) and CH1:CL constant region 
heterodimeric models.  Peaks related solely by hinge motion would have 
similar alpha and beta angles and potentially different gamma (Crowther 
convention Eulerian angles).  Historical note: this was back in the days 
when it was possible to remember the names of all the Fab fragments that 
were in PDB and their respective IDs.


This ploy was more important in the days before Phaser or Molrep, which 
will now gleefully try a long list of rotation function peaks for you 
quite quickly, so manually parsing the list of rotation function peaks 
is rather unnecessary.  And perhaps counter-productive.



Split your molecule apart at the hinge, giving fragment1 and fragment2. 
 Attempt to find both fragments independently.  Choose the one that 
gives the best results: TFZ score or LLG score or discrimination between 
possible space groupr or whatever you like.  Then, attempt to find the 
*other* fragment in the context of that first solution.



Phil Jeffrey
Princeton






On 10/5/14 3:34 AM, Luzuokun wrote:

Dear all,
I’m doing molecular replacement using Phaser. My protein is predicted to
have two domain with a “hinge” linking them. The model sequence identity
is 0.27. But the MR result is poor. I’ve tried other programme (Molrep,
MrBump, Balbes,,,_.) But no improvement was observed. I think that this
is due to the “open” or “closed” conformation around the hinge. I was
told that I could place the Z axis along the hinge
(http://xray0.princeton.edu/~phil/Facility/Guides/MolecularReplacement.html),
  could anyone tell me more details about how to do next?

Thanks!
Lu Zuokun

Re: [ccp4bb] Molecular Replacement model preparation

[Scott Thomas Walsh]

Hi Phil,

Thank you for the input.  I would like to get CCP4ers input.  I deal with 
multiple domain
cytokine receptors in a manner very similar to antibody molecules.

Have people have more correct solutions searching for 1 domain sequentially and 
then other
domains OR searching for multiple domains all at once?  I am curious to hear 
peoples’ experiences on
this topic?

Cheers,

Scott


Scott T. R. Walsh, PhD
Assistant Professor
University of Maryland
IBBR/CBMG
3127E CARB-2
9600 Gudelsky Drive
Rockville, MD  20850  USA
phone: (240) 314-6478
fax: (240) 314-6225
email: swals...@umd.edu


On Oct 6, 2014, at 2:11 PM, Phil Jeffrey pjeff...@princeton.edu wrote:

 That document is fairly old and is in dire need of revision to reflect the 
 modern arsenal of programs.
 
 Nevertheless:
 Putting the hinge axis along Z was a trick told to me by Steven Sheriff back 
 in the days when we worked on Fab structures - which after all are classical 
 examples of hinged molecules.  One would search with separate domain 
 fragments - split either side of the hinge - and the Z-orientation trick 
 makes it easier to spot pairs of peaks from each search model that are 
 related to each other.  In the Fab world we searched with Fv models (VH:VL 
 heterodimer) and CH1:CL constant region heterodimeric models.  Peaks related 
 solely by hinge motion would have similar alpha and beta angles and 
 potentially different gamma (Crowther convention Eulerian angles).  
 Historical note: this was back in the days when it was possible to remember 
 the names of all the Fab fragments that were in PDB and their respective IDs.
 
 This ploy was more important in the days before Phaser or Molrep, which will 
 now gleefully try a long list of rotation function peaks for you quite 
 quickly, so manually parsing the list of rotation function peaks is rather 
 unnecessary.  And perhaps counter-productive.
 
 
 Split your molecule apart at the hinge, giving fragment1 and fragment2.  
 Attempt to find both fragments independently.  Choose the one that gives the 
 best results: TFZ score or LLG score or discrimination between possible space 
 groupr or whatever you like.  Then, attempt to find the *other* fragment in 
 the context of that first solution.
 
 
 Phil Jeffrey
 Princeton
 
 
 
 
 
 
 On 10/5/14 3:34 AM, Luzuokun wrote:
 Dear all,
 I’m doing molecular replacement using Phaser. My protein is predicted to
 have two domain with a “hinge” linking them. The model sequence identity
 is 0.27. But the MR result is poor. I’ve tried other programme (Molrep,
 MrBump, Balbes,,,_.) But no improvement was observed. I think that this
 is due to the “open” or “closed” conformation around the hinge. I was
 told that I could place the Z axis along the hinge
 (http://xray0.princeton.edu/~phil/Facility/Guides/MolecularReplacement.html),
  could anyone tell me more details about how to do next?
 
 Thanks!
 Lu Zuokun

[ccp4bb] Molecular Replacement model preparation

[Luzuokun]

Dear all,
I’m doing molecular replacement using Phaser. My protein is predicted 
to have two domain with a “hinge” linking them. The model sequence identity is 
0.27. But the MR result is poor. I’ve tried other programme (Molrep, MrBump, 
Balbes,,,_.) But no improvement was observed. I think that this is due to the 
“open” or “closed” conformation around the hinge. I was told that I could place 
the Z axis along the hinge 
(http://xray0.princeton.edu/~phil/Facility/Guides/MolecularReplacement.html),  
could anyone tell me more details about how to do next?

Thanks!
Lu Zuokun

Re: [ccp4bb] Molecular Replacement model preparation

[David Schuller]

Since there is a hinge, you could try searching with the two domains 
separately.


On 10/05/14 03:34, Luzuokun wrote:

Dear all,
I’m doing molecular replacement using Phaser. My protein is predicted 
to have two domain with a “hinge” linking them. The model sequence 
identity is 0.27. But the MR result is poor. I’ve tried other 
programme (Molrep, MrBump, Balbes,,,_.) But no improvement was 
observed. I think that this is due to the “open” or “closed” 
conformation around the hinge. I was told that I could place the Z 
axis along the hinge 
(http://xray0.princeton.edu/~phil/Facility/Guides/MolecularReplacement.html 
http://xray0.princeton.edu/%7Ephil/Facility/Guides/MolecularReplacement.html), 
 could anyone tell me more details about how to do next?


Thanks!
Lu Zuokun



--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

Re: [ccp4bb] AW: [ccp4bb] Molecular Replacement using low sequence identity templates

[Eleanor Dodson]

I dont know about LLG scores - they seem extremely variable depending
on the degree of sequence similarity you assign.
However when you get an R/Rfree of 40%/47% that is a pretty good sign
that at least something is correct.

It isnt clear whether that is after you have placed 2 copies of the
second component?
Anyway - maybe try again with the refined component 2 and look for
component 1 again?
Eleanor



On 18 October 2013 15:51,  herman.schreu...@sanofi.com wrote:
 Dear Jan,

 There are a few things a would do in this case. The first is to check the 
 processing to make sure the space group is really C2 and, although unlikely, 
 not some other space group.

 The second thing would be to try to place the first component. From your 
 email it is not clear to me whether or not you were able to place the first 
 component after the second component had been placed. In your case, I would 
 give both components to phaser and ask phaser to first place component 2 and 
 then component 1.

 It might be that the correct solution gets rejected because of clashes, so I 
 would also try to trim the first component, or to increase the number of 
 allowed clashes in Phaser. Although you expect two copies of your 
 heterodimer, you may have a crystal with a high solvent content and only  one 
 dimer in the asymmetric unit. In this case the crystal packing should make 
 sense i.e. continuous crystal contacts in all three dimensions.

 Best,
 Herman


 -Ursprüngliche Nachricht-
 Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan 
 Félix
 Gesendet: Freitag, 18. Oktober 2013 13:17
 An: CCP4BB@JISCMAIL.AC.UK
 Betreff: [ccp4bb] Molecular Replacement using low sequence identity templates

 Dear all,
 I have a question regarding Molecular Replacement using low sequence identity 
 templates.

 I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex 
 (space group C 1 2 1, no twinning detected using xtriage). For the first 
 component homologs are available, but for the other the best found template 
 only has 20 % sequence similarity.
 Strangely, I cannot place the first component directly, but the second 
 component can be placed (after trimming the template with chainsaw) using 
 phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 
 NCS copies of the heterodimer are expected based on the unit cell parameters, 
 only 1 copy of the second component gets placed.
 If I try to place the first component based on the .sol file of the first MR 
 solution, the TFZ score for the second placement is only about 3.5, but if I 
 then try to place this second MR solution (2
 components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000.

 However, none of the MR solutions I obtained seems to refine in PHENIX. Using 
 autobuild does not lower the R/Rfree values which seem to get stuck at an 
 R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated annealing, 
 DEN-refinement, morphing and MR-rosetta, but nothing seems to improve the 
 model..
 Also, in every MR solution only half of the asymmetric unit seems to be 
 filled, but phaser fails to place more units..As I am seriously starting to 
 doubt the actual content of the crystals,  I tried Nearest Cell to search for 
 similar space group, but without any hits.

 So here is my question.  Is it possible to get TFZ/LLG values this high in C 
 1 2 1 with a completely incorrect model by chance, or can I assume that this 
 MR solution points out that what I think is in the crystal is actually there?
 And secondly, as I am a bit stuck here, are there any new strategies I can 
 try to tackle this problem?
 Off course, experimental phasing is an option, but the crystals grew slowly 
 over e few months and I only had 1 drop with 1 crystal, so reproducing the 
 crystals might be though..

 Thanks for any tips and best regards,

 Jan

Re: [ccp4bb] [ccp4bb] Molecular Replacement using low sequence identity templates

[Randy Read]

Hi Eleanor,

Yes, the initial LLG scores in Phaser are highly dependent on the assigned 
sequence identity, which is translated into an initial estimate of the 
effective RMSD of the model.  However, the latest versions of Phaser refine the 
RMSD estimate at the end of the job and, assuming that two runs find the same 
solution and the refinement manages to converge (which it usually does these 
days), the LLG at the end should be pretty reproducible regardless of the 
assigned sequence identity.

Best wishes,

Randy

On 21 Oct 2013, at 14:42, Eleanor Dodson eleanor.dod...@york.ac.uk wrote:

 I dont know about LLG scores - they seem extremely variable depending
 on the degree of sequence similarity you assign.
 However when you get an R/Rfree of 40%/47% that is a pretty good sign
 that at least something is correct.
 
 It isnt clear whether that is after you have placed 2 copies of the
 second component?
 Anyway - maybe try again with the refined component 2 and look for
 component 1 again?
 Eleanor
 
 
 
 On 18 October 2013 15:51,  herman.schreu...@sanofi.com wrote:
 Dear Jan,
 
 There are a few things a would do in this case. The first is to check the 
 processing to make sure the space group is really C2 and, although unlikely, 
 not some other space group.
 
 The second thing would be to try to place the first component. From your 
 email it is not clear to me whether or not you were able to place the first 
 component after the second component had been placed. In your case, I would 
 give both components to phaser and ask phaser to first place component 2 and 
 then component 1.
 
 It might be that the correct solution gets rejected because of clashes, so I 
 would also try to trim the first component, or to increase the number of 
 allowed clashes in Phaser. Although you expect two copies of your 
 heterodimer, you may have a crystal with a high solvent content and only  
 one dimer in the asymmetric unit. In this case the crystal packing should 
 make sense i.e. continuous crystal contacts in all three dimensions.
 
 Best,
 Herman
 
 
 -Ursprüngliche Nachricht-
 Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan 
 Félix
 Gesendet: Freitag, 18. Oktober 2013 13:17
 An: CCP4BB@JISCMAIL.AC.UK
 Betreff: [ccp4bb] Molecular Replacement using low sequence identity templates
 
 Dear all,
 I have a question regarding Molecular Replacement using low sequence 
 identity templates.
 
 I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex 
 (space group C 1 2 1, no twinning detected using xtriage). For the first 
 component homologs are available, but for the other the best found template 
 only has 20 % sequence similarity.
 Strangely, I cannot place the first component directly, but the second 
 component can be placed (after trimming the template with chainsaw) using 
 phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 
 NCS copies of the heterodimer are expected based on the unit cell 
 parameters, only 1 copy of the second component gets placed.
 If I try to place the first component based on the .sol file of the first MR 
 solution, the TFZ score for the second placement is only about 3.5, but if I 
 then try to place this second MR solution (2
 components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000.
 
 However, none of the MR solutions I obtained seems to refine in PHENIX. 
 Using autobuild does not lower the R/Rfree values which seem to get stuck at 
 an R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated 
 annealing, DEN-refinement, morphing and MR-rosetta, but nothing seems to 
 improve the model..
 Also, in every MR solution only half of the asymmetric unit seems to be 
 filled, but phaser fails to place more units..As I am seriously starting to 
 doubt the actual content of the crystals,  I tried Nearest Cell to search 
 for similar space group, but without any hits.
 
 So here is my question.  Is it possible to get TFZ/LLG values this high in C 
 1 2 1 with a completely incorrect model by chance, or can I assume that this 
 MR solution points out that what I think is in the crystal is actually there?
 And secondly, as I am a bit stuck here, are there any new strategies I can 
 try to tackle this problem?
 Off course, experimental phasing is an option, but the crystals grew slowly 
 over e few months and I only had 1 drop with 1 crystal, so reproducing the 
 crystals might be though..
 
 Thanks for any tips and best regards,
 
 Jan

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] [ccp4bb] Molecular Replacement using low sequence identity templates

[Eleanor Dodson]

I guess my experience is out of date - please ignore comments on LLG!
Eleanor

On 21 October 2013 15:40, Randy Read rj...@cam.ac.uk wrote:
 Hi Eleanor,

 Yes, the initial LLG scores in Phaser are highly dependent on the assigned 
 sequence identity, which is translated into an initial estimate of the 
 effective RMSD of the model.  However, the latest versions of Phaser refine 
 the RMSD estimate at the end of the job and, assuming that two runs find the 
 same solution and the refinement manages to converge (which it usually does 
 these days), the LLG at the end should be pretty reproducible regardless of 
 the assigned sequence identity.

 Best wishes,

 Randy

 On 21 Oct 2013, at 14:42, Eleanor Dodson eleanor.dod...@york.ac.uk wrote:

 I dont know about LLG scores - they seem extremely variable depending
 on the degree of sequence similarity you assign.
 However when you get an R/Rfree of 40%/47% that is a pretty good sign
 that at least something is correct.

 It isnt clear whether that is after you have placed 2 copies of the
 second component?
 Anyway - maybe try again with the refined component 2 and look for
 component 1 again?
 Eleanor



 On 18 October 2013 15:51,  herman.schreu...@sanofi.com wrote:
 Dear Jan,

 There are a few things a would do in this case. The first is to check the 
 processing to make sure the space group is really C2 and, although 
 unlikely, not some other space group.

 The second thing would be to try to place the first component. From your 
 email it is not clear to me whether or not you were able to place the first 
 component after the second component had been placed. In your case, I would 
 give both components to phaser and ask phaser to first place component 2 
 and then component 1.

 It might be that the correct solution gets rejected because of clashes, so 
 I would also try to trim the first component, or to increase the number of 
 allowed clashes in Phaser. Although you expect two copies of your 
 heterodimer, you may have a crystal with a high solvent content and only  
 one dimer in the asymmetric unit. In this case the crystal packing should 
 make sense i.e. continuous crystal contacts in all three dimensions.

 Best,
 Herman


 -Ursprüngliche Nachricht-
 Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan 
 Félix
 Gesendet: Freitag, 18. Oktober 2013 13:17
 An: CCP4BB@JISCMAIL.AC.UK
 Betreff: [ccp4bb] Molecular Replacement using low sequence identity 
 templates

 Dear all,
 I have a question regarding Molecular Replacement using low sequence 
 identity templates.

 I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex 
 (space group C 1 2 1, no twinning detected using xtriage). For the first 
 component homologs are available, but for the other the best found template 
 only has 20 % sequence similarity.
 Strangely, I cannot place the first component directly, but the second 
 component can be placed (after trimming the template with chainsaw) using 
 phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 
 NCS copies of the heterodimer are expected based on the unit cell 
 parameters, only 1 copy of the second component gets placed.
 If I try to place the first component based on the .sol file of the first 
 MR solution, the TFZ score for the second placement is only about 3.5, but 
 if I then try to place this second MR solution (2
 components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000.

 However, none of the MR solutions I obtained seems to refine in PHENIX. 
 Using autobuild does not lower the R/Rfree values which seem to get stuck 
 at an R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated 
 annealing, DEN-refinement, morphing and MR-rosetta, but nothing seems to 
 improve the model..
 Also, in every MR solution only half of the asymmetric unit seems to be 
 filled, but phaser fails to place more units..As I am seriously starting to 
 doubt the actual content of the crystals,  I tried Nearest Cell to search 
 for similar space group, but without any hits.

 So here is my question.  Is it possible to get TFZ/LLG values this high in 
 C 1 2 1 with a completely incorrect model by chance, or can I assume that 
 this MR solution points out that what I think is in the crystal is actually 
 there?
 And secondly, as I am a bit stuck here, are there any new strategies I can 
 try to tackle this problem?
 Off course, experimental phasing is an option, but the crystals grew slowly 
 over e few months and I only had 1 drop with 1 crystal, so reproducing the 
 crystals might be though..

 Thanks for any tips and best regards,

 Jan

 --
 Randy J. Read
 Department of Haematology, University of Cambridge
 Cambridge Institute for Medical Research  Tel: + 44 1223 336500
 Wellcome Trust/MRC Building   Fax: + 44 1223 336827
 Hills RoadE-mail: rj...@cam.ac.uk
 Cambridge CB2 0XY, U.K

[ccp4bb] Molecular Replacement using low sequence identity templates

[Jan Félix]

Dear all,
I have a question regarding Molecular Replacement using low sequence  
identity templates.


I have a 2.7 Angstrom dataset of a heterodimeric protein-protein  
complex (space group C 1 2 1, no twinning detected using xtriage). For  
the first component homologs are available, but for the other the best  
found template only has 20 % sequence similarity.
Strangely, I cannot place the first component directly, but the second  
component can be placed (after trimming the template with chainsaw)  
using phaser with a TFZ score of 12 and a LLG of about 1200. Although  
at least 2 NCS copies of the heterodimer are expected based on the  
unit cell parameters, only 1 copy of the second component gets placed.
If I try to place the first component based on the .sol file of the  
first MR solution, the TFZ score for the second placement is only  
about 3.5, but if I then try to place this second MR solution (2  
components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about  
1000.


However, none of the MR solutions I obtained seems to refine in  
PHENIX. Using autobuild does not lower the R/Rfree values which seem  
to get stuck at an R/Rfree of 0.40/0.47. I have tried trimming off  
loops, simulated annealing, DEN-refinement, morphing and MR-rosetta,  
but nothing seems to improve the model..
Also, in every MR solution only half of the asymmetric unit seems to  
be filled, but phaser fails to place more units..As I am seriously  
starting to doubt the actual content of the crystals,  I tried Nearest  
Cell to search for similar space group, but without any hits.


So here is my question.  Is it possible to get TFZ/LLG values this  
high in C 1 2 1 with a completely incorrect model by chance, or can I  
assume that this MR solution points out that what I think is in the  
crystal is actually there?
And secondly, as I am a bit stuck here, are there any new strategies I  
can try to tackle this problem?
Off course, experimental phasing is an option, but the crystals grew  
slowly over e few months and I only had 1 drop with 1 crystal, so  
reproducing the crystals might be though..


Thanks for any tips and best regards,

Jan

[ccp4bb] AW: [ccp4bb] Molecular Replacement using low sequence identity templates

[Herman . Schreuder]

Dear Jan,

There are a few things a would do in this case. The first is to check the 
processing to make sure the space group is really C2 and, although unlikely, 
not some other space group. 

The second thing would be to try to place the first component. From your email 
it is not clear to me whether or not you were able to place the first component 
after the second component had been placed. In your case, I would give both 
components to phaser and ask phaser to first place component 2 and then 
component 1.

It might be that the correct solution gets rejected because of clashes, so I 
would also try to trim the first component, or to increase the number of 
allowed clashes in Phaser. Although you expect two copies of your heterodimer, 
you may have a crystal with a high solvent content and only  one dimer in the 
asymmetric unit. In this case the crystal packing should make sense i.e. 
continuous crystal contacts in all three dimensions.

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan Félix
Gesendet: Freitag, 18. Oktober 2013 13:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Molecular Replacement using low sequence identity templates

Dear all,
I have a question regarding Molecular Replacement using low sequence identity 
templates.

I have a 2.7 Angstrom dataset of a heterodimeric protein-protein complex (space 
group C 1 2 1, no twinning detected using xtriage). For the first component 
homologs are available, but for the other the best found template only has 20 % 
sequence similarity.
Strangely, I cannot place the first component directly, but the second 
component can be placed (after trimming the template with chainsaw) using 
phaser with a TFZ score of 12 and a LLG of about 1200. Although at least 2 NCS 
copies of the heterodimer are expected based on the unit cell parameters, only 
1 copy of the second component gets placed.
If I try to place the first component based on the .sol file of the first MR 
solution, the TFZ score for the second placement is only about 3.5, but if I 
then try to place this second MR solution (2
components) as a whole I get a RFZ of 8, TFZ of 16 and a LLG of about 1000.

However, none of the MR solutions I obtained seems to refine in PHENIX. Using 
autobuild does not lower the R/Rfree values which seem to get stuck at an 
R/Rfree of 0.40/0.47. I have tried trimming off loops, simulated annealing, 
DEN-refinement, morphing and MR-rosetta, but nothing seems to improve the 
model..
Also, in every MR solution only half of the asymmetric unit seems to be filled, 
but phaser fails to place more units..As I am seriously starting to doubt the 
actual content of the crystals,  I tried Nearest Cell to search for similar 
space group, but without any hits.

So here is my question.  Is it possible to get TFZ/LLG values this high in C 1 
2 1 with a completely incorrect model by chance, or can I assume that this MR 
solution points out that what I think is in the crystal is actually there?
And secondly, as I am a bit stuck here, are there any new strategies I can try 
to tackle this problem?
Off course, experimental phasing is an option, but the crystals grew slowly 
over e few months and I only had 1 drop with 1 crystal, so reproducing the 
crystals might be though..

Thanks for any tips and best regards,

Jan

Re: [ccp4bb] molecular replacement problem.

[Eleanor Dodson]

Umm - this is tricky.
First of all you need to reindex the C2221 data into the P21 cell - do you
know the operator?
 then expand that data set to spacegroup P21. There is a cad option to do
this..
Then add that FreeR to the re-processed P21 data.
Eleanor


On 24 March 2013 14:37, Appu kumar appu.kum...@gmail.com wrote:

 Sorry for the misconception. Yes i am expanding the space group from
 merged mtz file.  Actually i have enough number of images collected. when i
 indexed, integrate, and scale the data in either C2221 or P 21, it fetches
 the  overall 98% completeness. But when i am trying to reindex the data
 from C2221 to P21 keeping the Rfree flag of C2221, the completeness of data
 reduced drastically to 40%. This is what i am not getting. I am a beginner
 so i have to read a lot which i am doing also, but i had few  practical
 confusion which i shared  and off course  i am getting good response. Thank
 you all for your kind response  and educating me on the problem i faced.
 Thank you all for your valuable response.

 On 24 March 2013 15:15, vellieux frederic.velli...@ibs.fr wrote:

  Hello,

 Here we deal with symmetry and the unique part of reciprocal space (the
 reciprocal space asymmetric unit so to speak).

 C222(1) has eight asymmetric units (international tables, space group 20);

 P2(1) only has two. Assuming that Friedel's law does apply, then the
 minimum rotation range to collect a non-redundant data set (one observation
 per reflection) is 90 degrees, provided that the crystal is correctly and
 perfectly aligned. Normally with our current data collection methods where
 the crystal is randomly oriented, we would collect more than 90 degrees
 (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR
 beamline where you cannot really check during data collection how well the
 crystal fares during exposure to the X-rays - shoot first, think later.

 The reciprocal space asymmetric unit in C222(1) is smaller.

 I assume that what you are doing is to take the reduced data set file (an
 MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will
 not cover the monoclinic reciprocal space asymmetric unit by doing so.

 The way to do it is to take the file from processing, before
 (crystallographic symmetry) merging of the equivalents, and perform the
 scaling and merging in the P2(1) space group. Or reprocess the data frames
 in P2(1) if you have lost the unmerged data file.

 Now of course this will still give you a poor completeness if you have
 used a strategy to optimize data collection in the orthorhombic space group
 (you won't have collected enough data then for good completeness in the
 monoclinic space group).

 I hope this is clear !

 HTH,

 Fred.


 On 24/03/13 11:20, Appu kumar wrote:

 I run the phenix.xtriage to evaluate the twining but it suggest no
 twining. When i reindex from C2221 to P21, the completeness of data reduced
 from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31
 for 2.2 resolution. I do not understand why the completeness of data
 reduced so much on reindexing. please Can anyone explain this phenomenon.
 Thank you

 On 24 March 2013 13:30, Matthias Zebisch 
 matthias.zebi...@bbz.uni-leipzig.de wrote:

  the p21 c2221 ambivalence can mean severe twinning (i had a similar
 case just now - try several crystals from the same condition) !
 What do the twinning statistics suggest?

 cheers, Matthias

 -
 Dr. Matthias Zebisch
 Division of Structural Biology,
 Wellcome Trust Centre for Human Genetics,
 University of Oxford,
 Roosevelt Drive,
 Oxford OX3 7BN, UK

 Phone (+44) 1865 287549;
 Fax (+44) 1865 287547
 Email matth...@strubi.ox.ac.uk
 Website http://www.strubi.ox.ac.uk
 -

   On 3/24/2013 7:46 AM, Appu kumar wrote:

 Thank you for the quick reply. After molecular replacement , i have done
 only few cycle of refinement in refmac. I have not done any solvent
 modification or NCS averaging. I have initially indexed the data in C2221
 but Rfree was not decreasing so i reindexed the data in  data in P121 space
 group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
 i found large space but no density. structure of Ligand binding domain is
 almost identical with 90% identity in sequence. I am stuck with this
 problem and don't know how to process further.
 Please give me your valuable suggestion. I will appreciate your effort.
 Thank you
 Appu

 On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

  I am not sure that I have a complete sense of the issue at hand since
 some of the information needed to think your issue through is missing in
 your email. For example, to what high resolution cut-off were the data
 measured? What resolution limits were used for the MR search? How do the
 unit cell dimensions and space group in the two cases compare?

  I am guessing the ligand binding 

Re: [ccp4bb] molecular replacement problem.

[Appu kumar]

Thank you for the quick reply. After molecular replacement , i have done
only few cycle of refinement in refmac. I have not done any solvent
modification or NCS averaging. I have initially indexed the data in C2221
but Rfree was not decreasing so i reindexed the data in  data in P121 space
group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
i found large space but no density. structure of Ligand binding domain is
almost identical with 90% identity in sequence. I am stuck with this
problem and don't know how to process further.
Please give me your valuable suggestion. I will appreciate your effort.
Thank you
Appu

On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

 I am not sure that I have a complete sense of the issue at hand since some
 of the information needed to think your issue through is missing in your
 email. For example, to what high resolution cut-off were the data measured?
 What resolution limits were used for the MR search? How do the unit cell
 dimensions and space group in the two cases compare?

 I am guessing the ligand binding domain in your protein has the identical
 sequence to that of the published ligand binding domain that you use as a
 template in your MR search. In any case, here are a couple of my thoughts:

 (1) It might be worth setting up different runs of MR with different
 numbers for expected copies (not just two copies but also one copy and
 three copies just in case you have one of the extreme cases of solvent
 content)?

 (2) If the MR solution is correct and there is physical room for a DNA
 binding domain in your lattice (check by displaying symmetry mates),
 perhaps the DNA binding domain is disordered. In that case (and if all
 attempts with current data fail), you may have to crystallize the protein
 in presence of DNA.


 Good luck!
 Raji




 On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.com wrote:

 Dear members,

   I am doing a molecular replacement of a
 transcription factor whose ligand binding structure(24000 Da) is available
 in PDB but not for the DNA binding(13000 Da). When i am searching for the
 two copies from ligand binding domain as a template model, i am getting
 very good solution but i am not getting any density for the DNA binding
 domain to build up in density. The space gorup is P 1 21 1 (4) and unit
 cell parameters are Unit Cell:   57.43   69.36  105.99   90.00   90.00
 90.00. Please guide me how to get the complete model structure. Table below
 show the matthews statistics

  For estimated molecular weight   37000.
 Nmol/asym  Matthews Coeff  %solvent   P(2.20) P(tot)
 _
   1 5.7178.46 0.00 0.01
   2 2.8556.91 0.62 0.70
   3 1.9035.37 0.37 0.29
   4 1.4313.82 0.00 0.00
 _


 The phaser molecular replacement gives the following table.
 istogram of relative frequencies of VM values
--
Frequency of most common VM value normalized to 1
VM values plotted in increments of 1/VM (0.02)

 --- relative frequency ---
 0.0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1.0
 |||||||||||
10.00 -
 8.33 -
 7.14 -
 6.25 -
 5.56 -
 5.00 -
 4.55 -
 4.17 -
 3.85 --
 3.57 ---
 3.33 --
 3.12 --
 2.94  (COMPOSITION*1)
 2.78 ---
 2.63 
 2.50 -
 2.38 
 2.27 --
 2.17 ---
 2.08 --
 2.00 --
 1.92 ---
 1.85 ---
 1.79 ---
 1.72 -
 1.67 -
 1.61 -
 1.56 -
 1.52 -
 1.47 * (COMPOSITION*2)
 1.43 -
 1.39 -
 1.35 -
 1.32 -
 1.28 -
 1.25 -

 $TABLE : Cell Content Analysis:
 $SCATTER
 :N*Composition vs Probability:0|3x0|1:1,2:
 $$
 N*Composition Probability
 $$ loggraph $$
 1 0.306066
 2 0.00141804
 $$

Most probable VM for resolution = 2.27817
Most probable MW of protein in asu for resolution = 92664.2

 Thank a lot in advance





 --
 Raji Edayathumangalam
 Instructor in Neurology, Harvard Medical School
 Research Associate, Brigham and Women's Hospital
 Visiting Research Scholar, Brandeis University

Re: [ccp4bb] molecular replacement problem.

[Appu kumar]

I run the phenix.xtriage to evaluate the twining but it suggest no twining.
When i reindex from C2221 to P21, the completeness of data reduced from 95
% to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2
resolution. I do not understand why the completeness of data reduced so
much on reindexing. please Can anyone explain this phenomenon.
Thank you

On 24 March 2013 13:30, Matthias Zebisch 
matthias.zebi...@bbz.uni-leipzig.de wrote:

  the p21 c2221 ambivalence can mean severe twinning (i had a similar case
 just now - try several crystals from the same condition) !
 What do the twinning statistics suggest?

 cheers, Matthias

 -
 Dr. Matthias Zebisch
 Division of Structural Biology,
 Wellcome Trust Centre for Human Genetics,
 University of Oxford,
 Roosevelt Drive,
 Oxford OX3 7BN, UK

 Phone (+44) 1865 287549;
 Fax (+44) 1865 287547
 Email matth...@strubi.ox.ac.uk
 Website http://www.strubi.ox.ac.uk
 -

 On 3/24/2013 7:46 AM, Appu kumar wrote:

 Thank you for the quick reply. After molecular replacement , i have done
 only few cycle of refinement in refmac. I have not done any solvent
 modification or NCS averaging. I have initially indexed the data in C2221
 but Rfree was not decreasing so i reindexed the data in  data in P121 space
 group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
 i found large space but no density. structure of Ligand binding domain is
 almost identical with 90% identity in sequence. I am stuck with this
 problem and don't know how to process further.
 Please give me your valuable suggestion. I will appreciate your effort.
 Thank you
 Appu

 On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

  I am not sure that I have a complete sense of the issue at hand since
 some of the information needed to think your issue through is missing in
 your email. For example, to what high resolution cut-off were the data
 measured? What resolution limits were used for the MR search? How do the
 unit cell dimensions and space group in the two cases compare?

  I am guessing the ligand binding domain in your protein has the
 identical sequence to that of the published ligand binding domain that you
 use as a template in your MR search. In any case, here are a couple of my
 thoughts:

  (1) It might be worth setting up different runs of MR with different
 numbers for expected copies (not just two copies but also one copy and
 three copies just in case you have one of the extreme cases of solvent
 content)?

  (2) If the MR solution is correct and there is physical room for a DNA
 binding domain in your lattice (check by displaying symmetry mates),
 perhaps the DNA binding domain is disordered. In that case (and if all
 attempts with current data fail), you may have to crystallize the protein
 in presence of DNA.


  Good luck!
 Raji




 On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.comwrote:

 Dear members,

   I am doing a molecular replacement of a
 transcription factor whose ligand binding structure(24000 Da) is available
 in PDB but not for the DNA binding(13000 Da). When i am searching for the
 two copies from ligand binding domain as a template model, i am getting
 very good solution but i am not getting any density for the DNA binding
 domain to build up in density. The space gorup is P 1 21 1 (4) and unit
 cell parameters are Unit Cell:   57.43   69.36  105.99   90.00   90.00
 90.00. Please guide me how to get the complete model structure. Table below
 show the matthews statistics

  For estimated molecular weight   37000.
 Nmol/asym  Matthews Coeff  %solvent   P(2.20) P(tot)
 _
   1 5.7178.46 0.00 0.01
   2 2.8556.91 0.62 0.70
   3 1.9035.37 0.37 0.29
   4 1.4313.82 0.00 0.00
 _


 The phaser molecular replacement gives the following table.
 istogram of relative frequencies of VM values
--
Frequency of most common VM value normalized to 1
VM values plotted in increments of 1/VM (0.02)

 --- relative frequency ---
 0.0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1.0
 |||||||||||
10.00 -
 8.33 -
 7.14 -
 6.25 -
 5.56 -
 5.00 -
 4.55 -
 4.17 -
 3.85 --
 3.57 ---
 3.33 --
 3.12 --
 2.94  (COMPOSITION*1)
 2.78 ---
 2.63 
 2.50 -
 2.38 
 2.27 

Re: [ccp4bb] molecular replacement problem.

[vellieux]

Hello,

Here we deal with symmetry and the unique part of reciprocal space (the 
reciprocal space asymmetric unit so to speak).


C222(1) has eight asymmetric units (international tables, space group 20);

P2(1) only has two. Assuming that Friedel's law does apply, then the 
minimum rotation range to collect a non-redundant data set (one 
observation per reflection) is 90 degrees, provided that the crystal is 
correctly and perfectly aligned. Normally with our current data 
collection methods where the crystal is randomly oriented, we would 
collect more than 90 degrees (180 degrees, or 360 degrees with the 
Pilatus detectors on an intense SR beamline where you cannot really 
check during data collection how well the crystal fares during exposure 
to the X-rays - shoot first, think later.


The reciprocal space asymmetric unit in C222(1) is smaller.

I assume that what you are doing is to take the reduced data set file 
(an MTZ file probably) and reduce the symmetry from C222(1) to P2(1). 
You will not cover the monoclinic reciprocal space asymmetric unit by 
doing so.


The way to do it is to take the file from processing, before 
(crystallographic symmetry) merging of the equivalents, and perform the 
scaling and merging in the P2(1) space group. Or reprocess the data 
frames in P2(1) if you have lost the unmerged data file.


Now of course this will still give you a poor completeness if you have 
used a strategy to optimize data collection in the orthorhombic space 
group (you won't have collected enough data then for good completeness 
in the monoclinic space group).


I hope this is clear !

HTH,

Fred.

On 24/03/13 11:20, Appu kumar wrote:
I run the phenix.xtriage to evaluate the twining but it suggest no 
twining. When i reindex from C2221 to P21, the completeness of data 
reduced from 95 % to 35% whereas the map is very good and Rwork and 
Rfree are 26/31 for 2.2 resolution. I do not understand why the 
completeness of data reduced so much on reindexing. please Can anyone 
explain this phenomenon.

Thank you

On 24 March 2013 13:30, Matthias Zebisch 
matthias.zebi...@bbz.uni-leipzig.de 
mailto:matthias.zebi...@bbz.uni-leipzig.de wrote:


the p21 c2221 ambivalence can mean severe twinning (i had a
similar case just now - try several crystals from the same
condition) !
What do the twinning statistics suggest?

cheers, Matthias

-
Dr. Matthias Zebisch
Division of Structural Biology,
Wellcome Trust Centre for Human Genetics,
University of Oxford,
Roosevelt Drive,
Oxford OX3 7BN, UK

Phone (+44) 1865 287549;
Fax (+44) 1865 287547
emailmatth...@strubi.ox.ac.uk  mailto:matth...@strubi.ox.ac.uk
Websitehttp://www.strubi.ox.ac.uk
-

On 3/24/2013 7:46 AM, Appu kumar wrote:

Thank you for the quick reply. After molecular replacement , i
have done only few cycle of refinement in refmac. I have not done
any solvent modification or NCS averaging. I have initially
indexed the data in C2221 but Rfree was not decreasing so i
reindexed the data in  data in P121 space group keeping the Rfree
flag of C2221. While analysing the symmetry mates , i found large
space but no density. structure of Ligand binding domain is
almost identical with 90% identity in sequence. I am stuck with
this problem and don't know how to process further.
Please give me your valuable suggestion. I will appreciate your
effort.
Thank you
Appu

On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu
mailto:r...@brandeis.edu wrote:

Dear Appu,

I am not sure that I have a complete sense of the issue at
hand since some of the information needed to think your issue
through is missing in your email. For example, to what high
resolution cut-off were the data measured? What resolution
limits were used for the MR search? How do the unit cell
dimensions and space group in the two cases compare?

I am guessing the ligand binding domain in your protein has
the identical sequence to that of the published ligand
binding domain that you use as a template in your MR search.
In any case, here are a couple of my thoughts:

(1) It might be worth setting up different runs of MR with
different numbers for expected copies (not just two copies
but also one copy and three copies just in case you have one
of the extreme cases of solvent content)?

(2) If the MR solution is correct and there is physical room
for a DNA binding domain in your lattice (check by displaying
symmetry mates), perhaps the DNA binding domain is
disordered. In that case (and if all attempts with current
data fail), you may have to crystallize the protein in
presence of DNA.


Good luck!
Raji

Re: [ccp4bb] molecular replacement problem.

[Raji Edayathumangalam]

Dear Appu,

You want to be sure you have good reason to drop the space group from
C222(1) to P2(1). There may be many reasons why your Rfree may not drop
following refinement, especially if you only have one domain in your
protein located and just in case there are more molecules to locate in the
MR search.

For C222(1) data, did Xtriage suggest any alternate space groups? Also,
what program did you use for MR? If you used Phaser with your C222(1) data,
did you ask to search for alternate space groups? Did you check for
translational NCS?

If you want me to take a look at your data, I'd be happy to look at your
scaled data and MR results and try to help you out. If so, please email me
off the bulletin board.

Good luck!
Raji




On Sun, Mar 24, 2013 at 6:45 AM, vellieux frederic.velli...@ibs.fr wrote:

  Hello,

 Here we deal with symmetry and the unique part of reciprocal space (the
 reciprocal space asymmetric unit so to speak).

 C222(1) has eight asymmetric units (international tables, space group 20);

 P2(1) only has two. Assuming that Friedel's law does apply, then the
 minimum rotation range to collect a non-redundant data set (one observation
 per reflection) is 90 degrees, provided that the crystal is correctly and
 perfectly aligned. Normally with our current data collection methods where
 the crystal is randomly oriented, we would collect more than 90 degrees
 (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR
 beamline where you cannot really check during data collection how well the
 crystal fares during exposure to the X-rays - shoot first, think later.

 The reciprocal space asymmetric unit in C222(1) is smaller.

 I assume that what you are doing is to take the reduced data set file (an
 MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will
 not cover the monoclinic reciprocal space asymmetric unit by doing so.

 The way to do it is to take the file from processing, before
 (crystallographic symmetry) merging of the equivalents, and perform the
 scaling and merging in the P2(1) space group. Or reprocess the data frames
 in P2(1) if you have lost the unmerged data file.

 Now of course this will still give you a poor completeness if you have
 used a strategy to optimize data collection in the orthorhombic space group
 (you won't have collected enough data then for good completeness in the
 monoclinic space group).

 I hope this is clear !

 HTH,

 Fred.


 On 24/03/13 11:20, Appu kumar wrote:

 I run the phenix.xtriage to evaluate the twining but it suggest no
 twining. When i reindex from C2221 to P21, the completeness of data reduced
 from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31
 for 2.2 resolution. I do not understand why the completeness of data
 reduced so much on reindexing. please Can anyone explain this phenomenon.
 Thank you

 On 24 March 2013 13:30, Matthias Zebisch 
 matthias.zebi...@bbz.uni-leipzig.de wrote:

  the p21 c2221 ambivalence can mean severe twinning (i had a similar
 case just now - try several crystals from the same condition) !
 What do the twinning statistics suggest?

 cheers, Matthias

 -
 Dr. Matthias Zebisch
 Division of Structural Biology,
 Wellcome Trust Centre for Human Genetics,
 University of Oxford,
 Roosevelt Drive,
 Oxford OX3 7BN, UK

 Phone (+44) 1865 287549;
 Fax (+44) 1865 287547
 Email matth...@strubi.ox.ac.uk
 Website http://www.strubi.ox.ac.uk
 -

   On 3/24/2013 7:46 AM, Appu kumar wrote:

 Thank you for the quick reply. After molecular replacement , i have done
 only few cycle of refinement in refmac. I have not done any solvent
 modification or NCS averaging. I have initially indexed the data in C2221
 but Rfree was not decreasing so i reindexed the data in  data in P121 space
 group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
 i found large space but no density. structure of Ligand binding domain is
 almost identical with 90% identity in sequence. I am stuck with this
 problem and don't know how to process further.
 Please give me your valuable suggestion. I will appreciate your effort.
 Thank you
 Appu

 On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

  I am not sure that I have a complete sense of the issue at hand since
 some of the information needed to think your issue through is missing in
 your email. For example, to what high resolution cut-off were the data
 measured? What resolution limits were used for the MR search? How do the
 unit cell dimensions and space group in the two cases compare?

  I am guessing the ligand binding domain in your protein has the
 identical sequence to that of the published ligand binding domain that you
 use as a template in your MR search. In any case, here are a couple of my
 thoughts:

  (1) It might be worth setting up different runs of MR with different
 numbers for expected copies 

Re: [ccp4bb] molecular replacement problem.

[Appu kumar]

Sorry for the misconception. Yes i am expanding the space group from merged
mtz file.  Actually i have enough number of images collected. when i
indexed, integrate, and scale the data in either C2221 or P 21, it fetches
the  overall 98% completeness. But when i am trying to reindex the data
from C2221 to P21 keeping the Rfree flag of C2221, the completeness of data
reduced drastically to 40%. This is what i am not getting. I am a beginner
so i have to read a lot which i am doing also, but i had few  practical
confusion which i shared  and off course  i am getting good response. Thank
you all for your kind response  and educating me on the problem i faced.
Thank you all for your valuable response.

On 24 March 2013 15:15, vellieux frederic.velli...@ibs.fr wrote:

  Hello,

 Here we deal with symmetry and the unique part of reciprocal space (the
 reciprocal space asymmetric unit so to speak).

 C222(1) has eight asymmetric units (international tables, space group 20);

 P2(1) only has two. Assuming that Friedel's law does apply, then the
 minimum rotation range to collect a non-redundant data set (one observation
 per reflection) is 90 degrees, provided that the crystal is correctly and
 perfectly aligned. Normally with our current data collection methods where
 the crystal is randomly oriented, we would collect more than 90 degrees
 (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR
 beamline where you cannot really check during data collection how well the
 crystal fares during exposure to the X-rays - shoot first, think later.

 The reciprocal space asymmetric unit in C222(1) is smaller.

 I assume that what you are doing is to take the reduced data set file (an
 MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will
 not cover the monoclinic reciprocal space asymmetric unit by doing so.

 The way to do it is to take the file from processing, before
 (crystallographic symmetry) merging of the equivalents, and perform the
 scaling and merging in the P2(1) space group. Or reprocess the data frames
 in P2(1) if you have lost the unmerged data file.

 Now of course this will still give you a poor completeness if you have
 used a strategy to optimize data collection in the orthorhombic space group
 (you won't have collected enough data then for good completeness in the
 monoclinic space group).

 I hope this is clear !

 HTH,

 Fred.


 On 24/03/13 11:20, Appu kumar wrote:

 I run the phenix.xtriage to evaluate the twining but it suggest no
 twining. When i reindex from C2221 to P21, the completeness of data reduced
 from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31
 for 2.2 resolution. I do not understand why the completeness of data
 reduced so much on reindexing. please Can anyone explain this phenomenon.
 Thank you

 On 24 March 2013 13:30, Matthias Zebisch 
 matthias.zebi...@bbz.uni-leipzig.de wrote:

  the p21 c2221 ambivalence can mean severe twinning (i had a similar
 case just now - try several crystals from the same condition) !
 What do the twinning statistics suggest?

 cheers, Matthias

 -
 Dr. Matthias Zebisch
 Division of Structural Biology,
 Wellcome Trust Centre for Human Genetics,
 University of Oxford,
 Roosevelt Drive,
 Oxford OX3 7BN, UK

 Phone (+44) 1865 287549;
 Fax (+44) 1865 287547
 Email matth...@strubi.ox.ac.uk
 Website http://www.strubi.ox.ac.uk
 -

   On 3/24/2013 7:46 AM, Appu kumar wrote:

 Thank you for the quick reply. After molecular replacement , i have done
 only few cycle of refinement in refmac. I have not done any solvent
 modification or NCS averaging. I have initially indexed the data in C2221
 but Rfree was not decreasing so i reindexed the data in  data in P121 space
 group keeping the Rfree flag of C2221. While analysing the symmetry mates ,
 i found large space but no density. structure of Ligand binding domain is
 almost identical with 90% identity in sequence. I am stuck with this
 problem and don't know how to process further.
 Please give me your valuable suggestion. I will appreciate your effort.
 Thank you
 Appu

 On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote:

 Dear Appu,

  I am not sure that I have a complete sense of the issue at hand since
 some of the information needed to think your issue through is missing in
 your email. For example, to what high resolution cut-off were the data
 measured? What resolution limits were used for the MR search? How do the
 unit cell dimensions and space group in the two cases compare?

  I am guessing the ligand binding domain in your protein has the
 identical sequence to that of the published ligand binding domain that you
 use as a template in your MR search. In any case, here are a couple of my
 thoughts:

  (1) It might be worth setting up different runs of MR with different
 numbers for expected copies (not just two copies but also one copy and
 

[ccp4bb] molecular replacement problem.

[Appu kumar]

Dear members,

  I am doing a molecular replacement of a
transcription factor whose ligand binding structure(24000 Da) is available
in PDB but not for the DNA binding(13000 Da). When i am searching for the
two copies from ligand binding domain as a template model, i am getting
very good solution but i am not getting any density for the DNA binding
domain to build up in density. The space gorup is P 1 21 1 (4) and unit
cell parameters are Unit Cell:   57.43   69.36  105.99   90.00   90.00
90.00. Please guide me how to get the complete model structure. Table below
show the matthews statistics

 For estimated molecular weight   37000.
Nmol/asym  Matthews Coeff  %solvent   P(2.20) P(tot)
_
  1 5.7178.46 0.00 0.01
  2 2.8556.91 0.62 0.70
  3 1.9035.37 0.37 0.29
  4 1.4313.82 0.00 0.00
_


The phaser molecular replacement gives the following table.
istogram of relative frequencies of VM values
   --
   Frequency of most common VM value normalized to 1
   VM values plotted in increments of 1/VM (0.02)

--- relative frequency ---
0.0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1.0
|||||||||||
   10.00 -
8.33 -
7.14 -
6.25 -
5.56 -
5.00 -
4.55 -
4.17 -
3.85 --
3.57 ---
3.33 --
3.12 --
2.94  (COMPOSITION*1)
2.78 ---
2.63 
2.50 -
2.38 
2.27 --
2.17 ---
2.08 --
2.00 --
1.92 ---
1.85 ---
1.79 ---
1.72 -
1.67 -
1.61 -
1.56 -
1.52 -
1.47 * (COMPOSITION*2)
1.43 -
1.39 -
1.35 -
1.32 -
1.28 -
1.25 -

$TABLE : Cell Content Analysis:
$SCATTER
:N*Composition vs Probability:0|3x0|1:1,2:
$$
N*Composition Probability
$$ loggraph $$
1 0.306066
2 0.00141804
$$

   Most probable VM for resolution = 2.27817
   Most probable MW of protein in asu for resolution = 92664.2

Thank a lot in advance

Re: [ccp4bb] molecular replacement problem.

[Raji Edayathumangalam]

Dear Appu,

I am not sure that I have a complete sense of the issue at hand since some
of the information needed to think your issue through is missing in your
email. For example, to what high resolution cut-off were the data measured?
What resolution limits were used for the MR search? How do the unit cell
dimensions and space group in the two cases compare?

I am guessing the ligand binding domain in your protein has the identical
sequence to that of the published ligand binding domain that you use as a
template in your MR search. In any case, here are a couple of my thoughts:

(1) It might be worth setting up different runs of MR with different
numbers for expected copies (not just two copies but also one copy and
three copies just in case you have one of the extreme cases of solvent
content)?

(2) If the MR solution is correct and there is physical room for a DNA
binding domain in your lattice (check by displaying symmetry mates),
perhaps the DNA binding domain is disordered. In that case (and if all
attempts with current data fail), you may have to crystallize the protein
in presence of DNA.


Good luck!
Raji




On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.com wrote:

 Dear members,

   I am doing a molecular replacement of a
 transcription factor whose ligand binding structure(24000 Da) is available
 in PDB but not for the DNA binding(13000 Da). When i am searching for the
 two copies from ligand binding domain as a template model, i am getting
 very good solution but i am not getting any density for the DNA binding
 domain to build up in density. The space gorup is P 1 21 1 (4) and unit
 cell parameters are Unit Cell:   57.43   69.36  105.99   90.00   90.00
 90.00. Please guide me how to get the complete model structure. Table below
 show the matthews statistics

  For estimated molecular weight   37000.
 Nmol/asym  Matthews Coeff  %solvent   P(2.20) P(tot)
 _
   1 5.7178.46 0.00 0.01
   2 2.8556.91 0.62 0.70
   3 1.9035.37 0.37 0.29
   4 1.4313.82 0.00 0.00
 _


 The phaser molecular replacement gives the following table.
 istogram of relative frequencies of VM values
--
Frequency of most common VM value normalized to 1
VM values plotted in increments of 1/VM (0.02)

 --- relative frequency ---
 0.0  0.1  0.2  0.3  0.4  0.5  0.6  0.7  0.8  0.9  1.0
 |||||||||||
10.00 -
 8.33 -
 7.14 -
 6.25 -
 5.56 -
 5.00 -
 4.55 -
 4.17 -
 3.85 --
 3.57 ---
 3.33 --
 3.12 --
 2.94  (COMPOSITION*1)
 2.78 ---
 2.63 
 2.50 -
 2.38 
 2.27 --
 2.17 ---
 2.08 --
 2.00 --
 1.92 ---
 1.85 ---
 1.79 ---
 1.72 -
 1.67 -
 1.61 -
 1.56 -
 1.52 -
 1.47 * (COMPOSITION*2)
 1.43 -
 1.39 -
 1.35 -
 1.32 -
 1.28 -
 1.25 -

 $TABLE : Cell Content Analysis:
 $SCATTER
 :N*Composition vs Probability:0|3x0|1:1,2:
 $$
 N*Composition Probability
 $$ loggraph $$
 1 0.306066
 2 0.00141804
 $$

Most probable VM for resolution = 2.27817
Most probable MW of protein in asu for resolution = 92664.2

 Thank a lot in advance





-- 
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University

[ccp4bb] molecular replacement for flexible domain of protein

[Appu kumar]

Dear ccp4 user,
 I have problem in finding the phase for the
flexible DNA binding domain of LTTR protein. The unit cell parameters are ,Unit
Cell: 46.92 105.30 47.75 90.00 103.26 90.00, and space-Group P 1 21 1.
Molecular weight of protein is 36000Da. When i am running phaser it showing
that
Most probable MW of protein in asu for resolution = 49714.8. Could you
please guide me how to set the proper setting for finding the correct
rotation function and translation function setting to get the phases for
the full length protein. I m getting the phases for the ligand binding
domain but not for the DNA binding domain. Could you please suggest me some
tricks which would work to get the phase. Thanks a lot in advance
Appu

Re: [ccp4bb] molecular replacement for flexible domain of protein

[Herman . Schreuder]

Dear Appu,
 
you give very little details, so it is not possible to give very
specific advice. Here are some general points:
-the Mw of your protein is 36 kDa, what is the Mw of the ligand binding
domain and of the DNA binding domain? If the ligand binding domain is a
seizable part of the total, you may get enough phase information from
the ligand binding domain alone. This will depend on the resolution you
have. Did you have a look at the electron density maps, phased with the
ligand binding domain alone?
-I asume you used the DNA- and ligand binding domains as separate models
and not the full-length protein? If not, you should separate the domains
and remove the linker from your search model and run again molecular
replacement.
-Are there different models for the DNA binding domain available (from
other complexes, homologous proteins)? If so, you should try these as
well, or even try them all at once. How this is done is explained in the
phaser manual.
-The most probable Mw in the asu is the Mw which would give ~50%
solvent. If your Mw is different, this means that you either have a lot
of solvent (1 molecule in the asu) or little solvent (2 molecules in the
asu). You have to try both options.
 
Good luck!
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Appu kumar
Sent: Monday, February 18, 2013 10:52 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] molecular replacement for flexible domain of
protein


Dear ccp4 user,
 I have problem in finding the phase for
the flexible DNA binding domain of LTTR protein. The unit cell
parameters are ,Unit Cell: 46.92 105.30 47.75 90.00 103.26 90.00, and
space-Group P 1 21 1. Molecular weight of protein is 36000Da. When i am
running phaser it showing that 
Most probable MW of protein in asu for resolution = 49714.8.
Could you please guide me how to set the proper setting for finding the
correct rotation function and translation function setting to get the
phases for the full length protein. I m getting the phases for the
ligand binding domain but not for the DNA binding domain. Could you
please suggest me some tricks which would work to get the phase. Thanks
a lot in advance
Appu

[ccp4bb] Molecular replacement of chimeric protein

[RHYS GRINTER]

Hi All,

Thanks for the all the advice on my previous question regarding the likely 
photo-reduction of my crystals.

I've now collected a data set on these crystals. The protein is a chimera of 
two domains of known structure (Ferredoxin domain and Colicin domain), what's 
the best way to use all of this information in molecular replacement i.e. can 
you use two search models simultaneously in Phaser?

Thanks again

Rhys Grinter

Re: [ccp4bb] Molecular replacement

[Eleanor Dodson]

Thank you very much!
Eleanor



On 19 April 2012 19:43, Bret Wallace bretw...@gmail.com wrote:

 I noticed this missing when I first installed v. 6.2 as well.

 They noted this in the problems page.  You just need to replace the
 phaser_MR.tcl file with the updated version in the updates page.  I tried
 this earlier and it created a new line in the GUI to specify the packing
 criterion under Additional Parameters.  I think this is what you were
 referring to?

 Bret

Re: [ccp4bb] Molecular replacement

[Eleanor Dodson]

Several possibilities.

1) Phaser is very prone to reject solutions because of packing clashes..
 (PS How do you reset the packing limit in the current GUI?)
Running chainsaw before you begin the search can help - it prunes out
patches where the sequences don't match and gives a sensibly truncated
search model.

2) There is only one molecule - does it pack reasonably or are there great
holes in the map with suspicious density for a 2nd molecule?

I would refine the molecule you have and rebuild if possible, then use that
model to search again
Eleanor Dodson



On 19 April 2012 03:24, Ed Pozharski epozh...@umaryland.edu wrote:

 36% solvent sounds too low.  Most protein crystals are at ~50%.  On the
 other hand, if you assume one molecule, your solvent content jumps to
 68% - not unheard of, but somewhat high for 1.7A resolution dataset.

 But you have a good MR solution, just try to refine/rebuild and see what
 you have in the density.

 Is your protein a dimer by any chance?  Then you may have two dimers,
 only one is formed by crystal symmetry.  Thus you'd have 1.5 molecules
 in asu, which would result in solvent content of ~52% - just right.  If
 that is the case, run MR with the monomer.

 Cheers,

 Ed.

 On Thu, 2012-04-19 at 02:26 +0100, Krithika Sundaram wrote:
  Hi all,
 
  I am working on an oxidoreductase and having some trouble during
 molecular replacement.
 
  The resolution of the crystal is 1.7 A and the space group is I4122 (a =
 b = 121.086, c =156.93 and alpha = beta = gamma = 90).
 
  The cell content analysis results  predicted two molecules in the
 asymmetric unit and the solvent content as 36 %. The Matthew's coefficient
 is 1.94. The Wilson plot also looks fine.
 
  However, after molecular replacement (using Phaser) the result just
 gives me just a single molecule. ( RFZ = 8.5 TFZ =13.9 PAK = 0 LLG =189
 TFZ=18.0 LLG = 642)
 
  Any suggestions how to solve this problem?
 
  Thanks in advance.
 

Re: [ccp4bb] Molecular replacement

[Bret Wallace]

I noticed this missing when I first installed v. 6.2 as well.

They noted this in the problems page.  You just need to replace the 
phaser_MR.tcl file with the updated version in the updates page.  I tried this 
earlier and it created a new line in the GUI to specify the packing criterion 
under Additional Parameters.  I think this is what you were referring to?

Bret

Re: [ccp4bb] Molecular Replacement

[Eleanor Dodson]

I always start MR by running chainsaw - you need a pit file or fast or
blast output with the sequence alignment of the template and your new
protein.
chainsaw will edit the template to a) renumber residues taking account of
any insertions and deletions  b) prune and rename residues to the given
sequence. e.g. if you have PHE and template has TYR the OH atom will be
removed and the rest kept.
This will actually help your MR search - there should be fewer clashes..
And it most certainly helps during rebuilding..
Eleanor


On 18 April 2012 00:34, Uma Ratu rosiso2...@gmail.com wrote:

  Does that mean a different number of molecules in the asymmetric was
 found,

 With Phaser, 4 monomers. With AutoMR, 2 monomers.

 did you divide the molecule and find each part separately?

 Each monomer (by AutoMR) is composed of two chains. One chain is part
 of my target. The other chain matchs to the rest of my target.

 The original template .pdb is a tetramer with four monomers. I used
 one of the monomer as the template for the molecualr replacement in
 both Phaser and AutoMR. The seq file is the whole of my target.

 Provide more details like space group

 Space group is P21.

  whether the tetramer is crystallograhic or all in the asymmetric unit,

 The tetramer is crystallograhic

 Thank you

 Ros

 On 4/18/12, Edward A. Berry ber...@upstate.edu wrote:
One more question about Molecular Replacement. With Phaser, I got
tetramer conformation. With autoMR, it gave me dimer conformation.
Each molecuale was trucated into two chains.  The scores from both
 
  Does that mean a different number of molecules in the asymmetric
  was found, or just that they were arranged differently? If the latter,
  try generating symmetry-mates around one dimer and see if the tetramer
  is created. Ideally if the solutions are right they should be the
  same, within an arbitrary choice of asymmetric unit and origin.
 
  What does it mean, each molecule was . .  two chains? did you
  divide the molecule and find each part separately? Is you
  dimer a heterodimer? Provide more details like space group and
  whether the tetramer is crystallograhic or all in the asymmetric
  unit, and some expert may be able to provide suggestions.
 
 
  Uma Ratu wrote:
  Thank you very much for your inputs and comments.
 
  I am getting understand what is going on now.
 
  If your resolution is high (2.2 A or better?) and you have ARP/wARP
 
  Yes, the resolution is about 2A. I have ARP/wARP. Will give a try.
 
  One more question about Molecular Replacement. With Phaser, I got
  tetramer conformation. With autoMR, it gave me dimer conformation.
  Each molecuale was trucated into two chains.  The scores from both
  methods were high, as expected from 90% sequence identity between
  template and target.
 
  Thank you for advice
 
  Ros
 
 
  On 4/18/12, Edward A. Berryber...@upstate.edu  wrote:
  I would say yes. In any case you need to examine each mutated
  residue to be sure the correct conformation is chosen, so you might
  as well do the mutagenesis in coot or O.  The ccp4 chainsaw program
  is sometimes used to prepare models for MR, but it truncates the
 mutated
  residues to variable extent rather than mutating- the purpose is to
  improve
  chances of success, not to arrive at a model with the correct sequence.
 
  If your resolution is high (2.2 A or better?) and you have ARP/wARP
  installed, run A/W in the mode to improve an existing model, giving
 it
  your current model and the correct sequence. Not only will it build
  most of the mutated residues correctly, but in its role as a model
 bias
  remover it will fix or remove incorrect parts of the structure that
 may
  not be obvious in the initial maps.
 
  Uma Ratu wrote:
  Hello,
  I have a question about molecular replacement.
  I use Phaser or AutoMR to generate models of my target protein.
  Input .mtz is from X-ray diffraction. Template is from a known
  structure. I also set up seq file using my target protein. The
 sequence
  identity between template and my target protein is quite high, over
 90%.
  When I exam the ouputs, I found the sequence of the output .pdb is
  exactly same as the template.
  Is this normal for Molecular replacement?
  In order to have my target .pdb, I need to mutate the residues using
  coot?
  Thank you for advice
  Ros
 
 
 
 
 

Re: [ccp4bb] Molecular Replacement

[Ed Pozharski]

On Tue, 2012-04-17 at 17:49 -0400, Uma Ratu wrote:
 In order to have my target .pdb, I need to mutate the residues using
 coot? 

Others already recommended CHAINSAW to prepare the model.  Note that
coot has a nice feature under Extensions-All molecule... called [Post
MR] Fill partial residues  By default, CHAINSAW will truncate
non-conserved residues to gamma atom, so you will end up with some
weird-looking tryptophans.  See 

http://www.biop.ox.ac.uk/coot/doc/coot/Fill-Partial-Residues.html

for details.

IMPORTANT!  You still have to inspect the model manually, adjust some
side chains, build alternate conformers, fix the backbone where needed,
fill gaps etc.  But you already know this. 

Cheers,

Ed.

-- 
Coot verendus est

Re: [ccp4bb] Molecular Replacement

[Uma Ratu]

Ed:

Thank you very much for your advice and inputs

regards

Ros

On Wed, Apr 18, 2012 at 8:44 AM, Ed Pozharski epozh...@umaryland.eduwrote:

 On Tue, 2012-04-17 at 17:49 -0400, Uma Ratu wrote:
  In order to have my target .pdb, I need to mutate the residues using
  coot?

 Others already recommended CHAINSAW to prepare the model.  Note that
 coot has a nice feature under Extensions-All molecule... called [Post
 MR] Fill partial residues  By default, CHAINSAW will truncate
 non-conserved residues to gamma atom, so you will end up with some
 weird-looking tryptophans.  See

 http://www.biop.ox.ac.uk/coot/doc/coot/Fill-Partial-Residues.html

 for details.

 IMPORTANT!  You still have to inspect the model manually, adjust some
 side chains, build alternate conformers, fix the backbone where needed,
 fill gaps etc.  But you already know this.

 Cheers,

 Ed.

 --
 Coot verendus est

[ccp4bb] Molecular replacement

[Krithika Sundaram]

Hi all,
 
I am working on an oxidoreductase and having some trouble during molecular 
replacement.
 
The resolution of the crystal is 1.7 A and the space group is I4122 (a = b = 
121.086, c =156.93 and alpha = beta = gamma = 90). 
 
The cell content analysis results  predicted two molecules in the asymmetric 
unit and the solvent content as 36 %. The Matthew's coefficient is 1.94. The 
Wilson plot also looks fine.
 
However, after molecular replacement (using Phaser) the result just gives me 
just a single molecule. ( RFZ = 8.5 TFZ =13.9 PAK = 0 LLG =189 TFZ=18.0 LLG = 
642)
 
Any suggestions how to solve this problem?
 
Thanks in advance.
 

Re: [ccp4bb] Molecular replacement

[Ed Pozharski]

36% solvent sounds too low.  Most protein crystals are at ~50%.  On the
other hand, if you assume one molecule, your solvent content jumps to
68% - not unheard of, but somewhat high for 1.7A resolution dataset.

But you have a good MR solution, just try to refine/rebuild and see what
you have in the density.

Is your protein a dimer by any chance?  Then you may have two dimers,
only one is formed by crystal symmetry.  Thus you'd have 1.5 molecules
in asu, which would result in solvent content of ~52% - just right.  If
that is the case, run MR with the monomer.

Cheers,

Ed.

On Thu, 2012-04-19 at 02:26 +0100, Krithika Sundaram wrote:
 Hi all,
  
 I am working on an oxidoreductase and having some trouble during molecular 
 replacement.
  
 The resolution of the crystal is 1.7 A and the space group is I4122 (a = b = 
 121.086, c =156.93 and alpha = beta = gamma = 90). 
  
 The cell content analysis results  predicted two molecules in the asymmetric 
 unit and the solvent content as 36 %. The Matthew's coefficient is 1.94. The 
 Wilson plot also looks fine.
  
 However, after molecular replacement (using Phaser) the result just gives me 
 just a single molecule. ( RFZ = 8.5 TFZ =13.9 PAK = 0 LLG =189 TFZ=18.0 LLG = 
 642)
  
 Any suggestions how to solve this problem?
  
 Thanks in advance.
  

[ccp4bb] Molecular Replacement

[Uma Ratu]

Hello,

I have a question about molecular replacement.

I use Phaser or AutoMR to generate models of my target protein. Input
.mtz is from X-ray diffraction. Template is from a known structure. I also
set up seq file using my target protein. The sequence identity between
template and my target protein is quite high, over 90%.

When I exam the ouputs, I found the sequence of the output .pdb is exactly
same as the template.

Is this normal for Molecular replacement?

In order to have my target .pdb, I need to mutate the residues using coot?

Thank you for advice

Ros

Re: [ccp4bb] Molecular Replacement

[Edward A. Berry]

I would say yes. In any case you need to examine each mutated
residue to be sure the correct conformation is chosen, so you might
as well do the mutagenesis in coot or O.  The ccp4 chainsaw program
is sometimes used to prepare models for MR, but it truncates the mutated
residues to variable extent rather than mutating- the purpose is to improve
chances of success, not to arrive at a model with the correct sequence.

If your resolution is high (2.2 A or better?) and you have ARP/wARP
installed, run A/W in the mode to improve an existing model, giving it
your current model and the correct sequence. Not only will it build
most of the mutated residues correctly, but in its role as a model bias
remover it will fix or remove incorrect parts of the structure that may
not be obvious in the initial maps.

Uma Ratu wrote:

Hello,
I have a question about molecular replacement.
I use Phaser or AutoMR to generate models of my target protein.
Input .mtz is from X-ray diffraction. Template is from a known
structure. I also set up seq file using my target protein. The sequence
identity between template and my target protein is quite high, over 90%.
When I exam the ouputs, I found the sequence of the output .pdb is
exactly same as the template.
Is this normal for Molecular replacement?
In order to have my target .pdb, I need to mutate the residues using coot?
Thank you for advice
Ros

Re: [ccp4bb] Molecular Replacement

[Uma Ratu]

Thank you very much for your inputs and comments.

I am getting understand what is going on now.

 If your resolution is high (2.2 A or better?) and you have ARP/wARP

Yes, the resolution is about 2A. I have ARP/wARP. Will give a try.

One more question about Molecular Replacement. With Phaser, I got
tetramer conformation. With autoMR, it gave me dimer conformation.
Each molecuale was trucated into two chains.  The scores from both
methods were high, as expected from 90% sequence identity between
template and target.

Thank you for advice

Ros


On 4/18/12, Edward A. Berry ber...@upstate.edu wrote:
 I would say yes. In any case you need to examine each mutated
 residue to be sure the correct conformation is chosen, so you might
 as well do the mutagenesis in coot or O.  The ccp4 chainsaw program
 is sometimes used to prepare models for MR, but it truncates the mutated
 residues to variable extent rather than mutating- the purpose is to improve
 chances of success, not to arrive at a model with the correct sequence.

 If your resolution is high (2.2 A or better?) and you have ARP/wARP
 installed, run A/W in the mode to improve an existing model, giving it
 your current model and the correct sequence. Not only will it build
 most of the mutated residues correctly, but in its role as a model bias
 remover it will fix or remove incorrect parts of the structure that may
 not be obvious in the initial maps.

 Uma Ratu wrote:
 Hello,
 I have a question about molecular replacement.
 I use Phaser or AutoMR to generate models of my target protein.
 Input .mtz is from X-ray diffraction. Template is from a known
 structure. I also set up seq file using my target protein. The sequence
 identity between template and my target protein is quite high, over 90%.
 When I exam the ouputs, I found the sequence of the output .pdb is
 exactly same as the template.
 Is this normal for Molecular replacement?
 In order to have my target .pdb, I need to mutate the residues using coot?
 Thank you for advice
 Ros

Re: [ccp4bb] Molecular Replacement

[Uma Ratu]

 Does that mean a different number of molecules in the asymmetric was found,

With Phaser, 4 monomers. With AutoMR, 2 monomers.

did you divide the molecule and find each part separately?

Each monomer (by AutoMR) is composed of two chains. One chain is part
of my target. The other chain matchs to the rest of my target.

The original template .pdb is a tetramer with four monomers. I used
one of the monomer as the template for the molecualr replacement in
both Phaser and AutoMR. The seq file is the whole of my target.

Provide more details like space group

Space group is P21.

 whether the tetramer is crystallograhic or all in the asymmetric unit,

The tetramer is crystallograhic

Thank you

Ros

On 4/18/12, Edward A. Berry ber...@upstate.edu wrote:
   One more question about Molecular Replacement. With Phaser, I got
   tetramer conformation. With autoMR, it gave me dimer conformation.
   Each molecuale was trucated into two chains.  The scores from both

 Does that mean a different number of molecules in the asymmetric
 was found, or just that they were arranged differently? If the latter,
 try generating symmetry-mates around one dimer and see if the tetramer
 is created. Ideally if the solutions are right they should be the
 same, within an arbitrary choice of asymmetric unit and origin.

 What does it mean, each molecule was . .  two chains? did you
 divide the molecule and find each part separately? Is you
 dimer a heterodimer? Provide more details like space group and
 whether the tetramer is crystallograhic or all in the asymmetric
 unit, and some expert may be able to provide suggestions.


 Uma Ratu wrote:
 Thank you very much for your inputs and comments.

 I am getting understand what is going on now.

 If your resolution is high (2.2 A or better?) and you have ARP/wARP

 Yes, the resolution is about 2A. I have ARP/wARP. Will give a try.

 One more question about Molecular Replacement. With Phaser, I got
 tetramer conformation. With autoMR, it gave me dimer conformation.
 Each molecuale was trucated into two chains.  The scores from both
 methods were high, as expected from 90% sequence identity between
 template and target.

 Thank you for advice

 Ros


 On 4/18/12, Edward A. Berryber...@upstate.edu  wrote:
 I would say yes. In any case you need to examine each mutated
 residue to be sure the correct conformation is chosen, so you might
 as well do the mutagenesis in coot or O.  The ccp4 chainsaw program
 is sometimes used to prepare models for MR, but it truncates the mutated
 residues to variable extent rather than mutating- the purpose is to
 improve
 chances of success, not to arrive at a model with the correct sequence.

 If your resolution is high (2.2 A or better?) and you have ARP/wARP
 installed, run A/W in the mode to improve an existing model, giving it
 your current model and the correct sequence. Not only will it build
 most of the mutated residues correctly, but in its role as a model bias
 remover it will fix or remove incorrect parts of the structure that may
 not be obvious in the initial maps.

 Uma Ratu wrote:
 Hello,
 I have a question about molecular replacement.
 I use Phaser or AutoMR to generate models of my target protein.
 Input .mtz is from X-ray diffraction. Template is from a known
 structure. I also set up seq file using my target protein. The sequence
 identity between template and my target protein is quite high, over 90%.
 When I exam the ouputs, I found the sequence of the output .pdb is
 exactly same as the template.
 Is this normal for Molecular replacement?
 In order to have my target .pdb, I need to mutate the residues using
 coot?
 Thank you for advice
 Ros

[ccp4bb] molecular replacement of protein/DNA complex

[Wei Shi]

Hi all,

I have been trying to solve a protein-DNA complex structure using molecular
replacement. I suspect two copies of protein bind one piece of the DNA, and
the angle between the two copies of protein is somewhere between 130 to 180
degrees. I could get molecular replacement solution using a search model of
protein/DNA complex I made with deletion in part of the protein and with
the angle between the two proteins about 145 degrees. Only 1 copy of this
two-protein/one-DNA complex is expected in the ASU. Below is the statistics
for the solution.

RFZ=2.9 TFZ=6.9 PAK=0 LLG=111 TFZ==14.6 LLG=111

And when I open the pdb file and generate symmetry mates, no clashes and I
could see contacts between the end of DNA, but space instead of contacts
between layers of the protein/DNA complex. I suspect that the angle between
the two proteins I use in the search model is not quite right. No solution
if I search with DNA and protein separately. I am trying to find a way to
move the protein a little bit around the original solution and see whether
that can help me make the solution better. I just read that “ROTate AROUnd
option of the brute rotation serch” and “NMAPdb” might do this?  Does
anyone have any suggestions about what I could try? Thank you so much!

Best,
Wei

[ccp4bb] molecular replacement solution

[Careina Edgooms]

Dear CCP4 members

I have a confusion with molecular replacement. I wish to solve a monomeric 
protein in P21 where there are 2 monomers in asymmetric unit. I have a search 
model that also has 2 monomers in asymmetric unit. 


First I try using Phaser. I use only one chain of the monomer model and search 
for 2 copies. This gives a good solution that refines with low R-factor but it 
shows many clashes. In other words, the 2 monomers are too close together and 
there are clashes where they meet. Then I try using Phaser and both chains of 
the model and search for 1 copy. This does not give a solution and says 
asymmetric unit is too full. Then I try using MolRep with the 2 chain search 
model. It gives solution that looks better because you can see that the 2 
monomers do not physically clash but it refines very badly giving R factors of 
about 50%

So I am confused. What am I doing wrong? Please help me with suggestions
Many thanks
Careina



  

Re: [ccp4bb] molecular replacement solution

[Frederic VELLIEUX]

Quoting you:

I wish to solve a monomeric protein in P21 where there are 2 monomers in 
asymmetric unit.

How do you know that if you haven't solved the structure? Matthews coefficient 
calculations?

Remember that the solvent content in protein crystal structures can go from ca. 
15 % to ca. 85 %.

So what about having only one monomer in the asymmetric unit? What are the 
statistics given by Phaser then? Does the molecular replacement solution 
provided by 
Phaser for just one monomer explain the crystal (i.e. contacts in all 3 
directions of space so that the crystal can form, no void between rows of 
molecules).

The second possibility is having different orientations of surface loops (these 
surface loops would be clashing), so that you may have to trim them off before 
performing 
the molecular replacement calculations. Then you need to use the (improving) 
electron density in order to rebuild the surface loops in their correct 
positions.

HTH,

Fred.

(there are other possibilities of course, such as incorrect space group 
assignment)

 Message du 15/03/11 07:41
 De : Careina Edgooms 
 A : CCP4BB@JISCMAIL.AC.UK
 Copie à : 
 Objet : [ccp4bb] molecular replacement solution
 
 Dear CCP4 members
 
 I have a confusion with molecular replacement. I wish to solve a monomeric 
 protein in P21 where there are 2 monomers in asymmetric unit. I have a search 
 model that also has 2 monomers in asymmetric unit. 
 
 
 First I try using Phaser. I use only one chain of the monomer model and 
 search 
 for 2 copies. This gives a good solution that refines with low R-factor but 
 it 
 shows many clashes. In other words, the 2 monomers are too close together and 
 there are clashes where they meet. Then I try using Phaser and both chains of 
 the model and search for 1 copy. This does not give a solution and says 
 asymmetric unit is too full. Then I try using MolRep with the 2 chain search 
 model. It gives solution that looks better because you can see that the 2 
 monomers do not physically clash but it refines very badly giving R factors 
 of 
 about 50%
 
 So I am confused. What am I doing wrong? Please help me with suggestions
 Many thanks
 Careina
 
 
 
 

Re: [ccp4bb] molecular replacement solution

[David Schuller]

On 03/15/11 02:41, Careina Edgooms wrote:

Dear CCP4 members

I have a confusion with molecular replacement. I wish to solve a 
monomeric protein in P21 where there are 2 monomers in asymmetric unit.


How do you know that? What would the %solvent be with one monomer, and 
with two?


If, using Phaser, you stop with one molecule, how does the R-factor look 
after rigid body refinement?


Cheers,

--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

Re: [ccp4bb] molecular replacement solution

[Ed Pozharski]

On Mon, 2011-03-14 at 23:41 -0700, Careina Edgooms wrote:
 So I am confused. What am I doing wrong? Please help me with
 suggestions

Generally, it may be a good idea to post phaser log-file, etc. when
asking a question.  You may be able to get more specific suggestions
then - otherwise, all one can say is general things like are you sure
you have the right spacegroup?, what is the solvent content?, etc.

-- 
I'd jump in myself, if I weren't so good at whistling.
   Julian, King of Lemurs

Re: [ccp4bb] molecular replacement solution

[Francis E Reyes]

Is there NCS? (How's the self rotation look? How about the native  
patterson?)


Does your search model have flexible loops? You may have the right  
solution, but the flexible loops may be in a different conformation  
and cause clashes. Trim the search model to the conserved / structured  
domains.


Do you have *any* anomalous signal? (whether you collected Sulfur-SAD,  
you have a metalloprotein, etc). These can be useful in verifying the  
correctness of the MR solution.




I use only one chain of the monomer model



Why?


F

On Mar 15, 2011, at 12:41 AM, Careina Edgooms wrote:


Dear CCP4 members

I have a confusion with molecular replacement. I wish to solve a  
monomeric protein in P21 where there are 2 monomers in asymmetric  
unit. I have a search model that also has 2 monomers in asymmetric  
unit.


First I try using Phaser. I use only one chain of the monomer model  
and search for 2 copies. This gives a good solution that refines  
with low R-factor but it shows many clashes. In other words, the 2  
monomers are too close together and there are clashes where they  
meet. Then I try using Phaser and both chains of the model and  
search for 1 copy. This does not give a solution and says asymmetric  
unit is too full. Then I try using MolRep with the 2 chain search  
model. It gives solution that looks better because you can see that  
the 2 monomers do not physically clash but it refines very badly  
giving R factors of about 50%


So I am confused. What am I doing wrong? Please help me with  
suggestions

Many thanks
Careina




-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

Re: [ccp4bb] Molecular replacement question

[zhang yu]

Maybe there is a domain shift of your protein compared to the model. If this
is the case, try to do the MP with successive domains.

2010/9/13 Paul Holland pholl...@umd.edu

 Hello fellow crystallographers,

 I am trying molecular replacement for a protein crystal dataset that has
 very high sequence similarity to the search model with several predicted
 flexible loop regions; however, all attempts at finding a solution have not
 produce very ideal starting solutions using Phaser and Molrep (CC = 0.3 and
 Z-score = 5).  I am very confident that the unit cell parameters are C2
 84.027  120.565  108.272  90.00 104.71  90.00, and there appears to be no
 evidence of twinning.  The Matthews calculation predicts from anywhere from
 2-4 monomers in the ASU, and calculation of the SRF in Molrep does not
 identify any peaks in higher order symmetry except for the expected
 crystallographic two-fold for C2.  Below is the table from the calculated
 SRF in molrep.  Any advice would be greatly appreciated.

 #thetaphichi alpha   beta  gamma Rf
 Rf/sigma
 Sol_RF   1 0.000.000.000.000.000.00 870.5
21.59
 Sol_RF   258.61  -10.17  180.00  169.83 -117.23   10.17 162.5
  4.03
 Sol_RF   366.02   -0.00  180.00  180.00 -132.030.00 161.1
  4.00
 Sol_RF   458.42   -9.54  180.00  170.46 -116.859.54 159.8
  3.96
 Sol_RF   5   149.840.00  180.00 -180.00   60.320.00 156.0
  3.87
 Sol_RF   658.96   -5.52  180.00  174.48 -117.915.52 151.5
  3.76
 Sol_RF   765.59   20.95  180.00   20.95  131.18  159.05 143.9
  3.57
 Sol_RF   890.00  -98.96  180.000.00  180.00   17.92 142.9
  3.55
 Sol_RF   956.53   15.78  180.00   15.78  113.07  164.22 142.0
  3.52
 Sol_RF  1071.10  -19.94  180.00  160.06 -142.20   19.94 141.6
  3.51
 Sol_RF  1171.28   29.78  180.00   29.78  142.55  150.22 140.4
  3.48
 Sol_RF  1265.22  -15.88  180.00  164.12 -130.44   15.88 139.2
  3.45
 Sol_RF  1368.84   -0.00  180.00  180.00 -137.670.00 138.0
  3.42
 Sol_RF  1432.51 -180.00  180.00 -180.00   65.02   -0.00 137.9
  3.42
 Sol_RF  1575.02  -28.84  180.00  151.16 -150.04   28.84 134.7
  3.34
 Sol_RF  1671.69  -20.99  180.00  159.01 -143.37   20.99 133.0
  3.30
 Sol_RF  1792.13  101.46  179.93  102.35 -175.74   79.42 130.9
  3.25
 Sol_RF  18   107.89  144.73  179.79  145.06 -144.22   35.61 128.8
  3.19
 Sol_RF  1987.45  -78.19  180.00  101.81 -174.90   78.19 128.1
  3.18
 Sol_RF  2038.570.69   30.36  102.66  -18.79  -78.71 122.4
  3.04
 Sol_RF  2126.77  174.59  176.58  172.68   53.523.49 120.5
  2.99
 Sol_RF  22   116.66  178.08  175.143.49  126.48  187.32 120.5
  2.99
 Sol_RF  2375.56  -41.35  180.00  138.65 -151.12   41.35 119.8
  2.97
 Sol_RF  2466.12   36.35  180.00   36.35  132.24  143.65 116.6
  2.89
 Sol_RF  2583.87   71.62  180.00   71.62  167.74  108.38 114.7
  2.85
 Sol_RF  2669.24  -12.37  180.00  167.63 -138.48   12.37 112.3
  2.79
 Sol_RF  2759.75   15.26  172.297.64  119.07  157.12 112.2
  2.78
 Sol_RF  28   120.25 -164.74  172.29   22.88  119.07  172.36 112.2
  2.78
 Sol_RF  2996.68  -70.99  180.00  109.01 -166.63   70.99 110.9
  2.75
 Sol_RF  3063.23  -44.73  180.00  135.27 -126.47   44.73 108.9
  2.70

 Cheers,

 Paul Holland





-- 
Yu Zhang
HHMI associate
Waksman Institute
Rutgers University

Re: [ccp4bb] Molecular replacement question

[Donnie Berkholz]

On 09:50 Tue 14 Sep , Dirk Kostrewa wrote:
 I would spend some time in improving the search model, first. If there 
 are more than one possible search molecules in the PDB, I usually do a 
 structural alignment (ssm superposition) to get an idea about the 
 flexibility of the search molecules. Then I remove all parts that I 
 suspect to be flexible (usually N- and C-termini, long loops), until I 
 get a very compact search model.

Dirk,

If you aren't already doing this automatically, you could save some time 
next time around by using the MUSTANG-MR server 
http://pxgrid.med.monash.edu.au:8080/mustangserver/. It does the 
second part (finding conserved structural regions) automatically with a 
variety of RMSD cutoffs. We've recently had some success on a difficult 
case by coupling the SSM and MUSTANG-MR servers, using the top ~10 SSM 
results (up to ~3 Å RMSD on common regions) and a 1.4 Å MUSTANG cutoff.

-- 
Thanks,
Donnie

Donald S. Berkholz, Ph.D.
Research Fellow
James R. Thompson lab, Physiology  Biomedical Engineering
Grazia Isaya lab, Pediatric  Adolescent Medicine
Medical Sciences 2-66
Mayo Clinic College of Medicine
200 First Street SW
Rochester, MN 55905
office: 507-538-6924
cell: 612-991-1321


pgpgKe946izdO.pgp
Description: PGP signature

Re: [ccp4bb] Molecular replacement question

[Dirk Kostrewa]

 Dear Paul Holland,

I would spend some time in improving the search model, first. If there 
are more than one possible search molecules in the PDB, I usually do a 
structural alignment (ssm superposition) to get an idea about the 
flexibility of the search molecules. Then I remove all parts that I 
suspect to be flexible (usually N- and C-termini, long loops), until I 
get a very compact search model. You may try a model with no side-chain 
truncation, since in the hydrophobic core, there might be a similar 
isosteric packing of atoms. In addition, I would truncate the side 
chains either to the longest common chain (with ctruncate) if there is a 
good sequence alignment, or to poly-Ser or even poly-Ala. I would try 
these core-models as search models, with the idea that missing atoms 
lower the signal, but wrong atoms raise the noise and may produce wrong 
clashes. In case of several search models, where each individual model 
fails, you may try to run Phaser using all of them simultaneously as a 
search ensemble.
This model-pruning together with a search-ensemble of several PDB 
entries worked recently in a project, where otherwise all molecular 
replacement trials failed.


Good luck,

Dirk.

Am 13.09.10 16:52, schrieb Paul Holland:

Hello fellow crystallographers,

I am trying molecular replacement for a protein crystal dataset that has very 
high sequence similarity to the search model with several predicted flexible 
loop regions; however, all attempts at finding a solution have not produce very 
ideal starting solutions using Phaser and Molrep (CC = 0.3 and Z-score = 5).  I 
am very confident that the unit cell parameters are C2 84.027  120.565  108.272 
 90.00 104.71  90.00, and there appears to be no evidence of twinning.  The 
Matthews calculation predicts from anywhere from 2-4 monomers in the ASU, and 
calculation of the SRF in Molrep does not identify any peaks in higher order 
symmetry except for the expected crystallographic two-fold for C2.  Below is 
the table from the calculated SRF in molrep.  Any advice would be greatly 
appreciated.

#thetaphichi alpha   beta  gamma Rf 
Rf/sigma
Sol_RF   1 0.000.000.000.000.000.00 870.5   
   21.59
Sol_RF   258.61  -10.17  180.00  169.83 -117.23   10.17 162.5  4.03
Sol_RF   366.02   -0.00  180.00  180.00 -132.030.00 161.1  4.00
Sol_RF   458.42   -9.54  180.00  170.46 -116.859.54 159.8  3.96
Sol_RF   5   149.840.00  180.00 -180.00   60.320.00 156.0  3.87
Sol_RF   658.96   -5.52  180.00  174.48 -117.915.52 151.5  3.76
Sol_RF   765.59   20.95  180.00   20.95  131.18  159.05 143.9  3.57
Sol_RF   890.00  -98.96  180.000.00  180.00   17.92 142.9  3.55
Sol_RF   956.53   15.78  180.00   15.78  113.07  164.22 142.0  3.52
Sol_RF  1071.10  -19.94  180.00  160.06 -142.20   19.94 141.6  3.51
Sol_RF  1171.28   29.78  180.00   29.78  142.55  150.22 140.4  3.48
Sol_RF  1265.22  -15.88  180.00  164.12 -130.44   15.88 139.2  3.45
Sol_RF  1368.84   -0.00  180.00  180.00 -137.670.00 138.0  3.42
Sol_RF  1432.51 -180.00  180.00 -180.00   65.02   -0.00 137.9  3.42
Sol_RF  1575.02  -28.84  180.00  151.16 -150.04   28.84 134.7  3.34
Sol_RF  1671.69  -20.99  180.00  159.01 -143.37   20.99 133.0  3.30
Sol_RF  1792.13  101.46  179.93  102.35 -175.74   79.42 130.9  3.25
Sol_RF  18   107.89  144.73  179.79  145.06 -144.22   35.61 128.8  3.19
Sol_RF  1987.45  -78.19  180.00  101.81 -174.90   78.19 128.1  3.18
Sol_RF  2038.570.69   30.36  102.66  -18.79  -78.71 122.4  3.04
Sol_RF  2126.77  174.59  176.58  172.68   53.523.49 120.5  2.99
Sol_RF  22   116.66  178.08  175.143.49  126.48  187.32 120.5  2.99
Sol_RF  2375.56  -41.35  180.00  138.65 -151.12   41.35 119.8  2.97
Sol_RF  2466.12   36.35  180.00   36.35  132.24  143.65 116.6  2.89
Sol_RF  2583.87   71.62  180.00   71.62  167.74  108.38 114.7  2.85
Sol_RF  2669.24  -12.37  180.00  167.63 -138.48   12.37 112.3  2.79
Sol_RF  2759.75   15.26  172.297.64  119.07  157.12 112.2  2.78
Sol_RF  28   120.25 -164.74  172.29   22.88  119.07  172.36 112.2  2.78
Sol_RF  2996.68  -70.99  180.00  109.01 -166.63   70.99 110.9  2.75
Sol_RF  3063.23  -44.73  180.00  135.27 -126.47   44.73 108.9  2.70

Cheers,

Paul Holland




--

***
Dirk Kostrewa
Gene Center Munich, A5.07
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:+49-89-2180-76999
E-mail: kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de

Re: [ccp4bb] Molecular replacement question

[xaravich ivan]

Two questions.
What is the resolution of your data? what is the percentage sequence
identity?

even if you are confident of C2, try using PHASER with all space groups and
searching for 2-4 monomers.


Ivan

On Mon, Sep 13, 2010 at 7:52 AM, Paul Holland pholl...@umd.edu wrote:

 Hello fellow crystallographers,

 I am trying molecular replacement for a protein crystal dataset that has
 very high sequence similarity to the search model with several predicted
 flexible loop regions; however, all attempts at finding a solution have not
 produce very ideal starting solutions using Phaser and Molrep (CC = 0.3 and
 Z-score = 5).  I am very confident that the unit cell parameters are C2
 84.027  120.565  108.272  90.00 104.71  90.00, and there appears to be no
 evidence of twinning.  The Matthews calculation predicts from anywhere from
 2-4 monomers in the ASU, and calculation of the SRF in Molrep does not
 identify any peaks in higher order symmetry except for the expected
 crystallographic two-fold for C2.  Below is the table from the calculated
 SRF in molrep.  Any advice would be greatly appreciated.

 #thetaphichi alpha   beta  gamma Rf
 Rf/sigma
 Sol_RF   1 0.000.000.000.000.000.00 870.5
21.59
 Sol_RF   258.61  -10.17  180.00  169.83 -117.23   10.17 162.5
  4.03
 Sol_RF   366.02   -0.00  180.00  180.00 -132.030.00 161.1
  4.00
 Sol_RF   458.42   -9.54  180.00  170.46 -116.859.54 159.8
  3.96
 Sol_RF   5   149.840.00  180.00 -180.00   60.320.00 156.0
  3.87
 Sol_RF   658.96   -5.52  180.00  174.48 -117.915.52 151.5
  3.76
 Sol_RF   765.59   20.95  180.00   20.95  131.18  159.05 143.9
  3.57
 Sol_RF   890.00  -98.96  180.000.00  180.00   17.92 142.9
  3.55
 Sol_RF   956.53   15.78  180.00   15.78  113.07  164.22 142.0
  3.52
 Sol_RF  1071.10  -19.94  180.00  160.06 -142.20   19.94 141.6
  3.51
 Sol_RF  1171.28   29.78  180.00   29.78  142.55  150.22 140.4
  3.48
 Sol_RF  1265.22  -15.88  180.00  164.12 -130.44   15.88 139.2
  3.45
 Sol_RF  1368.84   -0.00  180.00  180.00 -137.670.00 138.0
  3.42
 Sol_RF  1432.51 -180.00  180.00 -180.00   65.02   -0.00 137.9
  3.42
 Sol_RF  1575.02  -28.84  180.00  151.16 -150.04   28.84 134.7
  3.34
 Sol_RF  1671.69  -20.99  180.00  159.01 -143.37   20.99 133.0
  3.30
 Sol_RF  1792.13  101.46  179.93  102.35 -175.74   79.42 130.9
  3.25
 Sol_RF  18   107.89  144.73  179.79  145.06 -144.22   35.61 128.8
  3.19
 Sol_RF  1987.45  -78.19  180.00  101.81 -174.90   78.19 128.1
  3.18
 Sol_RF  2038.570.69   30.36  102.66  -18.79  -78.71 122.4
  3.04
 Sol_RF  2126.77  174.59  176.58  172.68   53.523.49 120.5
  2.99
 Sol_RF  22   116.66  178.08  175.143.49  126.48  187.32 120.5
  2.99
 Sol_RF  2375.56  -41.35  180.00  138.65 -151.12   41.35 119.8
  2.97
 Sol_RF  2466.12   36.35  180.00   36.35  132.24  143.65 116.6
  2.89
 Sol_RF  2583.87   71.62  180.00   71.62  167.74  108.38 114.7
  2.85
 Sol_RF  2669.24  -12.37  180.00  167.63 -138.48   12.37 112.3
  2.79
 Sol_RF  2759.75   15.26  172.297.64  119.07  157.12 112.2
  2.78
 Sol_RF  28   120.25 -164.74  172.29   22.88  119.07  172.36 112.2
  2.78
 Sol_RF  2996.68  -70.99  180.00  109.01 -166.63   70.99 110.9
  2.75
 Sol_RF  3063.23  -44.73  180.00  135.27 -126.47   44.73 108.9
  2.70

 Cheers,

 Paul Holland

[ccp4bb] Molecular replacement question

[Paul Holland]

Hello fellow crystallographers,

I am trying molecular replacement for a protein crystal dataset that has very 
high sequence similarity to the search model with several predicted flexible 
loop regions; however, all attempts at finding a solution have not produce very 
ideal starting solutions using Phaser and Molrep (CC = 0.3 and Z-score = 5).  I 
am very confident that the unit cell parameters are C2 84.027  120.565  108.272 
 90.00 104.71  90.00, and there appears to be no evidence of twinning.  The 
Matthews calculation predicts from anywhere from 2-4 monomers in the ASU, and 
calculation of the SRF in Molrep does not identify any peaks in higher order 
symmetry except for the expected crystallographic two-fold for C2.  Below is 
the table from the calculated SRF in molrep.  Any advice would be greatly 
appreciated.

#thetaphichi alpha   beta  gamma Rf 
Rf/sigma
Sol_RF   1 0.000.000.000.000.000.00 870.5   
   21.59
Sol_RF   258.61  -10.17  180.00  169.83 -117.23   10.17 162.5  4.03
Sol_RF   366.02   -0.00  180.00  180.00 -132.030.00 161.1  4.00
Sol_RF   458.42   -9.54  180.00  170.46 -116.859.54 159.8  3.96
Sol_RF   5   149.840.00  180.00 -180.00   60.320.00 156.0  3.87
Sol_RF   658.96   -5.52  180.00  174.48 -117.915.52 151.5  3.76
Sol_RF   765.59   20.95  180.00   20.95  131.18  159.05 143.9  3.57
Sol_RF   890.00  -98.96  180.000.00  180.00   17.92 142.9  3.55
Sol_RF   956.53   15.78  180.00   15.78  113.07  164.22 142.0  3.52
Sol_RF  1071.10  -19.94  180.00  160.06 -142.20   19.94 141.6  3.51
Sol_RF  1171.28   29.78  180.00   29.78  142.55  150.22 140.4  3.48
Sol_RF  1265.22  -15.88  180.00  164.12 -130.44   15.88 139.2  3.45
Sol_RF  1368.84   -0.00  180.00  180.00 -137.670.00 138.0  3.42
Sol_RF  1432.51 -180.00  180.00 -180.00   65.02   -0.00 137.9  3.42
Sol_RF  1575.02  -28.84  180.00  151.16 -150.04   28.84 134.7  3.34
Sol_RF  1671.69  -20.99  180.00  159.01 -143.37   20.99 133.0  3.30
Sol_RF  1792.13  101.46  179.93  102.35 -175.74   79.42 130.9  3.25
Sol_RF  18   107.89  144.73  179.79  145.06 -144.22   35.61 128.8  3.19
Sol_RF  1987.45  -78.19  180.00  101.81 -174.90   78.19 128.1  3.18
Sol_RF  2038.570.69   30.36  102.66  -18.79  -78.71 122.4  3.04
Sol_RF  2126.77  174.59  176.58  172.68   53.523.49 120.5  2.99
Sol_RF  22   116.66  178.08  175.143.49  126.48  187.32 120.5  2.99
Sol_RF  2375.56  -41.35  180.00  138.65 -151.12   41.35 119.8  2.97
Sol_RF  2466.12   36.35  180.00   36.35  132.24  143.65 116.6  2.89
Sol_RF  2583.87   71.62  180.00   71.62  167.74  108.38 114.7  2.85
Sol_RF  2669.24  -12.37  180.00  167.63 -138.48   12.37 112.3  2.79
Sol_RF  2759.75   15.26  172.297.64  119.07  157.12 112.2  2.78
Sol_RF  28   120.25 -164.74  172.29   22.88  119.07  172.36 112.2  2.78
Sol_RF  2996.68  -70.99  180.00  109.01 -166.63   70.99 110.9  2.75
Sol_RF  3063.23  -44.73  180.00  135.27 -126.47   44.73 108.9  2.70

Cheers,

Paul Holland

Re: [ccp4bb] Molecular replacement question

[Eleanor Dodson]

Does your model structure form a dimer?  Maybe best to search with that 
model..

eleanor

Paul Holland wrote:

Hello fellow crystallographers,

I am trying molecular replacement for a protein crystal dataset that has very 
high sequence similarity to the search model with several predicted flexible 
loop regions; however, all attempts at finding a solution have not produce very 
ideal starting solutions using Phaser and Molrep (CC = 0.3 and Z-score = 5).  I 
am very confident that the unit cell parameters are C2 84.027  120.565  108.272 
 90.00 104.71  90.00, and there appears to be no evidence of twinning.  The 
Matthews calculation predicts from anywhere from 2-4 monomers in the ASU, and 
calculation of the SRF in Molrep does not identify any peaks in higher order 
symmetry except for the expected crystallographic two-fold for C2.  Below is 
the table from the calculated SRF in molrep.  Any advice would be greatly 
appreciated.

#thetaphichi alpha   beta  gamma Rf 
Rf/sigma
Sol_RF   1 0.000.000.000.000.000.00 870.5   
   21.59
Sol_RF   258.61  -10.17  180.00  169.83 -117.23   10.17 162.5  4.03
Sol_RF   366.02   -0.00  180.00  180.00 -132.030.00 161.1  4.00
Sol_RF   458.42   -9.54  180.00  170.46 -116.859.54 159.8  3.96
Sol_RF   5   149.840.00  180.00 -180.00   60.320.00 156.0  3.87
Sol_RF   658.96   -5.52  180.00  174.48 -117.915.52 151.5  3.76
Sol_RF   765.59   20.95  180.00   20.95  131.18  159.05 143.9  3.57
Sol_RF   890.00  -98.96  180.000.00  180.00   17.92 142.9  3.55
Sol_RF   956.53   15.78  180.00   15.78  113.07  164.22 142.0  3.52
Sol_RF  1071.10  -19.94  180.00  160.06 -142.20   19.94 141.6  3.51
Sol_RF  1171.28   29.78  180.00   29.78  142.55  150.22 140.4  3.48
Sol_RF  1265.22  -15.88  180.00  164.12 -130.44   15.88 139.2  3.45
Sol_RF  1368.84   -0.00  180.00  180.00 -137.670.00 138.0  3.42
Sol_RF  1432.51 -180.00  180.00 -180.00   65.02   -0.00 137.9  3.42
Sol_RF  1575.02  -28.84  180.00  151.16 -150.04   28.84 134.7  3.34
Sol_RF  1671.69  -20.99  180.00  159.01 -143.37   20.99 133.0  3.30
Sol_RF  1792.13  101.46  179.93  102.35 -175.74   79.42 130.9  3.25
Sol_RF  18   107.89  144.73  179.79  145.06 -144.22   35.61 128.8  3.19
Sol_RF  1987.45  -78.19  180.00  101.81 -174.90   78.19 128.1  3.18
Sol_RF  2038.570.69   30.36  102.66  -18.79  -78.71 122.4  3.04
Sol_RF  2126.77  174.59  176.58  172.68   53.523.49 120.5  2.99
Sol_RF  22   116.66  178.08  175.143.49  126.48  187.32 120.5  2.99
Sol_RF  2375.56  -41.35  180.00  138.65 -151.12   41.35 119.8  2.97
Sol_RF  2466.12   36.35  180.00   36.35  132.24  143.65 116.6  2.89
Sol_RF  2583.87   71.62  180.00   71.62  167.74  108.38 114.7  2.85
Sol_RF  2669.24  -12.37  180.00  167.63 -138.48   12.37 112.3  2.79
Sol_RF  2759.75   15.26  172.297.64  119.07  157.12 112.2  2.78
Sol_RF  28   120.25 -164.74  172.29   22.88  119.07  172.36 112.2  2.78
Sol_RF  2996.68  -70.99  180.00  109.01 -166.63   70.99 110.9  2.75
Sol_RF  3063.23  -44.73  180.00  135.27 -126.47   44.73 108.9  2.70

Cheers,

Paul Holland

 

Re: [ccp4bb] Molecular replacement question

[Ed Pozharski]

On Mon, 2010-09-13 at 15:52 +0100, Paul Holland wrote:
 that has very high sequence similarity to the search model

How high exactly?

-- 
I'd jump in myself, if I weren't so good at whistling.
   Julian, King of Lemurs

Re: [ccp4bb] Molecular replacement question

[Roger Rowlett]

Here is how I would approach this:


  Use Phaser to search with monomers, or
dimers if you suspect that the biological unit is composed of dimers
and have reasonable guess at a dimer search model.

  Also consider searching with N- and
C-terminal truncated search models. Sometimes a wayward N- or
C-terminal helix or loop makes a solution impossible to find because of
packing issues.

  No joy? Don't give up yet, try EPMR. It is
especially good at finding multiple chains in the ASU. If searching for
multiple copies of protein in the ASU, set the search criterion for
CC=1 to force an exhaustive search for partial solutions. (With the
default setting, EPMR gives up too early in our experience). We have
found that in difficult cases, you can use some or all of your
high-resolution data to improve the search in EPMR. (It is slow as all
get-out when you do this, but sometimes worth the wait. It's only CPU
cycles...) The resolution range you use in EPMR can have a profound
impact on the ability to find a solution.

  Examine any and all candidate solutions for
packing in the ASU. You may have to relax the packing criteria for
Phaser and EPMR to allow the solution to be found. If any of these look
like they pack reasonably well, they *might* be good starting points
for density modification. Partial solutions may give you some hints
about how many protein units you need in the ASU.
  We've had good luck in a recent "hard nut to
crack" using a marginal Phaser solution that packed well, followed by
DM using Parrot and autobuilding with Buccaneer. The basic orientation
of the proteins in the ASU were properly predicted by Phaser (8
monomers or 4 dimers--only the dimer search worked), but the initial
map was uninterpretable before DM. I think the rms difference between
the search model and final solution in this case was about 1.5 A.

Cheers.


Paul Holland wrote:

  Hello fellow crystallographers,

I am trying molecular replacement for a protein crystal dataset that has very high sequence similarity to the search model with several predicted flexible loop regions; however, all attempts at finding a solution have not produce very ideal starting solutions using Phaser and Molrep (CC = 0.3 and Z-score = 5).  I am very confident that the unit cell parameters are C2 84.027  120.565  108.272  90.00 104.71  90.00, and there appears to be no evidence of twinning.  The Matthews calculation predicts from anywhere from 2-4 monomers in the ASU, and calculation of the SRF in Molrep does not identify any peaks in higher order symmetry except for the expected crystallographic two-fold for C2.  Below is the table from the calculated SRF in molrep.  Any advice would be greatly appreciated.

#thetaphichi alpha   beta  gamma Rf Rf/sigma
Sol_RF   1 0.000.000.000.000.000.00 870.5  21.59
Sol_RF   258.61  -10.17  180.00  169.83 -117.23   10.17 162.5  4.03
Sol_RF   366.02   -0.00  180.00  180.00 -132.030.00 161.1  4.00
Sol_RF   458.42   -9.54  180.00  170.46 -116.859.54 159.8  3.96
Sol_RF   5   149.840.00  180.00 -180.00   60.320.00 156.0  3.87
Sol_RF   658.96   -5.52  180.00  174.48 -117.915.52 151.5  3.76
Sol_RF   765.59   20.95  180.00   20.95  131.18  159.05 143.9  3.57
Sol_RF   890.00  -98.96  180.000.00  180.00   17.92 142.9  3.55
Sol_RF   956.53   15.78  180.00   15.78  113.07  164.22 142.0  3.52
Sol_RF  1071.10  -19.94  180.00  160.06 -142.20   19.94 141.6  3.51
Sol_RF  1171.28   29.78  180.00   29.78  142.55  150.22 140.4  3.48
Sol_RF  1265.22  -15.88  180.00  164.12 -130.44   15.88 139.2  3.45
Sol_RF  1368.84   -0.00  180.00  180.00 -137.670.00 138.0  3.42
Sol_RF  1432.51 -180.00  180.00 -180.00   65.02   -0.00 137.9  3.42
Sol_RF  1575.02  -28.84  180.00  151.16 -150.04   28.84 134.7  3.34
Sol_RF  1671.69  -20.99  180.00  159.01 -143.37   20.99 133.0  3.30
Sol_RF  1792.13  101.46  179.93  102.35 -175.74   79.42 130.9  3.25
Sol_RF  18   107.89  144.73  179.79  145.06 -144.22   35.61 128.8  3.19
Sol_RF  1987.45  -78.19  180.00  101.81 -174.90   78.19 128.1  3.18
Sol_RF  2038.570.69   30.36  102.66  -18.79  -78.71 122.4  3.04
Sol_RF  2126.77  174.59  176.58  172.68   53.523.49 120.5  2.99
Sol_RF  22   116.66  178.08  175.143.49  126.48  187.32 120.5  2.99
Sol_RF  2375.56  -41.35  180.00  138.65 -151.12   41.35 119.8  2.97
Sol_RF  2466.12   36.35  180.00   36.35  132.24  143.65 116.6  2.89
Sol_RF  2583.87   71.62  180.00   71.62  167.74  108.38 114.7  2.85
Sol_RF  2669.24  -12.37  180.00  167.63 -138.48   12.37 112.3  2.79
Sol_RF  2759.75   15.26  172.297.64  119.07  157.12 112.2  2.78

Re: [ccp4bb] Molecular replacement question

[Maia Cherney]

Try balbes from G. Murshudov's website. It will find proper search model 
and use proper truncations automatically. In addition, it will put in 
your sequence.


Paul Holland wrote:

Hello fellow crystallographers,

I am trying molecular replacement for a protein crystal dataset that has very 
high sequence similarity to the search model with several predicted flexible 
loop regions; however, all attempts at finding a solution have not produce very 
ideal starting solutions using Phaser and Molrep (CC = 0.3 and Z-score = 5).  I 
am very confident that the unit cell parameters are C2 84.027  120.565  108.272 
 90.00 104.71  90.00, and there appears to be no evidence of twinning.  The 
Matthews calculation predicts from anywhere from 2-4 monomers in the ASU, and 
calculation of the SRF in Molrep does not identify any peaks in higher order 
symmetry except for the expected crystallographic two-fold for C2.  Below is 
the table from the calculated SRF in molrep.  Any advice would be greatly 
appreciated.

#thetaphichi alpha   beta  gamma Rf 
Rf/sigma
Sol_RF   1 0.000.000.000.000.000.00 870.5   
   21.59
Sol_RF   258.61  -10.17  180.00  169.83 -117.23   10.17 162.5  4.03
Sol_RF   366.02   -0.00  180.00  180.00 -132.030.00 161.1  4.00
Sol_RF   458.42   -9.54  180.00  170.46 -116.859.54 159.8  3.96
Sol_RF   5   149.840.00  180.00 -180.00   60.320.00 156.0  3.87
Sol_RF   658.96   -5.52  180.00  174.48 -117.915.52 151.5  3.76
Sol_RF   765.59   20.95  180.00   20.95  131.18  159.05 143.9  3.57
Sol_RF   890.00  -98.96  180.000.00  180.00   17.92 142.9  3.55
Sol_RF   956.53   15.78  180.00   15.78  113.07  164.22 142.0  3.52
Sol_RF  1071.10  -19.94  180.00  160.06 -142.20   19.94 141.6  3.51
Sol_RF  1171.28   29.78  180.00   29.78  142.55  150.22 140.4  3.48
Sol_RF  1265.22  -15.88  180.00  164.12 -130.44   15.88 139.2  3.45
Sol_RF  1368.84   -0.00  180.00  180.00 -137.670.00 138.0  3.42
Sol_RF  1432.51 -180.00  180.00 -180.00   65.02   -0.00 137.9  3.42
Sol_RF  1575.02  -28.84  180.00  151.16 -150.04   28.84 134.7  3.34
Sol_RF  1671.69  -20.99  180.00  159.01 -143.37   20.99 133.0  3.30
Sol_RF  1792.13  101.46  179.93  102.35 -175.74   79.42 130.9  3.25
Sol_RF  18   107.89  144.73  179.79  145.06 -144.22   35.61 128.8  3.19
Sol_RF  1987.45  -78.19  180.00  101.81 -174.90   78.19 128.1  3.18
Sol_RF  2038.570.69   30.36  102.66  -18.79  -78.71 122.4  3.04
Sol_RF  2126.77  174.59  176.58  172.68   53.523.49 120.5  2.99
Sol_RF  22   116.66  178.08  175.143.49  126.48  187.32 120.5  2.99
Sol_RF  2375.56  -41.35  180.00  138.65 -151.12   41.35 119.8  2.97
Sol_RF  2466.12   36.35  180.00   36.35  132.24  143.65 116.6  2.89
Sol_RF  2583.87   71.62  180.00   71.62  167.74  108.38 114.7  2.85
Sol_RF  2669.24  -12.37  180.00  167.63 -138.48   12.37 112.3  2.79
Sol_RF  2759.75   15.26  172.297.64  119.07  157.12 112.2  2.78
Sol_RF  28   120.25 -164.74  172.29   22.88  119.07  172.36 112.2  2.78
Sol_RF  2996.68  -70.99  180.00  109.01 -166.63   70.99 110.9  2.75
Sol_RF  3063.23  -44.73  180.00  135.27 -126.47   44.73 108.9  2.70

Cheers,

Paul Holland

 



  

[ccp4bb] molecular replacement

[Xianhui Wu]

Dear All,

   I am trying to resolve a protein structure with the use of Molecular
Replacement. However, some part of protein are overlap in the interface of
homodimer. Would someone please give me suggestions? Thank you!

-- 
Best regards,
WuXH

Re: [ccp4bb] molecular replacement

[Vellieux Frederic]

Hi there,

If there are a few clashes (acceptable - usually surface loops that 
enter the surface part of another molecule or subunit) then you can 
delete the parts of loops that usually have poor density and that clash, 
refine, compute a new map and see if the loop can be traced (usually it 
can be).


If there are really severe clashes, then your solution is not a proper 
solution.


Also, have you checked the packing of the molecules in the crystal? Do 
you have contacts (in the 3 directions of space) that explain the 
crystal itself, with solvent regions or channels? This is also 
indicative of a real solution or if your solution to the MR problem is 
garbage.


Further, if you can have a search model (located somewhere in the PDB) 
that is already a homodimer, then the molecular replacement calculations 
should be repeated. Better results simply because there are more 
vectors involved.


Fred.

Xianhui Wu wrote:

Dear All,
 
   I am trying to resolve a protein structure with the use of 
Molecular Replacement. However, some part of protein are overlap in 
the interface of homodimer. Would someone please give me suggestions? 
Thank you!


--
Best regards,
WuXH

Re: [ccp4bb] molecular replacement

[Eleanor Dodson]

  Hmm - that is odd.
You may have the wrong SG in the mtz file. Try MOLREP or PHASER with the 
option to try all spacegroups consistewnt with the pointgroup.


Eleanor

intekhab alam wrote:

Hi all
I am trying to do molecular replacement with low resolution (4Å) using
Molrep and Phaser.

Overall R-factor is 11.3%, completeness 95.4%, I/sigma 2, and Chi^2 0.95.

Identities between my protein and templates were more than 80%.

I couldn’t get correct solution.

Rotation function, translation score, and contrast were low, and they had no
significance, though I changed the range of resolution.

Molrep suggested solution coordinates clashed with symmetry molecules.

I tried MR after remove clashed regions, but another clashes happened.

In the case of phaser, there were many clashes, too.

Please, give me any suggestion.
Should I concern about any options when I run MR programs?

Hope you guys will be interested to answer!!
Thanks in advance

[ccp4bb] molecular replacement

[intekhab alam]

Hi all
I am trying to do molecular replacement with low resolution (4Å) using
Molrep and Phaser.

Overall R-factor is 11.3%, completeness 95.4%, I/sigma 2, and Chi^2 0.95.

Identities between my protein and templates were more than 80%.

I couldn’t get correct solution.

Rotation function, translation score, and contrast were low, and they had no
significance, though I changed the range of resolution.

Molrep suggested solution coordinates clashed with symmetry molecules.

I tried MR after remove clashed regions, but another clashes happened.

In the case of phaser, there were many clashes, too.

Please, give me any suggestion.
Should I concern about any options when I run MR programs?

Hope you guys will be interested to answer!!
Thanks in advance


-- 
INTEKHAB ALAM
LABORATORY OF STRUCTURAL BIOINFORMATICS
KOREA UNIVERSITY, SEOUL

Re: [ccp4bb] molecular replacement

[Tim Gruene]

Dear  intekhab,

a few suggestions:
- are you sure of the space group or might there be alternatives?
- is you protein globular or modular, i.e., is it worth running MR with stable
  subdomains one after the other?
- try a poly-alanine model (e.g. chainsaw can do this for you, pdbset probably,
  too)
- did you get overloads? If so, collect a low resolution pass to get correct
  intensities even at the poor resolution end.
- what is your low resolution limit? Did you use the default or could you extend
  it?
- did your crystal(s) suffer from radiation damage? It might be worth collection
  only a complete data set with not too high multiplicity.

Good luck, Tim

On Fri, May 21, 2010 at 08:01:10PM +0900, intekhab alam wrote:
 Hi all
 I am trying to do molecular replacement with low resolution (4Å) using
 Molrep and Phaser.
 
 Overall R-factor is 11.3%, completeness 95.4%, I/sigma 2, and Chi^2 0.95.
 
 Identities between my protein and templates were more than 80%.
 
 I couldn’t get correct solution.
 
 Rotation function, translation score, and contrast were low, and they had no
 significance, though I changed the range of resolution.
 
 Molrep suggested solution coordinates clashed with symmetry molecules.
 
 I tried MR after remove clashed regions, but another clashes happened.
 
 In the case of phaser, there were many clashes, too.
 
 Please, give me any suggestion.
 Should I concern about any options when I run MR programs?
 
 Hope you guys will be interested to answer!!
 Thanks in advance
 
 
 -- 
 INTEKHAB ALAM
 LABORATORY OF STRUCTURAL BIOINFORMATICS
 KOREA UNIVERSITY, SEOUL

-- 
--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A



signature.asc
Description: Digital signature

Re: [ccp4bb] molecular replacement

[Vineet Gaur]

Also check if you have correctly estimated no. of molecules in asymmetric
unit.

On Fri, May 21, 2010 at 4:58 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:

 Dear  intekhab,

 a few suggestions:
 - are you sure of the space group or might there be alternatives?
 - is you protein globular or modular, i.e., is it worth running MR with
 stable
  subdomains one after the other?
 - try a poly-alanine model (e.g. chainsaw can do this for you, pdbset
 probably,
  too)
 - did you get overloads? If so, collect a low resolution pass to get
 correct
  intensities even at the poor resolution end.
 - what is your low resolution limit? Did you use the default or could you
 extend
  it?
 - did your crystal(s) suffer from radiation damage? It might be worth
 collection
  only a complete data set with not too high multiplicity.

 Good luck, Tim

 On Fri, May 21, 2010 at 08:01:10PM +0900, intekhab alam wrote:
  Hi all
  I am trying to do molecular replacement with low resolution (4Å) using
  Molrep and Phaser.
 
  Overall R-factor is 11.3%, completeness 95.4%, I/sigma 2, and Chi^2 0.95.
 
  Identities between my protein and templates were more than 80%.
 
  I couldn’t get correct solution.
 
  Rotation function, translation score, and contrast were low, and they had
 no
  significance, though I changed the range of resolution.
 
  Molrep suggested solution coordinates clashed with symmetry molecules.
 
  I tried MR after remove clashed regions, but another clashes happened.
 
  In the case of phaser, there were many clashes, too.
 
  Please, give me any suggestion.
  Should I concern about any options when I run MR programs?
 
  Hope you guys will be interested to answer!!
  Thanks in advance
 
 
  --
  INTEKHAB ALAM
  LABORATORY OF STRUCTURAL BIOINFORMATICS
  KOREA UNIVERSITY, SEOUL

 --
 --
 Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen

 GPG Key ID = A46BEE1A


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 Version: GnuPG v1.4.9 (GNU/Linux)

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 XCPdN9n9Km6Gdn2SK+jv8WY=
 =ncth
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Re: [ccp4bb] molecular replacement

[Phoebe Rice]

I may be a pessimist, but I'd focus my efforts on getting
experimental phases.  It can be hard to tell a correct
molecular replacement solution from garbage with that kind of
data, and it can be a real time sink because its hard to know
when to quit.
If you can get an SeMet data set, even it it doesn't provide
enough phasing power to crack the problem, you can use the
positions of Se peaks in anomalous diff maps to check possible
molecular replacement solutions.
  Good luck!
Phoebe Rice

 Original message 
Date: Fri, 21 May 2010 20:23:23 +0530
From: Vineet Gaur vineetgaur1...@gmail.com  
Subject: Re: [ccp4bb] molecular replacement  
To: CCP4BB@JISCMAIL.AC.UK

   Also check if you have correctly estimated no. of
   molecules in asymmetric unit.

   On Fri, May 21, 2010 at 4:58 PM, Tim Gruene
   t...@shelx.uni-ac.gwdg.de wrote:

 Dear  intekhab,

 a few suggestions:
 - are you sure of the space group or might there
 be alternatives?
 - is you protein globular or modular, i.e., is it
 worth running MR with stable
  subdomains one after the other?
 - try a poly-alanine model (e.g. chainsaw can do
 this for you, pdbset probably,
  too)
 - did you get overloads? If so, collect a low
 resolution pass to get correct
  intensities even at the poor resolution end.
 - what is your low resolution limit? Did you use
 the default or could you extend
  it?
 - did your crystal(s) suffer from radiation
 damage? It might be worth collection
  only a complete data set with not too high
 multiplicity.

 Good luck, Tim
 On Fri, May 21, 2010 at 08:01:10PM +0900, intekhab
 alam wrote:
  Hi all
  I am trying to do molecular replacement with low
 resolution (4Å) using
  Molrep and Phaser.
 
  Overall R-factor is 11.3%, completeness 95.4%,
 I/sigma 2, and Chi^2 0.95.
 
  Identities between my protein and templates were
 more than 80%.
 
  I couldn’t get correct solution.
 
  Rotation function, translation score, and
 contrast were low, and they had no
  significance, though I changed the range of
 resolution.
 
  Molrep suggested solution coordinates clashed
 with symmetry molecules.
 
  I tried MR after remove clashed regions, but
 another clashes happened.
 
  In the case of phaser, there were many clashes,
 too.
 
  Please, give me any suggestion.
  Should I concern about any options when I run MR
 programs?
 
  Hope you guys will be interested to answer!!
  Thanks in advance
 
 
  --
  INTEKHAB ALAM
  LABORATORY OF STRUCTURAL BIOINFORMATICS
  KOREA UNIVERSITY, SEOUL

 --
 --
 Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen

 GPG Key ID = A46BEE1A

 -BEGIN PGP SIGNATURE-
 Version: GnuPG v1.4.9 (GNU/Linux)


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 XCPdN9n9Km6Gdn2SK+jv8WY=
 =ncth
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Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry  Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123

RNA is really nifty
DNA is over fifty
We have put them 
  both in one book
Please do take a 
  really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp

[ccp4bb] Molecular Replacement

[Muhammed bashir Khan]

Dear All;

We have solved a crystal structrure of protein at 1.8 A. I have now
another crystal of the same protein in aother unit cell,for the new
crystal type resolution is 3.6 A but when I use our structure as a seach
model It does't give any solution.

Any suggestion would be highly appreciated.


-- 
Muhammad Bashir Khan
Department for Biomolecular Structural Chemistry
Max F. Perutz Laboratories
University of Vienna
Campus Vienna Biocenter 5
A-1030 Vienna
Austria

Phone: +43(1)427752224
Fax: +43(1)42779522

Re: [ccp4bb] Molecular Replacement

[Tim Gruene]

Dear Muhammed,

that's not uncommon. Your protein might undergo domain movements or 
contain flexible parts.


if your structure contains any loops / floppy regions (with high 
B-values), you can exclude them from the search model.


If it is composed of domains, chop it into pieces and search with one 
after the other, starting from the largest one.


Tim

--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A


On Mon, 18 Jan 2010, Muhammed bashir Khan wrote:


Dear All;

We have solved a crystal structrure of protein at 1.8 A. I have now
another crystal of the same protein in aother unit cell,for the new
crystal type resolution is 3.6 A but when I use our structure as a seach
model It does't give any solution.

Any suggestion would be highly appreciated.


--
Muhammad Bashir Khan
Department for Biomolecular Structural Chemistry
Max F. Perutz Laboratories
University of Vienna
Campus Vienna Biocenter 5
A-1030 Vienna
Austria

Phone: +43(1)427752224
Fax: +43(1)42779522

Re: [ccp4bb] Molecular Replacement

[Christian Biertuempfel]

Dear Muhammed,
In case you have used phaser for MR you could try to either decrease
sequence identity or increase the rmsd of the search model (both ways
are equivalent). This allows also to account for flexibility/movements
in your target protein like Tim explained.

Cheers,
christian



Muhammed bashir Khan wrote:
 Dear All;
 
 We have solved a crystal structrure of protein at 1.8 A. I have now
 another crystal of the same protein in aother unit cell,for the new
 crystal type resolution is 3.6 A but when I use our structure as a seach
 model It does't give any solution.
 
 Any suggestion would be highly appreciated.
 
 


___

Dr. Christian Biertümpfel
Laboratory of Molecular Biology

NIDDK/National Institutes of Health  phone: +1 301 402 4647
9000 Rockville Pike, Bldg. 5, Rm. B1-03  fax:   +1 301 496 0201
Bethesda, MD 20892-0580
USA
___

[ccp4bb] molecular replacement in phaser

[Lisa Wang]

Hi all,
I got one data about 3.0 A, belong to C2 space group. There are two protein
molecules and one 18-nt dsRNA per ASU. The structure of  last 100aa
(C-terminal) has been reported, and 400 aa at N-terminalhe has homology
structure with sequence identiy 30%. I try to solve it by MR with phaser.
I did rotation and tranlation with 18-nt ideal dsRNA first, then search with
C-terminal model. I got about 20 sol after seaching dsRNA with TFZ 8-10.0. I
use all of them to serch the first c-terminal, and second C-terminal. Ding
this process,  LLG keep on increasing, and TFZ is higher than 10. But all
have serious clash. It looks like all the sol are wrong.

Please give me a hand.

Re: [ccp4bb] molecular replacement in phaser

[Christian Biertuempfel]

Hi Lisa,
There are many things you can try and the phaser manual gives a lot of
useful information what to do in difficult cases. From my experience, it
is quite difficult to find solutions for MR with nucleic acids. I
recommend to search only for protein. As a side effect you can use this
approach as an unbiased quality check. If your solution is correct you
expect to see additional density coming up for RNA (assumed that the RNA
is ordered).
Try different protein models containing either C-term or N-term or both.
Homology modeling can help a lot to generate a good search model. Try
also to give higher values for rms differences (or lower sequence
identity) between search model and target. Do you allow too many clashes
as a packing criterion?

Cheers,
christian

p.s. Of course, check that you work in the right space group.


Lisa Wang wrote:
 Hi all,
 I got one data about 3.0 A, belong to C2 space group. There are two
 protein molecules and one 18-nt dsRNA per ASU. The structure of  last
 100aa  (C-terminal) has been reported, and 400 aa at N-terminalhe has
 homology structure with sequence identiy 30%. I try to solve it by MR
 with phaser.
 I did rotation and tranlation with 18-nt ideal dsRNA first, then search
 with C-terminal model. I got about 20 sol after seaching dsRNA with TFZ
 8-10.0. I use all of them to serch the first c-terminal, and second
 C-terminal. Ding this process,  LLG keep on increasing, and TFZ is
 higher than 10. But all have serious clash. It looks like all the sol
 are wrong.
  
 Please give me a hand.

Re: [ccp4bb] molecular replacement in phaser

[Phoebe Rice]

We've had good luck (sometimes) searching for nucleic acids 
with multiple small pieces - say ask it to find several 5bp 
chunks of DNA rather than one 20bp duplex.  I think Bill 
Scott will agree?
I have a hunch that searching for the protein 1st, at least 
at modest resolutions, may confuse the software because 
the DNA is denser. 
Of course, every case has its own idiosyncracies.
  Phoebe

 Original message 
Date: Tue, 10 Nov 2009 14:19:43 -0500
From: Christian Biertuempfel biertumpf...@niddk.nih.gov  
Subject: Re: [ccp4bb] molecular replacement in phaser  
To: CCP4BB@JISCMAIL.AC.UK

Hi Lisa,
There are many things you can try and the phaser manual 
gives a lot of
useful information what to do in difficult cases. From my 
experience, it
is quite difficult to find solutions for MR with nucleic 
acids. I
recommend to search only for protein. As a side effect you 
can use this
approach as an unbiased quality check. If your solution 
is correct you
expect to see additional density coming up for RNA (assumed 
that the RNA
is ordered).
Try different protein models containing either C-term or N-
term or both.
Homology modeling can help a lot to generate a good search 
model. Try
also to give higher values for rms differences (or lower 
sequence
identity) between search model and target. Do you allow too 
many clashes
as a packing criterion?

Cheers,
christian

p.s. Of course, check that you work in the right space 
group.


Lisa Wang wrote:
 Hi all,
 I got one data about 3.0 A, belong to C2 space group. 
There are two
 protein molecules and one 18-nt dsRNA per ASU. The 
structure of  last
 100aa  (C-terminal) has been reported, and 400 aa at N-
terminalhe has
 homology structure with sequence identiy 30%. I try to 
solve it by MR
 with phaser.
 I did rotation and tranlation with 18-nt ideal dsRNA 
first, then search
 with C-terminal model. I got about 20 sol after seaching 
dsRNA with TFZ
 8-10.0. I use all of them to serch the first c-terminal, 
and second
 C-terminal. Ding this process,  LLG keep on increasing, 
and TFZ is
 higher than 10. But all have serious clash. It looks like 
all the sol
 are wrong.
  
 Please give me a hand.
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry  Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123

RNA is really nifty
DNA is over fifty
We have put them 
  both in one book
Please do take a 
  really good look
http://www.rsc.org/shop/books/2008/9780854042722.asp

[ccp4bb] Molecular replacement with merohedarlly twinned data

[Pietro Roversi]

Dear all,
when searching in Molecular Replacement
against say perfectly merohedrally twinned data (i.e. an alpha=0.5 twin)
do I understand it right that I should expect two sets of solutions
that are equivalent under the twin operator (bar xtal symmetry and/or
origin shifts)?

Thanks!

Ciao

Pietro
-- 
Pietro Roversi
EP Abraham Fellow in Biochemistry - Lincoln College - Oxford
Sir William Dunn School of Pathology, Oxford University
South Parks Road, Oxford OX1 3RE, England UK
Tel. 0044-1865-275385

Re: [ccp4bb] Molecular replacement with merohedarlly twinned data

[Randy Read]

Yes, but if the twin fraction deviates even slightly from 1/2, you may  
only get one solution from the search.  If you want, you can generate  
the other one and see how happy the program is.


Regards,

Randy

On 11 Sep 2009, at 15:12, Pietro Roversi wrote:


Dear all,
when searching in Molecular Replacement
against say perfectly merohedrally twinned data (i.e. an alpha=0.5  
twin)

do I understand it right that I should expect two sets of solutions
that are equivalent under the twin operator (bar xtal symmetry and/or
origin shifts)?

Thanks!

Ciao

Pietro
--
Pietro Roversi
EP Abraham Fellow in Biochemistry - Lincoln College - Oxford
Sir William Dunn School of Pathology, Oxford University
South Parks Road, Oxford OX1 3RE, England UK
Tel. 0044-1865-275385


--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www- 
structmed.cimr.cam.ac.uk

[ccp4bb] Molecular Replacement of a protein complex

[Daniel Bonsor]

My first time posting to the CCP4BB board and I am very sure it will not be
my last. I have a general and specific question concerning molecular
replacements of protein-protein complexes. I have a good dataset (2.5A, 98%
complete), which has been integrated and scaled. Pointless suggested the
pointgroup P222, in agreement with imosflm and HKL2000. For the spacegroup
it could not decide between P2221 or P21221.

Spacegroup TotProb SysAbsProb Reindex Conditions

P 2 2 21 ( 17)0.889  0.900 00l: l=2n (zone 3)
   P 21 2 21 ( 18)0.889  0.900 h00: h=2n, 00l:
l=2n (zones 1,3)
..
 P 2 2 2 ( 16)0.057  0.057 
P 21 2 2 ( 17)0.057  0.057 h00: h=2n (zone 1)
..
   P 2 21 21 ( 18)0.039  0.040 0k0: k=2n, 00l:
l=2n (zones 2,3)
  P 21 21 21 ( 19)0.039  0.040 h00: h=2n, 0k0:
k=2n, 00l: l=2n (zones 1,2,3)

I reindexed to both space groups and proceeded with Mathews Coeff. Assuming
a 1:1 complex had formed (MW = 39000) it predicts 1 complex per ASU. MolRep
using the larger protein (MW=27000) found the protein with a contrast of
3.33 (p2221) and 2.61 (p21221). However repeating MolRep for the smaller
protein in both spacegroups failed to locate it. Contrast was low, 1.11
(p2221) and 1.10 (p21221). 

I reran Mathews Coeff in case the second protein was not their (though gels
of crystals show both proteins) and it predicted 2 proteins per ASU. MolRep
looking for 2 of the larger protein generated contrasts of 1.99 (p2221) and
2.27 (p21221). 

Using Refmac5, I performed a rigid body refinement of all 4 cases with
details summarised below

p2221 1proteinper ASU - Proteins are arranged in layers separated by a
55Angstrom space. Some electron density is present but nothing that could be
generate a polypeptide. Perhaps two or three amino acids here and there.
Density fits the protein well. Rfac, 0.527, Rfree 0.523

p21221 1proteinper ASU - Proteins are again arranged in layers separated by
a 55Angstrom space. Again some electron density is present that could
accommodate two or three amino acids. Density also fits the protein well.
Rfac, 0.527, Rfree 0.512

p2221 2proteinper ASU -  No layers are observed. Density fits the proteins
very poorly. Rfac, 0.541, Rfree 0.546

p21221 2proteinper ASU -  No layers are observed. Density fits the proteins
very poorly. Rfac, 0.540, Rfree 0.547

My questions are this; In general what is the best procedure for molecular
replacement and refinement of protein-protein complex where the Kd is 1uM
and weaker and it is difficult to observe one of the proteins; In this case
I believe the second protein is their, though either the starting model is
poor (80% identical) or a gross conformational change of the protein has
taken place (which is unlikely based upon similar complexes that have been
solved). I am also concerned about Rfree being smaller than Rfac. Should I
continue with these two data set and try modeling alanine in the electron
density I can observe and see if further density is generated on further
refinement? Or should I go back and look at alternative space groups?

Thanks in advance for you opinions, suggestions and help

Daniel A. Bonsor,
Boston Biomedical Research Institute,
64 Grove Street,
Watertown,
MA 02472 USA

Re: [ccp4bb] Molecular Replacement of a small peptide

[Mark J. van Raaij]

Dear Allen, Sue,
we also have had some luck with ACORN, but as our peptide did not have  
alpha-helical structure, the postdoc on the project used a 4-aa  
beta-turn fragment instead. I can put you in contact with him for more  
details. As I understand it, ACORN uses a mixed MR/direct methods  
approach.
We had also tried extensive MR before, but without luck - it appears  
peptides often are too different for it.

Mark


Quoting s...@email.arizona.edu:


Have you tried acorn in ccp4?  I've had it work well at this resolution,
especially if the protein/peptide has some alpha helical content.  We used
acorn to solve a small cro protein that we couldn't get molecular replacement
to work with by using a 5-residue ideal poly-ala helix as the starting model.

Sue

Quoting Sickmier, Allen sickm...@amgen.com:

I am trying to do molecular replacement on a small peptide (less   
than 40 AA) and have not had any success using phaser. Are there   
any tricks or better programs for really small peptides?
The data is great 1.1 A, ~35% solvent, and two molecules in the   
ASU. I have tried all the standard stuff, changing resolution cut   
off etc.   I may move to sulfur phasing at this resolution but I   
would like to get the MR to work.



Allen

Re: [ccp4bb] Molecular Replacement of a small peptide

[George M. Sheldrick]

If you have 1.1A data and your structure is not too large, ab initio 
direct methods (e.g. with SHELXD) will probably solve the structure. I 
would be happy to advise if you wish to try this.

George

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582


On Fri, 19 Jun 2009, Sickmier, Allen wrote:

 I am trying to do molecular replacement on a small peptide (less than 40 AA) 
 and have not had any success using phaser. Are there any tricks or better 
 programs for really small peptides?
 The data is great 1.1 A, ~35% solvent, and two molecules in the ASU. I have 
 tried all the standard stuff, changing resolution cut off etc.   I may move 
 to sulfur phasing at this resolution but I would like to get the MR to work.
 
 
 Allen
 
 
 
 
 

Re: [ccp4bb] Molecular Replacement of a small peptide

[Nicholas M Glykos]

Hi Allen,

With such low solvent content (and corresponding tight packing), you may 
want to give 'Queen of spades (Qs)' a try. The reason being that Qs is not 
(at least directly) a Patterson-based method (and does not assume that 
intra-molecular vectors are topologically segregated). If you expect that 
your model is good, do not hesitate to give it a try with even 2A data 
(but also try 3A ;-). If you have access to a dual- or quad-core machine, 
do compile the MPI version (see 
http://norma.mbg.duth.gr/index.php?id=about:benchmarks:qsmpi for results 
on relatively recent hardware).


My twocents,
Nicholas


On Fri, 19 Jun 2009, Sickmier, Allen wrote:

 I am trying to do molecular replacement on a small peptide (less than 40 
 AA) and have not had any success using phaser. Are there any tricks or 
 better programs for really small peptides? The data is great 1.1 A, ~35% 
 solvent, and two molecules in the ASU. I have tried all the standard 
 stuff, changing resolution cut off etc.  I may move to sulfur phasing at 
 this resolution but I would like to get the MR to work.
 
 
 Allen

-- 


  Dr Nicholas M. Glykos, Department of Molecular Biology
 and Genetics, Democritus University of Thrace, University Campus,
  Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620,
Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/

[ccp4bb] Molecular Replacement of a small peptide

[Sickmier, Allen]

I am trying to do molecular replacement on a small peptide (less than 40 AA) 
and have not had any success using phaser. Are there any tricks or better 
programs for really small peptides?
The data is great 1.1 A, ~35% solvent, and two molecules in the ASU. I have 
tried all the standard stuff, changing resolution cut off etc.   I may move to 
sulfur phasing at this resolution but I would like to get the MR to work.


Allen

Re: [ccp4bb] Molecular Replacement of a small peptide

[suer]

Have you tried acorn in ccp4?  I've had it work well at this resolution,
especially if the protein/peptide has some alpha helical content.  We used
acorn to solve a small cro protein that we couldn't get molecular replacement
to work with by using a 5-residue ideal poly-ala helix as the starting model.

Sue

Quoting Sickmier, Allen sickm...@amgen.com:

I am trying to do molecular replacement on a small peptide (less than 
40 AA) and have not had any success using phaser. Are there any 
tricks or better programs for really small peptides?
The data is great 1.1 A, ~35% solvent, and two molecules in the ASU. 
I have tried all the standard stuff, changing resolution cut off etc. 
  I may move to sulfur phasing at this resolution but I would like to 
get the MR to work.



Allen

Re: [ccp4bb] Molecular Replacement of a small peptide

[Randy Read]

I've had good luck using Phaser on test cases like that, but haven't  
had access to a case that was previously unsolved.  But in principle  
it should work, if the model is good enough.


It sounds like you've thought about the resolution already, so this is  
probably redundant.  Anyway, the default of 2.5A is good for normal  
sized molecules, but for very small molecules there might not be  
enough observations, so you would want to change the default.  If 2.5A  
data would be suitable for a protein of 200 residues, then 1.5A would  
give about the same number of reflections for a molecule of 40  
residues.  (Another way to look at this is that it will give about the  
same ratio between the diameter of the molecule and the resolution  
limit.)  So I'd try 1.5A first, and if that didn't work I'd probably  
try 1.1 and even 2.0 for completeness.


Another thing is that a peptide may not be as well conserved in  
structure as you'd expect from the sequence, compared to a medium- 
sized protein.  So you might want to increase the RMS error deduced  
from the sequence identity.  (Of course, the data at a resolution  
higher than a bit more than 2 times the estimated RMS error hardly  
contribute to the likelihood function, so this may counteract the use  
of higher resolution data to some extent.)


If the peptide is really flexible so that the model doesn't fit, then  
that might explain why you can't solve the structure.  As well,  
structures with low solvent tend to be a bit harder.


Is your peptide reasonably spherical in shape, or is it asymmetric?   
The current default in Phaser probably doesn't sample the angles for  
highly asymmetric molecules finely enough to avoid rotating them too  
much perpendicular to their long direction, so you might want to  
increase the sampling in the rotation search.


As you imply, if a crystal diffracts to 1.1A, then you'll probably  
have a decent anomalous signal from the intrinsic sulfur atoms.  Once  
you've got a license for SHELXD, HySS or SnB, you can probably  
complete the structure in a variety of ways -- with atomic resolution  
data, you can even ask Phaser (now using the SAD target instead of the  
molecular replacement modes) to complete with S and C atoms, and it  
will probably build a complete model.


Best wishes,

Randy Read

On 19 Jun 2009, at 17:26, Sickmier, Allen wrote:

I am trying to do molecular replacement on a small peptide (less  
than 40 AA) and have not had any success using phaser. Are there any  
tricks or better programs for really small peptides?
The data is great 1.1 A, ~35% solvent, and two molecules in the ASU.  
I have tried all the standard stuff, changing resolution cut off  
etc.   I may move to sulfur phasing at this resolution but I would  
like to get the MR to work.


Allen







--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www- 
structmed.cimr.cam.ac.uk

[ccp4bb] molecular replacement and twinning problems

[Kay Diederichs]

Dear all,

we have crystals that nicely diffract to 1.7 A (sharp spots), with the 
following characteristics and findings:
a) the data appear as P212121, with axes 117.2  133.6  138.3 (if reduced 
in P1, the largest deviation of any angle from 90° is 0.2°); the odd 
screw-axis reflections are mostly indistinguishable from noise; the data 
do not scale significantly better in P2/P21 (any setting) or P1.
b) there is a good model available, with coords known from a complex of 
this protein with another one; two molecules of this model would give 
64% solvent in P212121 which appears reasonable for a membrane protein
c) the structure cannot be solved with this model in P212121, nor can it 
be solved in P222, P2122, P2212, P2221, P21212, P22121, P21221
d) we conclude that the true space group must be P2 or P21 (with one of 
the three possible settings), with almost-perfect twinning. Or it is P1 
with tetartohedral twinning. There are thus still six + one possibilities.

e) MOLREP tells us
 --- Check Patterson for pseudo-translation ---
   PST_limit :   0.125 of origin peak
 INFO: pseudo-translation was detected.
Origin Patterson peak: P,P/sig :57.748   257.690
1 Patterson. peak: p,P/sig :28.773   128.395
2 Patterson peak : P,P/sig :16.55173.856
3 Patterson peak : P,P/sig : 8.50237.936
Peak 1: trans.vector /ort/ : 0.01155.68869.399
trans.vector /frac/: 0.000 0.416 0.500
Peak 2: trans.vector /ort/ :58.55466.863 0.000
trans.vector /frac/: 0.500 0.500 0.000
Peak 3: trans.vector /ort/ :58.56511.38569.399
trans.vector /frac/: 0.500 0.085 0.500
 INFO:  translation vector of peak 1 will be used.

Two molecules (for the orthorhombic spacegroups) may produce only one 
pseudo-translation vector. As there is more than one strong 
pseudo-translation vector, I conclude that we have at least 3 molecules 
in the ASU (consistent with monoclinic).


f) we've calculated all seven molecular replacement searches of d) in 
MOLREP. The contrast is very high in all cases. However, Refmac 
rigid-body refinement of the solutions, with Twin refinement 
activated, gives about 51% R and the same for Rfree (give or take 1 %), 
in all cases.


I'm wondering: how reliable is a rotation search in the presence of 
perfect twinning? Is there any molecular replacement program that can 
take a given twinning operator into account in the rotation and 
translation search?


Any other hints what to try?

best,

Kay
--
Kay Diederichshttp://strucbio.biologie.uni-konstanz.de
email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183
Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz

This e-mail is digitally signed. If your e-mail client does not have the
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Re: [ccp4bb] molecular replacement and twinning problems

[Eleanor Dodson]

You dont mention any twinning tests?
The L test, now part of the newest ctruncate is pretty good at detecting 
twinning even with the NCS translation.

And SFCHECK  does a good job too.
If these are inconclusive I would not assume twinning.

Usually you can get solutions for MR with twinned data, but I  havent 
much experience of the signal quality..

 Can you solve it in P1 then sort out the spacegroup later?
 Eleanor



Kay Diederichs wrote:

Dear all,

we have crystals that nicely diffract to 1.7 A (sharp spots), with the 
following characteristics and findings:
a) the data appear as P212121, with axes 117.2  133.6  138.3 (if 
reduced in P1, the largest deviation of any angle from 90° is 0.2°); 
the odd screw-axis reflections are mostly indistinguishable from 
noise; the data do not scale significantly better in P2/P21 (any 
setting) or P1.
b) there is a good model available, with coords known from a complex 
of this protein with another one; two molecules of this model would 
give 64% solvent in P212121 which appears reasonable for a membrane 
protein
c) the structure cannot be solved with this model in P212121, nor can 
it be solved in P222, P2122, P2212, P2221, P21212, P22121, P21221
d) we conclude that the true space group must be P2 or P21 (with one 
of the three possible settings), with almost-perfect twinning. Or it 
is P1 with tetartohedral twinning. There are thus still six + one 
possibilities.

e) MOLREP tells us
 --- Check Patterson for pseudo-translation ---
   PST_limit :   0.125 of origin peak
 INFO: pseudo-translation was detected.
Origin Patterson peak: P,P/sig :57.748   257.690
1 Patterson. peak: p,P/sig :28.773   128.395
2 Patterson peak : P,P/sig :16.55173.856
3 Patterson peak : P,P/sig : 8.50237.936
Peak 1: trans.vector /ort/ : 0.01155.68869.399
trans.vector /frac/: 0.000 0.416 0.500
Peak 2: trans.vector /ort/ :58.55466.863 0.000
trans.vector /frac/: 0.500 0.500 0.000
Peak 3: trans.vector /ort/ :58.56511.38569.399
trans.vector /frac/: 0.500 0.085 0.500
 INFO:  translation vector of peak 1 will be used.

Two molecules (for the orthorhombic spacegroups) may produce only one 
pseudo-translation vector. As there is more than one strong 
pseudo-translation vector, I conclude that we have at least 3 
molecules in the ASU (consistent with monoclinic).


f) we've calculated all seven molecular replacement searches of d) in 
MOLREP. The contrast is very high in all cases. However, Refmac 
rigid-body refinement of the solutions, with Twin refinement 
activated, gives about 51% R and the same for Rfree (give or take 1 
%), in all cases.


I'm wondering: how reliable is a rotation search in the presence of 
perfect twinning? Is there any molecular replacement program that can 
take a given twinning operator into account in the rotation and 
translation search?


Any other hints what to try?

best,

Kay

Re: [ccp4bb] molecular replacement and twinning problems

[Kay Diederichs]

Eleanor Dodson schrieb:

You dont mention any twinning tests?


sorry, I forgot to mention that the twinning tests do not show twinning.

Rather, the actual curves in the Cumulative distribution of H lie on 
the not-twinned-at-all (i.e opposite) side of the alpha=0 curve (see 
plot below). But I'm pretty sure that this is due to the 
pseudo-translation (almost centering) which results in a high proportion 
of very weak reflections - contrary to what you get with twinning.


That's what I get from sfcheck, when run in P212121:
 Perfect twinning test I^2/I^2 :  3.1699
 Partial Twinning test:
-h,+l,+k
 Polar angles:  135.00  -89.99  179.99
 Alpha(twin fraction),Npair,Ior,Tol :-0.109  1625882 0.030

--- Partial Twinning Test : H = !I(h1)-I(h2)!/(I(h1)+I(h2)) ---

   Alpha(twinning fraction) = 1/2 - H

 Reflection related to hkl :
-h,+l,+k

  Cumulative distribution of H

   0  0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9  1
   *---+---+---+---+---+---+---+---+---+---+
O+++.+  .   .   .   .   .   .   .   .   !
O ++.+  . + .   .   .   .   .   .   .   !
!O +++  +   .  +.   .   .   .   .   .   !
0.10!-O-++-+--+-+---!
! O .++ +   .+  .   .   .+  .   .   .   !
!  O. + + + .  +.   .   .   . + .   .   !
!   O  +.+  +   . + .   .   .   .  +.   !
0.20!---O---+-+--+--+---+ alpha=0.4
!   .O  .+ +.  +.   .  +.   .   .   .   +
!   . O . + +   .+  .   .+  .   .   .   +
!   . O .  +. + . + .   .   +   .   .   +
0.30!--O+--++-+-+
!   .   O   .+  +   . + .   .   .+  .   +
!   .   O   . + .+  .  +.   .   .  +.   +
!   .   .O  .  +.  +.   .+  .   .   . + +
0.40!-O-+---+--++ alpha=0.3
!   .   .  O.   .+  .+  .   +   .   .   +
!   .   .  O.   . + .  +.   . + .   .   +
!   .   .   O   .  +.   +   .   +   .   +
0.50!O--++---+--+
!   .   .   .O  .   .+  . + .   .  +.   +
!   .   .   . O .   . + .   +   .   .+  +
!   .   .   .  O.   .  +.   .+  .   . + +
0.60!---O---+-+-+ alpha=0.2
!   .   .   .   .O  .   .+  .  +.   .   +
!   .   .   .   . O .   . + .   .+  .   +
!   .   .   .   .  O.   .  +.   . + .   +
0.70!--O+--++
!   .   .   .   .   O   .   .+  .   +   +
!   .   .   .   .   .O  .   . + .   .+  +
!   .   .   .   .   .  O.   .  +.   .  ++
0.80!---O---+---+ alpha=0.1
!   .   .   .   .   .   .O  .   .+  .   +
!   .   .   .   .   .   . O .   . + .   +
!   .   .   .   .   .   .  O.   .  +.   +
0.90!O--+---+
!   .   .   .   .   .   .   .  O.   .+  +
!   .   .   .   .   .   .   .   .O  . + +
!   .   .   .   .   .   .   .   .  O.  ++
H  *---+---+---+---+---+---+---+---+---+---* alpha=0
   0  0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9  1

Of course this plot looks _very_ different if I run it in P1 because 
then sfcheck uses -h,k,-l as twinning operator - it then looks like 
perfect twinning.



The L test, now part of the newest ctruncate is pretty good at detecting 
twinning even with the NCS translation.


this is the L test from ctruncate 1.0.0 : 30/10/08 run in P212121 :

$TABLE: L test for twinning:
$GRAPHS: cumulative distribution function for |L|:0|1x0|1:1,2,3,4:
$$ |L| Observed Expected_untwinned Expected_twinned $$
$$
0.00 0.00 0.00 0.00
0.05 0.048142 0.05 0.074938
0.10 0.093721 0.10 0.149500
0.15 0.139205 0.15 0.223312
0.20 0.184799 0.20 0.296000
0.25 0.230867 0.25 0.367188
0.30 0.277173 0.30 0.436500
0.35 0.323650 0.35 0.503563
0.40 0.370747 0.40 0.568000
0.45 0.418233 0.45 0.629437
0.50 0.466569 0.50 0.687500
0.55 0.515615 0.55 0.741812
0.60 0.565706 0.60 0.792000
0.65 0.616860 0.65 0.837688
0.70 0.669232 0.70 0.878500
0.75 0.723200 0.75 0.914062
0.80 0.779144 0.80 0.944000
0.85 0.836994 0.85 0.967938
0.90 0.896933 0.90 0.985500
0.95 0.957486 0.95 0.996313
1.00 1.00 1.00 1.00
$$



And SFCHECK  does a good job too.
If these are inconclusive I would not assume twinning.

Usually you can get solutions for MR with twinned data, but I  havent 
much experience of the signal quality..

 Can you solve it in P1 then sort out the spacegroup later?


I tried; this is the full story:
I ran molrep (version 10.2.12 from CCP4 6.1.0) in P1, with NMON=8. It 
uses the pseudo-translation vector and thus places 4 times 2 molecules. 
In the fast mode (standard RF and TF without rigid body refinement) 
the contrast is 1.93/1.77/1.87/14.72 for the 1st/2nd/3rd/4th pair of 
molecules. However the result is different if I run it in medium 
(contrast=2.82/4.56/1.55/3.10) or slow (2.87/11.47/2.01/2.59) mode, 
and molrep only writes out 4 molecules instead of 8, in these two modes.


I therefore 

Re: [ccp4bb] molecular replacement and twinning problems

[Eleanor Dodson]

Well - there s nothing there to indicate twinning, although as you say 
it is hard to be sure with a pseudo translation...


The contrast doesnt look brilliant, but then it often doesnt..
What about phaser? If it gives a contrast between one spacegroup and the 
others I always think that is a good sign..

It will take a while though..
 Eleanor

Phil Evans program othercell suggests you could reindex to get two axes 
equal.. And that is a prequisite for twinning .


C 1 2/m 1  192.3 192.3 117.2  90.0  90.0  92.01.98  [k-l,k+l,h]
 Same cell
   [-k+l,-k-l,h]


Eleanor

Kay Diederichs wrote:

Eleanor Dodson schrieb:

You dont mention any twinning tests?


sorry, I forgot to mention that the twinning tests do not show twinning.

Rather, the actual curves in the Cumulative distribution of H lie on 
the not-twinned-at-all (i.e opposite) side of the alpha=0 curve (see 
plot below). But I'm pretty sure that this is due to the 
pseudo-translation (almost centering) which results in a high 
proportion of very weak reflections - contrary to what you get with 
twinning.


That's what I get from sfcheck, when run in P212121:
 Perfect twinning test I^2/I^2 :  3.1699
 Partial Twinning test:
-h,+l,+k
 Polar angles:  135.00  -89.99  179.99
 Alpha(twin fraction),Npair,Ior,Tol :-0.109  1625882 0.030

--- Partial Twinning Test : H = !I(h1)-I(h2)!/(I(h1)+I(h2)) ---

   Alpha(twinning fraction) = 1/2 - H

 Reflection related to hkl :
-h,+l,+k

  Cumulative distribution of H

   0  0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9  1
   *---+---+---+---+---+---+---+---+---+---+
O+++.+  .   .   .   .   .   .   .   .   !
O ++.+  . + .   .   .   .   .   .   .   !
!O +++  +   .  +.   .   .   .   .   .   !
0.10!-O-++-+--+-+---!
! O .++ +   .+  .   .   .+  .   .   .   !
!  O. + + + .  +.   .   .   . + .   .   !
!   O  +.+  +   . + .   .   .   .  +.   !
0.20!---O---+-+--+--+---+ alpha=0.4
!   .O  .+ +.  +.   .  +.   .   .   .   +
!   . O . + +   .+  .   .+  .   .   .   +
!   . O .  +. + . + .   .   +   .   .   +
0.30!--O+--++-+-+
!   .   O   .+  +   . + .   .   .+  .   +
!   .   O   . + .+  .  +.   .   .  +.   +
!   .   .O  .  +.  +.   .+  .   .   . + +
0.40!-O-+---+--++ alpha=0.3
!   .   .  O.   .+  .+  .   +   .   .   +
!   .   .  O.   . + .  +.   . + .   .   +
!   .   .   O   .  +.   +   .   +   .   +
0.50!O--++---+--+
!   .   .   .O  .   .+  . + .   .  +.   +
!   .   .   . O .   . + .   +   .   .+  +
!   .   .   .  O.   .  +.   .+  .   . + +
0.60!---O---+-+-+ alpha=0.2
!   .   .   .   .O  .   .+  .  +.   .   +
!   .   .   .   . O .   . + .   .+  .   +
!   .   .   .   .  O.   .  +.   . + .   +
0.70!--O+--++
!   .   .   .   .   O   .   .+  .   +   +
!   .   .   .   .   .O  .   . + .   .+  +
!   .   .   .   .   .  O.   .  +.   .  ++
0.80!---O---+---+ alpha=0.1
!   .   .   .   .   .   .O  .   .+  .   +
!   .   .   .   .   .   . O .   . + .   +
!   .   .   .   .   .   .  O.   .  +.   +
0.90!O--+---+
!   .   .   .   .   .   .   .  O.   .+  +
!   .   .   .   .   .   .   .   .O  . + +
!   .   .   .   .   .   .   .   .  O.  ++
H  *---+---+---+---+---+---+---+---+---+---* alpha=0
   0  0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9  1

Of course this plot looks _very_ different if I run it in P1 because 
then sfcheck uses -h,k,-l as twinning operator - it then looks like 
perfect twinning.



The L test, now part of the newest ctruncate is pretty good at 
detecting twinning even with the NCS translation.


this is the L test from ctruncate 1.0.0 : 30/10/08 run in P212121 :

$TABLE: L test for twinning:
$GRAPHS: cumulative distribution function for |L|:0|1x0|1:1,2,3,4:
$$ |L| Observed Expected_untwinned Expected_twinned $$
$$
0.00 0.00 0.00 0.00
0.05 0.048142 0.05 0.074938
0.10 0.093721 0.10 0.149500
0.15 0.139205 0.15 0.223312
0.20 0.184799 0.20 0.296000
0.25 0.230867 0.25 0.367188
0.30 0.277173 0.30 0.436500
0.35 0.323650 0.35 0.503563
0.40 0.370747 0.40 0.568000
0.45 0.418233 0.45 0.629437
0.50 0.466569 0.50 0.687500
0.55 0.515615 0.55 0.741812
0.60 0.565706 0.60 0.792000
0.65 0.616860 0.65 0.837688
0.70 0.669232 0.70 0.878500
0.75 0.723200 0.75 0.914062
0.80 0.779144 0.80 0.944000
0.85 0.836994 0.85 0.967938
0.90 0.896933 0.90 0.985500
0.95 0.957486 0.95 0.996313
1.00 1.00 1.00 1.00
$$



And SFCHECK  does a good job too.
If these are inconclusive I would not assume twinning.

Usually you can get solutions for MR with twinned data, 

Re: [ccp4bb] Molecular replacement using a partial molecular replacement solution

[Herman . Schreuder]

Hi Mo,
Phaser sometimes rejects solutions because of clashes in the crystal
packing, which may be due to differences between the search model and
the target molecule. I would certainly recommend to play around with the
PACK keyword to see whether Phaser will find solutions if more clashes
are allowed.
 
Best regards,
Herman




From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On
Behalf Of Mo Wong
Sent: Monday, January 19, 2009 5:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Molecular replacement using a partial
molecular replacement solution


Hello,

My problem: I have poorly phased 3.5A data which suggests 6
molecules per ASU, and using MolRep with the experimental phases
(search for model in the map) I have good solutions 3 of them. There
is a lot of empty electron density which needs to be filled with more
copies of the molecule. I have looked for NCS operators as I know this
will improve the map and help with model fitting (the Self Rot function
suggests I have at least 2-fold symmetry), but no luck yet. My current
focus is on using the 3-molecule partial solution as a starting point,
but since I'm not getting anywhere fast, I though I'd post to the
bulletin board.

Can someone please either point me to a MolRep script that
allows me to fix the known solutions, use the experimental phases, and
search for (3?) more copies of the model, or tell if there is something
wrong with the following Phaser scripts below (is it necessary to
apply an operator to the MolRep solution before reading into Phaser?).

Thanks!

#
phaser  eof

MODE MR_FRF

HKLIN overall_best_denmod_map_coeffs.mtz
LABIN F = FP SIGF = SIGFP

ENSEMBLE trimer PDBFILE trimer.pdb IDENTITY 0.25
ENSEMBLE monomer PDBFILE monomer.pdb IDENTITY 0.25
COMPOSITION PROTEIN SEQUENCE trimer.seq NUM 1
COMPOSITION PROTEIN SEQUENCE monomer.seq NUM 3

SOLU 6DIM ENSE trimer EULER 0 0 0 FRAC 0 0 0

SEARCH ENSEMBLE monomer NUM 1

ROOT AUTO_monomer

eof
#
phaser  eof

MODE MR_FTF

HKLIN overall_best_denmod_map_coeffs.mtz
LABIN F = FP SIGF = SIGFP

ENSEMBLE trimer PDBFILE trimer.pdb IDENTITY 0.25
ENSEMBLE monomer PDBFILE monomer.pdb IDENTITY 0.25
COMPOSITION PROTEIN SEQUENCE trimer.seq NUM 1
COMPOSITION PROTEIN SEQUENCE monomer.seq NUM 3

@AUTO_monomer.rlist

ROOT AUTO_monomer2

eof
#

[ccp4bb] Molecular replacement using a partial molecular replacement solution

[Mo Wong]

Hello,

My problem: I have poorly phased 3.5A data which suggests 6 molecules per
ASU, and using MolRep with the experimental phases (search for model in the
map) I have good solutions 3 of them. There is a lot of empty electron
density which needs to be filled with more copies of the molecule. I have
looked for NCS operators as I know this will improve the map and help with
model fitting (the Self Rot function suggests I have at least 2-fold
symmetry), but no luck yet. My current focus is on using the 3-molecule
partial solution as a starting point, but since I'm not getting anywhere
fast, I though I'd post to the bulletin board.

Can someone please either point me to a MolRep script that allows me to fix
the known solutions, use the experimental phases, and search for (3?) more
copies of the model, or tell if there is something wrong with the following
Phaser scripts below (is it necessary to apply an operator to the MolRep
solution before reading into Phaser?).

Thanks!

#
phaser  eof

MODE MR_FRF

HKLIN overall_best_denmod_map_coeffs.mtz
LABIN F = FP SIGF = SIGFP

ENSEMBLE trimer PDBFILE trimer.pdb IDENTITY 0.25
ENSEMBLE monomer PDBFILE monomer.pdb IDENTITY 0.25
COMPOSITION PROTEIN SEQUENCE trimer.seq NUM 1
COMPOSITION PROTEIN SEQUENCE monomer.seq NUM 3

SOLU 6DIM ENSE trimer EULER 0 0 0 FRAC 0 0 0

SEARCH ENSEMBLE monomer NUM 1

ROOT AUTO_monomer

eof
#
phaser  eof

MODE MR_FTF

HKLIN overall_best_denmod_map_coeffs.mtz
LABIN F = FP SIGF = SIGFP

ENSEMBLE trimer PDBFILE trimer.pdb IDENTITY 0.25
ENSEMBLE monomer PDBFILE monomer.pdb IDENTITY 0.25
COMPOSITION PROTEIN SEQUENCE trimer.seq NUM 1
COMPOSITION PROTEIN SEQUENCE monomer.seq NUM 3

@AUTO_monomer.rlist

ROOT AUTO_monomer2

eof
#

Re: [ccp4bb] Molecular replacement using a partial molecular replacement solution

[Klaus Piontek]

Wong,

did you look for pseudo-symmetry? For example for pseudo-translation, 
which you can deduce from a native Patterson.


Good luck,

Klaus

Mo Wong wrote:

Hello,

My problem: I have poorly phased 3.5A data which suggests 6 molecules per
ASU, and using MolRep with the experimental phases (search for model in the
map) I have good solutions 3 of them. There is a lot of empty electron
density which needs to be filled with more copies of the molecule. I have
looked for NCS operators as I know this will improve the map and help with
model fitting (the Self Rot function suggests I have at least 2-fold
symmetry), but no luck yet. My current focus is on using the 3-molecule
partial solution as a starting point, but since I'm not getting anywhere
fast, I though I'd post to the bulletin board.

Can someone please either point me to a MolRep script that allows me to fix
the known solutions, use the experimental phases, and search for (3?) more
copies of the model, or tell if there is something wrong with the following
Phaser scripts below (is it necessary to apply an operator to the MolRep
solution before reading into Phaser?).

Thanks!

#
phaser  eof

MODE MR_FRF

HKLIN overall_best_denmod_map_coeffs.mtz
LABIN F = FP SIGF = SIGFP

ENSEMBLE trimer PDBFILE trimer.pdb IDENTITY 0.25
ENSEMBLE monomer PDBFILE monomer.pdb IDENTITY 0.25
COMPOSITION PROTEIN SEQUENCE trimer.seq NUM 1
COMPOSITION PROTEIN SEQUENCE monomer.seq NUM 3

SOLU 6DIM ENSE trimer EULER 0 0 0 FRAC 0 0 0

SEARCH ENSEMBLE monomer NUM 1

ROOT AUTO_monomer

eof
#
phaser  eof

MODE MR_FTF

HKLIN overall_best_denmod_map_coeffs.mtz
LABIN F = FP SIGF = SIGFP

ENSEMBLE trimer PDBFILE trimer.pdb IDENTITY 0.25
ENSEMBLE monomer PDBFILE monomer.pdb IDENTITY 0.25
COMPOSITION PROTEIN SEQUENCE trimer.seq NUM 1
COMPOSITION PROTEIN SEQUENCE monomer.seq NUM 3

@AUTO_monomer.rlist

ROOT AUTO_monomer2

eof
#

  



--
Dr. Klaus Piontek
Albert-Ludwigs-University Freiburg
Institute of Organic Chemistry and Biochemistry, Room 401 H 
Albertstrasse 21
D-79104 Freiburg 
Germany

Phone: ++49-761-203-6036
Fax: ++49-761-203-8714
Email: klaus.pion...@ocbc.uni-freiburg.de
Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/

Re: [ccp4bb] Molecular Replacement at low resolution

[Preben Morth]

Hi Junyu

It is possible at 6 Å resolution, what I would try is to cut your data 
and try searching in different resolution ranges 20-7 or 25-6.5 , if you 
are uncertain about your search model, try with a higher RMS value try 
going up to 2.0. If you have any data, even at very low resolution, with 
heavy atom soaks try to see if you can locate your heavy atom scatterers 
in the anomalous difference Fourier map using your MR model phases. 
Wrong space group, incomplete data, poor search models and low 
resolution :-) will make your life hard. Going back to the lab and try 
getting some experimental phases and better crystals,  is probably where 
most of your time should be spend at this stage.


good luck
Preben


Junyu Xiao wrote:

Hi,

Does anyone have experience with molecular replacement at low 
resolution? I have a 6Å dataset with probably 3-4 monomers per ASU. 
Phaser doesn't seem to give me clear results. Can I get some advice? 
Any comment will be appreciated. 


Thanks a lot,
Junyu


=
Junyu Xiao
Department of Biological Chemistry,
University of Michigan

Lab address: 
3163 Life Sciences Institute, 
University of Michigan, 
210 Washtenaw Avenue

Ann Arbor, MI, 48109-2216
Phone: 734-615-2078
==






--
Jens Preben Morth, Ph.D
Aarhus University
Department of Molecular Biology
Gustav Wieds Vej 10 C
DK - 8000 Aarhus C
Tel. +45 8942 5025, Fax. +45 8612 3178
[EMAIL PROTECTED]
website: http://person.au.dk/da/[EMAIL PROTECTED]

[ccp4bb] Molecular Replacement at low resolution

[Junyu Xiao]

Hi,

Does anyone have experience with molecular replacement at low  
resolution? I have a 6Å dataset with probably 3-4 monomers per ASU.  
Phaser doesn't seem to give me clear results. Can I get some advice?  
Any comment will be appreciated.


Thanks a lot,
Junyu


=
Junyu Xiao
Department of Biological Chemistry,
University of Michigan

Lab address:
3163 Life Sciences Institute,
University of Michigan,
210 Washtenaw Avenue
Ann Arbor, MI, 48109-2216
Phone: 734-615-2078
==

Re: [ccp4bb] Molecular replacement of a multidomain protein

[Lucas Bleicher]

I've had a very good experience with MrBump:

http://www.ccp4.ac.uk/MrBUMP/

Not only because of the program itself, which was able
to find an unexpected template for the problematic
chain (the first one was straightforward in Phaser),
but also because of great support from Martyn  Ron.
It's definitely worth a try.

Lucas

--- Anjali Mehta [EMAIL PROTECTED] escreveu:

 Dear All,
 I am working with a Bifunctional protein of
 molecular weight ~60 kDa.
 I have a 3.3 angstrom native dataset.  The matthews
 number show there are 6
 molecules in the asymmetric unit.
 The structures of the individual domains are already
 known from prokaryotes.
 The sequence identity with the known structures are
 about 30%.
 I have tried molecular replacement using the two
 parts as models
 respectively with CNS, MOLREP, PHASER etc. However I
 always get the solution
 for one domain. I have also tried to fix that domain
 and find the other one.
 But none of the programs can find a solution.
 I am trying to model build the correct sequence of
 one domain using a
 density modified (using CNS), NCS averaged (using
 RAVE) map but the map does
 not look very good. The side chains are not clear.
 That might be due to the
 fact that I am only having a partial model.
 Any suggestion will be appreciated.
 Thanks.
 Ms. Anjali Mehta
 



  Abra sua conta no Yahoo! Mail, o único sem limite de espaço para 
armazenamento!
http://br.mail.yahoo.com/

Re: [ccp4bb] Molecular replacement of a multidomain protein

[Roger Rowlett]

EPMR (now Open-EPMR, http://www.epmr.info) is an excellent alternative 
for finding difficult, high-dimensional MR solutions. Experiment with 
various resolution limits. We solved an asymmetric unit with 3 
difficult-to-place dimers of low sequence homology by gradually 
increasing the high-resolution limit of the structure factor data used. 
If you have a partial static solution, it can look for additional 
protein chain placements. It is strongly recommended for your type of 
problem that you set the correlation coefficient threshold for a good 
solution to 1.00 and not use the defaults. This will force EPMR to do an 
exhaustive search.


Cheers,

--

Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [EMAIL PROTECTED]


Lucas Bleicher wrote:

I've had a very good experience with MrBump:

http://www.ccp4.ac.uk/MrBUMP/

Not only because of the program itself, which was able
to find an unexpected template for the problematic
chain (the first one was straightforward in Phaser),
but also because of great support from Martyn  Ron.
It's definitely worth a try.

Lucas

--- Anjali Mehta [EMAIL PROTECTED] escreveu:

  

Dear All,
I am working with a Bifunctional protein of
molecular weight ~60 kDa.
I have a 3.3 angstrom native dataset.  The matthews
number show there are 6
molecules in the asymmetric unit.
The structures of the individual domains are already
known from prokaryotes.
The sequence identity with the known structures are
about 30%.
I have tried molecular replacement using the two
parts as models
respectively with CNS, MOLREP, PHASER etc. However I
always get the solution
for one domain. I have also tried to fix that domain
and find the other one.
But none of the programs can find a solution.
I am trying to model build the correct sequence of
one domain using a
density modified (using CNS), NCS averaged (using
RAVE) map but the map does
not look very good. The side chains are not clear.
That might be due to the
fact that I am only having a partial model.
Any suggestion will be appreciated.
Thanks.
Ms. Anjali Mehta






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Re: [ccp4bb] Molecular replacement of a multidomain protein

[Meyer, Peter]

Hi,

A few things you might try (or that you may have tried, but didn't mention):

1. Run the search models through chainsaw with their sequence alignments before 
searching (or just use poly-alanine versions of the search models), and reset 
the B-factor of the search models to the B-factor of the dataset (or your 
favorite number, the important part is that it's constant).

2. You mentioned that you get a solution for one domain.  Is this a single 
Rotational/Translational solution set for 6 copies of this one search model, or 
something else?  Assuming it is, you could try with fewer copies of your search 
model.  If there are in fact 6 copies, and you search for 4, there should be 
density for all 6 in the maps phased from from the 4-model solution (assuming 
the MR solution is correct); but if it's the other way around there may be 
problems finding a solution.  You may also want to try a range of combinations 
for searching (1 domain A + 1 domain B, 2 domain A + 1 domain B, 1 domain A + 2 
domain B, etc).

3. It might be better not to bother with density modification for a 3.3 
Angstrom model-phased dataset, especially if you have the possibility of using 
NCS.

Good luck,

Pete

-Original Message-
From: CCP4 bulletin board on behalf of Anjali Mehta
Sent: Thu 3/6/2008 7:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Molecular replacement of a multidomain protein
 
Dear All,
I am working with a Bifunctional protein of molecular weight ~60 kDa.
I have a 3.3 angstrom native dataset.  The matthews number show there are 6
molecules in the asymmetric unit.
The structures of the individual domains are already known from prokaryotes.
The sequence identity with the known structures are about 30%.
I have tried molecular replacement using the two parts as models
respectively with CNS, MOLREP, PHASER etc. However I always get the solution
for one domain. I have also tried to fix that domain and find the other one.
But none of the programs can find a solution.
I am trying to model build the correct sequence of one domain using a
density modified (using CNS), NCS averaged (using RAVE) map but the map does
not look very good. The side chains are not clear. That might be due to the
fact that I am only having a partial model.
Any suggestion will be appreciated.
Thanks.
Ms. Anjali Mehta