[ccp4bb] nonspecific interactions with GST beads

[Peter Hsu]

Hi all,

I've recently switched over to using a GST system for purifying my proteins. 
Although I can get enough protein out of a few liters, I've always been finding 
that when I take some of the beads to run on a gel, I always get a decent 
amount of protein just stuck on there that won't elute off no matter what. I 
don't know what proportion is just sticking, but I would love to get better 
yields. 

I'm purifying using a HEPES based buffer at pH8.0 with 350mM NaCl in the 
buffer. Does anyone else have any ideas as to what I can add to my buffers to 
help reduce the amount of sticking?

Thanks,
Peter

Re: [ccp4bb] completeness in scala

[case]

On Tue, May 15, 2012, Toth, Eric wrote:

> In sports, maximal effort is considered to be 110%, so you're actually
> 9.9% short of getting everything you could out of that crystal at its
> resolution limit.  All things considered, that's not bad.

In (U.S.) politics, the standard continues to be the 1000% support given
by presidential candidate George McGovern to vice-presidential candidate
Thomas Eagleton, shortly before dropping him from the ticket.

Sounds to me like you need to re-examine the statistics of this particular
crystal to try to see why it is diffracting so poorly.

...dave case

Re: [ccp4bb] completeness in scala

[Toth, Eric]

In sports, maximal effort is considered to be 110%, so you're actually 9.9% 
short of getting everything you could out of that crystal at its resolution 
limit.  All things considered, that's not bad.


_
Eric A. Toth, Ph.D. 
Assistant Professor 
Department of Biochemistry and Molecular Biology 
Marlene and Stewart Greenebaum Cancer Center 
University of Maryland School of Medicine 
108 North Greene St. 
Baltimore, MD 21201 

Email: et...@som.umaryland.edu 
Phone: x-410-706-5345 
Fax: x-410-706-8297
http://medschool.umaryland.edu/FACULTYRESEARCHPROFILE/viewprofile.aspx?id=8032
http://crystal.umaryland.edu 


 A Third Century
    Where Discovery Transforms Medicine



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed 
Pozharski
Sent: Tuesday, May 15, 2012 1:51 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] completeness in scala

Just a curiosity - I have a dataset at 1.45A for which SCALA reports the 
highest resolution shell completeness at 100.1%.  I am impressed :-)


--
"Hurry up before we all come back to our senses!"
   Julian, King of Lemurs

[ccp4bb] completeness in scala

[Ed Pozharski]

Just a curiosity - I have a dataset at 1.45A for which SCALA reports the
highest resolution shell completeness at 100.1%.  I am impressed :-)


-- 
"Hurry up before we all come back to our senses!"
   Julian, King of Lemurs

Re: [ccp4bb] Strange Density

[Sanishvili, Ruslan]

Hi Rhis,
It may have been suggested already but still...
X-ray fluorescence spectra can often tell you what elements you may be dealing 
with. Spectra won't tell anything about binding specifics, but any extra bit of 
information could help. Any descent synchrotron MX beamline, equipped for 
MAD/SAD phasing, should be able to help. Of course, spectroscopy beamlines 
would be even better.
Cheers,
N.

Ruslan Sanishvili (Nukri), Ph.D.

GM/CA-CAT
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RHYS 
GRINTER
Sent: Tuesday, May 15, 2012 9:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Strange Density

Dear Community,

As I'm a relatively new to protein crystallography this might turn out to be an 
obvious question, however.

I'm working on the structure of a enzyme requiring Ca2+ for activity and with 
calcium coordinated in the active site by Asp and 2x backbone carbonyl groups, 
in a crystal structure with Ca in the crystallisation conditions 
(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg). 
When Ca is omitted from the crystallizing conditions and a divalent chelator 
(EGTA) is added the crystals are of significantly lower resolution (3.13A). 
Refinement of this data reveals density for a molecule coordinated by the Ca 
coordinating Asp and backbone, however this density is significantly further 
away (3.4-3.8A) too far away for water or a strongly coordinated divalent 
cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). The 
density is also much weaker than for Ca in the previous model disappearing at 
3.5 sigma.

The crystallisation conditions for the Ca free condition is:

0.1M Tris/Bicine buffer [pH 8.5]
8% PEG 8000
30% Ethylene Glycol
1mM EGTA

The protein was purified by nickel affinity/SEC and dialysed into: 
20mM NaCl 
20mM Tris [pH 8.0]


A colleague suggested that sulphate or phosphate could fit at these distances, 
but these ions have not been added at any stage of the crystallisation process. 


Could anyone give me some insight into what this density might represent?

Thanks in advance,

Rhys Grinter
PhD Candidate
University of Glasgow

[ccp4bb] how to ignore spot overlap in imosflm?

[Xinghua Qin]

Dear all,
 
Thanks for all of the suggestions, It helps me a lot as I am a newcomer to the 
structural world.
Increasing the "Profile Tolerance" parametersas Dr Harry Powell has pointed out 
 can increase the completeness by ten percent (from 50% to 60%). I will try 
other people's advice soon.
 
Although the completeness is quite low (just 50%), now I have determined the 
structure (Rfree=0.31, Rfactor=0.24, resolution=2.6). I will look into the map 
with COOT, and try to get the structure more beautiful.
 
Thanks again
 
Best wishes
 
Xinghua Qin
--
Xinghua Qin
State Key Laboratory of Plant Physiology and biochemistry 
College of Biological Sciences
China Agricultural University
No.2, Yuan Ming Yuan West Road
Haidian District, Beijing, China 100193
Tel: +86-10-62732672
E-mail: xinghua...@126.com


At 2012-05-14 11:48:58,"Zhijie Li"  wrote:

Hi Xinghua,
 
The total intensity of each reflection needs to be accurately quantitated in 
order to calculate the structure factors. Not only the dots need to be well 
separated in the 3D reciprocal space, but also a small area around the dots are 
often needed to calculate the background for subtraction. That is why when two 
dots are getting too close, the programs will reject both dots. The first thing 
you need to do is to inspect the images reported with large number of overlaps 
to see if the dots are really overlapping or just close to each other. If the 
dots are barely touching or just too close to each other, you can manipulate 
the SEPERATION parameter to force the program to take the closely spaced spots. 
But keep in mind that you may get less accurate integration by doing so. If 
many spots are really touching each other, normally we won't force the programs 
to use them. Then the proper remedy is to move the detector farther and collect 
the dataset again (also, try to optimize your f!
 reezing to get the mosaicity as low as possible).
 
For how to play with the mosflm parameters, please read here: 
http://www.mrc-lmb.cam.ac.uk/harry/cgi-bin/keyword2.cgi?SEPARATION. What you 
need is probably CLOSE.
 
The hazard of high percentage of overlaps:
If the overlaps are only scattered in a whole dataset, it is OK, even if they 
make up 5-10% or even 20% of the whole dataset. It will only give you a lower 
completeness, which is not too detrimental to the structure solution. However, 
if large, continuous regions in the dataset are missing, that will cause you to 
have poorly defined regions in the calculated map, often seen as featureless 
stripes or layers in the map. Unfortunately, when you have closely spaced 
reflections, the latter is often the case. The proper solution is to collect 
the data at a greater detector distance to resolve the spots (after taking the 
test images, both imosflm and HKL2000 can simulate the collection run to help 
you to decide what distance you need). In cases that you have a long unit cell 
(>200A), the first thing you need to do is to align the long edge of the Unix 
cell with the rotational axis of the pin. In the difficult cases, you probably 
even need to shoot multiple crystals and combine the !
 datasets to get enough completeness.
 
Zhijie
 


From:Xinghua Qin
Sent: Sunday, May 13, 2012 10:22 PM
To:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] how to ignore spot overlap in imosflm?


Dear CCP4ers,
 
We collected a diffraction dataset with high percentage of spot overlaps, It 
would be so kind to tell me how to ignore spot overlap in imosflm and explain 
the hazard of high percentage of spot overlaps.
Thanks in advance.
 
Best wishes
 
Xinghua Qin
--
Xinghua Qin
State Key Laboratory of Plant Physiology and biochemistry 
College of Biological Sciences
China Agricultural University
No.2, Yuan Ming Yuan West Road
Haidian District, Beijing, China 100193
Tel: +86-10-62732672
E-mail: xinghua...@126.com

Re: [ccp4bb] Strange Density

[Dale Tronrud]

   Your holo structure has a Ca++ and three water molecules that have not been
built into your low resolution apo map.  These atoms are not expected to be
resolved at 3 A resolution, so I would expect them to appear as a large, 
misshapened,
blob.  Your screenshot only shows one contour level.  It is quite possible that
the highest density value is not at the center of the blob.

   You might have a lower occupancy Ca++ atom at the site and the image is 
confused
by the low resolution.  Remember, even if the concentration of Ca++ is lower in
this mother liquor any Ca++ that binds will bind exactly as it does in the
fully occupied case.  A weakly binding Ca++ site will not bind before the 
strongly
binding site.

   I would first look to see what your holo map looks like when it's resolution 
is
truncated to 3 A.  This will give you a sense of what a Ca++ binding in this 
site
would look like.  You could try refining a model with the Ca++ and water 
molecules,
with lower occupancy, and see what the residual difference map looks like.  You 
will,
of course, have to have strong restraints on the geometry to hold this model 
together
at 3 A resolution, but fortunately you have a higher resolution model to base 
these
restraints on.

   The PDB file is a statement of your belief of what is in the crystal.  Don't 
waste
your time refining models that don't make chemical sense.  An ion floating in 
space
with no ligands is not a reasonable model so even if it "fits" the density it 
can't
be correct.

   There a multiple ways of justifying the model of a crystal and others on the 
list
will likely have different ideas for the criteria that should be used.  My 
belief is
that you know the holo model and the most likely outcome of your Ca++ extraction
experiment (in a Bayesian prior sense) is a lower occupancy binding of the Ca++ 
and
its water molecules.  If you build and refine that model and the difference map 
is
acceptable you can say that this model is consistent with your experiment.  If 
there
is residual density then you can conclude that something is replacing the Ca++,
but untangling superimposed, partial occupancy, models at 3.1 A resolution is
extremely difficult.  I think all you will be able to say is that "something
replaces the Ca++ but it cannot be identified".

   Not everything can be identified in a 3 A map.  Not everything can be 
identified
in a 1 A map.  Your job is to say "these parts I understand and these parts I 
don't".

Dale Tronrud

On 05/15/12 07:51, RHYS GRINTER wrote:
> Dear Community,
> 
> As I'm a relatively new to protein crystallography this might turn out to be 
> an obvious question, however.
> 
> I'm working on the structure of a enzyme requiring Ca2+ for activity and with 
> calcium coordinated in the active site by Asp and 2x backbone carbonyl 
> groups, in a crystal structure with Ca in the crystallisation conditions 
> (http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg). 
> When Ca is omitted from the crystallizing conditions and a divalent chelator 
> (EGTA) is added the crystals are of significantly lower resolution (3.13A). 
> Refinement of this data reveals density for a molecule coordinated by the Ca 
> coordinating Asp and backbone, however this density is significantly further 
> away (3.4-3.8A) too far away for water or a strongly coordinated divalent 
> cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). 
> The density is also much weaker than for Ca in the previous model 
> disappearing at 3.5 sigma.
> 
> The crystallisation conditions for the Ca free condition is:
> 
> 0.1M Tris/Bicine buffer [pH 8.5]
> 8% PEG 8000
> 30% Ethylene Glycol
> 1mM EGTA
> 
> The protein was purified by nickel affinity/SEC and dialysed into: 
> 20mM NaCl 
> 20mM Tris [pH 8.0]
> 
> 
> A colleague suggested that sulphate or phosphate could fit at these 
> distances, but these ions have not been added at any stage of the 
> crystallisation process. 
> 
> 
> Could anyone give me some insight into what this density might represent?
> 
> Thanks in advance,
> 
> Rhys Grinter
> PhD Candidate
> University of Glasgow

[ccp4bb] multiple sequence alignments..should be done structurally if possible

[Paul Kraft]

One must remember that multiple sequences alignments are not just alignment of 
letters...they are amino acids with three dimensional (four if you count time) 
coordinates.
The alignments can be very subjective and are best done in conjunction with a 
structural alignment of overlapping structures. Often it is best to choose PDB 
structures with the most diverse sequences in a particular structural family 
using VAST and/or DALI to align sequences, paying careful attention to 
alignment of prolines and glycines, knowing that loops are most always 
conserved, alpha helices are almost always alpha helices, strands almost 
always strands, and the connecting chain lengths may vary. 
Be patient :-)

Dr. Paul Kraft
Structural Biologist
cell 586-596-2770
email: haresea...@yahoo.com

Re: [ccp4bb] How to refine CNS output file in refmac

[Garib N Murshudov]

Dear Vidisha

The easiest way is to convert DNA names to standard pdb v3 names. You can do it 
using molprobity. The probelem is that GUA may have different meaning than G 
(RNA) or DG (DNA).

regards
Garib

On 15 May 2012, at 16:37, tipu Lall wrote:

> Dear CCP4 users,
>  
> I am a beginner in this field. It would be kind of all of you if can help me 
> how to move ahead with my problem.
>  
> I have an output file from CNS of a binary complex (DNA-protein) and I am 
> trying to refine it with refmac5 (restrain refinement) and job is failing 
> with error messages (written below)
>  
> Before restrain refinement, I converted the CNS output file (pdb) from 
> Codconv of CCP4 to pdb format.
>  
> Thanks in advance
> Vidisha
>  
>   INFO: link is found (not be used) dist=   3.003 ideal_dist=   2.400
> ch:CC   res:   1  GUA  at:C2' .->ch:Ca   res:   2  GUA  
> at:O2P .
>   INFO: link is found (not be used) dist=   2.625 ideal_dist=   2.400
> ch:CC   res:   1  GUA  at:C3' .->ch:Ca   res:   2  GUA  
> at:P   .
>   INFO: link is found (not be used) dist=   2.797 ideal_dist=   2.400
> ch:CC   res:   1  GUA  at:C3' .->ch:Ca   res:   2  GUA  
> at:O2P .
>   INFO: link is found (not be used) dist=   1.606 ideal_dist=   2.400
> ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA  
> at:P   .
>   INFO: link is found (not be used) dist=   2.499 ideal_dist=   2.400
> ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA  
> at:O1P .
>   INFO: link is found (not be used) dist=   2.504 ideal_dist=   2.400
> ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA  
> at:O2P .
>   INFO: link is found (not be used) dist=   2.507 ideal_dist=   2.400
> ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA  
> at:O5' .
>   INFO: link is found (not be used) dist=   2.843 ideal_dist=   2.400
> ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA  
> at:C5' .
>   INFO: link is found (not be used) dist=   2.637 ideal_dist=   2.400
> ch:Ca   res:   2  GUA  at:C3' .->ch:Cb   res:   3  ADE  
> at:P   .
>   INFO: link is found (not be used) dist=   3.116 ideal_dist=   2.400
> ch:Ca   res:   2  GUA  at:C3' .->ch:Cb   res:   3  ADE  
> at:O1P .
>   INFO: link is found (not be used) dist=   1.604 ideal_dist=   2.400
> ch:Ca   res:   2  GUA  at:O3' .->ch:Cb   res:   3  ADE  
> at:P   .
>   INFO: link is found (not be used) dist=   2.504 ideal_dist=   2.400
> ch:Ca   res:   2  GUA  at:O3' .->ch:Cb   res:   3  ADE  
> at:O1P .
>   INFO: link is found (not be used) dist=   2.514 ideal_dist=   2.400
> ch:Ca   res:   2  GUA  at:O3' .->ch:Cb   res:   3  ADE  
> at:O2P .
>  
>   ATTENTION: atom:O4   GUA 1  CC   is missing in the structure
>   ATTENTION: atom:O3   GUA 1  CC   is missing in the structure
>   ATTENTION: atom:C3   GUA 1  CC   is missing in the structure
>   ATTENTION: atom:C1   GUA 1  CC   is missing in the structure
>   ATTENTION: atom:O1   GUA 1  CC   is missing in the structure
>   ATTENTION: atom:O2   GUA 1  CC   is missing in the structure
>  
> ===> Error: Fatal error. Cannot continue
>  

Dr Garib N Murshudov
Group Leader, MRC Laboratory of Molecular Biology
Hills Road 
Cambridge 
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk 
Web http://www.mrc-lmb.cam.ac.uk

[ccp4bb] How to refine CNS output file in refmac

[tipu Lall]

Dear CCP4 users,



I am a beginner in this field. It would be kind of all of you if can help
me how to move ahead with my problem.



I have an output file from CNS of a binary complex (DNA-protein) and I am
trying to refine it with refmac5 (restrain refinement) and job is failing
with error messages (written below)



Before restrain refinement, I converted the CNS output file (pdb) from
Codconv of CCP4 to pdb format.



Thanks in advance

Vidisha



  INFO: link is found (not be used) dist=   3.003 ideal_dist=   2.400

ch:CC   res:   1  GUA  at:C2' .->ch:Ca   res:   2  GUA
 at:O2P
.

  INFO: link is found (not be used) dist=   2.625 ideal_dist=   2.400

ch:CC   res:   1  GUA  at:C3' .->ch:Ca   res:   2  GUA
at:P   .

  INFO: link is found (not be used) dist=   2.797 ideal_dist=   2.400

ch:CC   res:   1  GUA  at:C3' .->ch:Ca   res:   2  GUA
 at:O2P
.

  INFO: link is found (not be used) dist=   1.606 ideal_dist=   2.400

ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA
at:P   .

  INFO: link is found (not be used) dist=   2.499 ideal_dist=   2.400

ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA
 at:O1P
.

  INFO: link is found (not be used) dist=   2.504 ideal_dist=   2.400

ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA
 at:O2P
.

  INFO: link is found (not be used) dist=   2.507 ideal_dist=   2.400

ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA
 at:O5'
.

  INFO: link is found (not be used) dist=   2.843 ideal_dist=   2.400

ch:CC   res:   1  GUA  at:O3' .->ch:Ca   res:   2  GUA
 at:C5'
.

  INFO: link is found (not be used) dist=   2.637 ideal_dist=   2.400

ch:Ca   res:   2  GUA  at:C3' .->ch:Cb   res:   3  ADE
at:P   .

  INFO: link is found (not be used) dist=   3.116 ideal_dist=   2.400

ch:Ca   res:   2  GUA  at:C3' .->ch:Cb   res:   3  ADE
 at:O1P
.

  INFO: link is found (not be used) dist=   1.604 ideal_dist=   2.400

ch:Ca   res:   2  GUA  at:O3' .->ch:Cb   res:   3  ADE
at:P   .

  INFO: link is found (not be used) dist=   2.504 ideal_dist=   2.400

ch:Ca   res:   2  GUA  at:O3' .->ch:Cb   res:   3  ADE
 at:O1P
.

  INFO: link is found (not be used) dist=   2.514 ideal_dist=   2.400

ch:Ca   res:   2  GUA  at:O3' .->ch:Cb   res:   3  ADE
 at:O2P
.



  ATTENTION: atom:O4   GUA 1  CC   is missing in the structure

  ATTENTION: atom:O3   GUA 1  CC   is missing in the structure

  ATTENTION: atom:C3   GUA 1  CC   is missing in the structure

  ATTENTION: atom:C1   GUA 1  CC   is missing in the structure

  ATTENTION: atom:O1   GUA 1  CC   is missing in the structure

  ATTENTION: atom:O2   GUA 1  CC   is missing in the structure



===> Error: Fatal error. Cannot continue

Re: [ccp4bb] Strange Density

[Ed Pozharski]

On Tue, 2012-05-15 at 15:51 +0100, RHYS GRINTER wrote:
> A colleague suggested that sulphate or phosphate could fit at these
> distances, but these ions have not been added at any stage of the
> crystallisation process. 
> 

I vaguely remember a report about 2-3 years ago at the ACA meeting of
phosphate contamination of PEGs from certain manufacturers.  But why
would phosphate (negatively charged) bind at the site that seems to
attract calcium (positively charged)?  Potassium?

Check if you have a peak at that position in anomalous difference map.
Maybe ICP-MS can give you an answer.

-- 
I don't know why the sacrifice thing didn't work.  
Science behind it seemed so solid.
Julian, King of Lemurs

Re: [ccp4bb] Strange Density

[Kelly Daughtry]

Try refining with: Na, Ca or Water at that position and compare the
resulting maps. That should provide you with the information you need.

It could be a weakly bound sodium, or calcium ion. It could be that
calcium was not fully removed by EGTA treatment.

Kelly
***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Tue, May 15, 2012 at 10:51 AM, RHYS GRINTER
 wrote:
> Dear Community,
>
> As I'm a relatively new to protein crystallography this might turn out to be 
> an obvious question, however.
>
> I'm working on the structure of a enzyme requiring Ca2+ for activity and with 
> calcium coordinated in the active site by Asp and 2x backbone carbonyl 
> groups, in a crystal structure with Ca in the crystallisation conditions 
> (http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg).
> When Ca is omitted from the crystallizing conditions and a divalent chelator 
> (EGTA) is added the crystals are of significantly lower resolution (3.13A). 
> Refinement of this data reveals density for a molecule coordinated by the Ca 
> coordinating Asp and backbone, however this density is significantly further 
> away (3.4-3.8A) too far away for water or a strongly coordinated divalent 
> cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). 
> The density is also much weaker than for Ca in the previous model 
> disappearing at 3.5 sigma.
>
> The crystallisation conditions for the Ca free condition is:
>
> 0.1M Tris/Bicine buffer [pH 8.5]
> 8% PEG 8000
> 30% Ethylene Glycol
> 1mM EGTA
>
> The protein was purified by nickel affinity/SEC and dialysed into:
> 20mM NaCl
> 20mM Tris [pH 8.0]
>
>
> A colleague suggested that sulphate or phosphate could fit at these 
> distances, but these ions have not been added at any stage of the 
> crystallisation process.
>
>
> Could anyone give me some insight into what this density might represent?
>
> Thanks in advance,
>
> Rhys Grinter
> PhD Candidate
> University of Glasgow

Re: [ccp4bb] Off topic about program for multiple protein sequence alignment

[First, Eric]

Dear Donghui,

Your implied question seems to be "How do I know whether my MSA is
good?"  Errors in MSAs arise from two factors.  First, due to
computational limitations, heuristic methods are used for most MSA
programs.  As a result, one can never be certain that a progressive MSA
method actually gives you the optimum alignment since not all of the
alignment space has been searched.  Having said that, some programs are
better than others at finding reasonable alignments (e.g. TCoffee is
better than ClustalW).  The second factor is the scoring system itself.
The choice of substitution matrix that you use depends on how divergent
your sequences are.  Ideally, one should try several different matrices
and see which ones work best.  The gap opening and extension penalties
are another matter, since they not only vary between protein families,
but within the protein sequence itself.  The bottom line is that no
matter what method you use, you need to look at the alignment and, if
possible, overlay the secondary structure for one or more of the
homologs to make sure that gaps and insertions occur in sensible places
(e.g. loops), signature sequences line up, etc.

With best regards,
Eric

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Tim Gruene
Sent: Tuesday, May 15, 2012 2:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic about program for multiple protein
sequence alignment

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Donghui,

even within one program (clustalw) you would get different results by
picking different weighting schemes (clustalx: Aligment->Alignment
Parameter -> Multiple Alignment Parameter: BLOSUM, PAM, Gonnet...).

As with any software I would assume the developers know best what they
are doing and recommend to stick to the defaults unless you know what
you are doing.

Regards,
Tim

On 05/15/12 08:02, wu donghui wrote:
> Dear all,
> 
> I want to know your suggestions about current protein sequence 
> alignment programs. It seems that different programs give different 
> alignment results such as from analysis of Clustal W and MULTALIN.
> Thanks for any input or comments.
> 
> Best regards,
> 
> Donghui
> 

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

GPG Key ID = A46BEE1A

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[ccp4bb] Strange Density

[RHYS GRINTER]

Dear Community,

As I'm a relatively new to protein crystallography this might turn out to be an 
obvious question, however.

I'm working on the structure of a enzyme requiring Ca2+ for activity and with 
calcium coordinated in the active site by Asp and 2x backbone carbonyl groups, 
in a crystal structure with Ca in the crystallisation conditions 
(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg). 
When Ca is omitted from the crystallizing conditions and a divalent chelator 
(EGTA) is added the crystals are of significantly lower resolution (3.13A). 
Refinement of this data reveals density for a molecule coordinated by the Ca 
coordinating Asp and backbone, however this density is significantly further 
away (3.4-3.8A) too far away for water or a strongly coordinated divalent 
cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). The 
density is also much weaker than for Ca in the previous model disappearing at 
3.5 sigma.

The crystallisation conditions for the Ca free condition is:

0.1M Tris/Bicine buffer [pH 8.5]
8% PEG 8000
30% Ethylene Glycol
1mM EGTA

The protein was purified by nickel affinity/SEC and dialysed into: 
20mM NaCl 
20mM Tris [pH 8.0]


A colleague suggested that sulphate or phosphate could fit at these distances, 
but these ions have not been added at any stage of the crystallisation process. 


Could anyone give me some insight into what this density might represent?

Thanks in advance,

Rhys Grinter
PhD Candidate
University of Glasgow

Re: [ccp4bb] dm: Error in opening input map file.

[Yu Feng]

Dear De-Feng Li,

Thank you for your reply. Actually, I already used the script. The script
and the log file are at the bottom of the email.

Best wishes,
Yu


On Tue, May 15, 2012 at 3:18 AM, lidefeng  wrote:

> Dear Yu Feng,
>
> You could try it in script, but not in the CCP4i.   The same
> problem could be found in DMMulti.
>
>   Your sincerely
>De-Feng Li
>2012-05-15
>
> De-Feng Li, Ph.D,
> National Laboratory of Biomacromolecules
> Institute of Biophysics, Chinese Academy of Sciences
> 15 Datun Road, Chaoyang District
> Beijing 100101, PR China
>
>
> === 2012-05-15 18:44:48 You writed in your letter:===
>
> >Dear CCP4ers,
> >
> >I have a problem when I use DM to do NCS averaging. If I input 9 NCS
> >averaging masks, DM works OK. However, if I input 10 NCS averaging masks,
> >DM can not open input map file. The masks should be OK because they are
> >generated by the same method. Do you have any idea how to solve the
> problem?
> >
> >Thank you in advance!
> >Yu
> >
> >
> >DM script is as below:
>
> >
> >/usr/share/CCP4-6.0.2/ccp4-6.2.0/bin/dm \
> >hklin
>
> >/home/crystal/Documents/Ecoli/datasets/F111062011/130_10/reprocess/DM/x_refine33_phase.mtz
> >\
> >ncsin1 E.msk   \
> >ncsin2 F1.msk   \
> >ncsin3 F2.msk   \
> >ncsin4 D4.msk   \
> >ncsin5 D5.msk   \
> >ncsin6 D3.msk   \
> >ncsin7 D2.msk   \
> >ncsin8 D1.msk   \
> >ncsin9 C5.msk   \
> >ncsin10 C6.msk   \
> >hklout x_dm.mtz < >SOLC 0.62
> >#RESOL 50 4.0
> >#NCSMASK SIZE 1
> >#NCSMASK UPDATE 3
> >#GRID 144 144 144
> >MODE SOLV hist AVER MULTI
> >combine weight 0.15
> >combine pert
> >scheme res from 4.5
> >NCYCLE 10
> >ncsmask overlap
> >AVER DOMAIN 1 REFINE
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 1 REFINE
> >ROTA MATRIX -0.26665 0.52763 0.80654 0.60893 -0.55642 0.56533 0.74706
> >0.64187 -0.17293
> >TRAN -31.41600 172.23766 -96.59856
> >
> >AVER DOMAIN 1 REFINE
> >ROTA MATRIX 0.82271 0.34200 -0.45406 -0.55866 0.63407 -0.53465 0.10506
> >0.69353 0.71272
> >TRAN -141.48692 39.40455 -17.81890
> >
> >AVER DOMAIN 1 REFINE
> >ROTA MATRIX -0.35001 0.06658 0.93438 0.03939 -0.99554 0.08569 0.93592
> >0.06679 0.34582
> >TRAN -75.12174 220.50938 35.16329
> >
> >AVER DOMAIN 2 REFIN
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 2 REFINE
> >ROTA MATRIX -0.42731 0.24363 0.87066 0.39149 -0.81818 0.42109 0.81494
> >0.52080 0.25424
> >TRAN -7.42336 185.84557 -68.83428
> >
> >AVER DOMAIN 2 REFINE
> >ROTA MATRIX -0.42918 0.04126 0.90228 0.01801 -0.99837 0.05422 0.90304
> >0.03952 0.42774
> >TRAN -74.51554 219.69878 39.63858
> >
> >AVER DOMAIN 2 REFINE
> >ROTA MATRIX 0.94716 0.32039 -0.01558 -0.30765 0.89358 -0.32691 -0.09082
> >0.31442 0.94493
> >TRAN -121.99072 28.41423 20.60830
> >
> >AVER DOMAIN 3 REFINE
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 3 REFINE
> >ROTA MATRIX -0.42590 0.08845 0.90044 -0.00887 -0.99557 0.09361 0.90473
> >0.03188 0.42480
> >TRAN -77.38764 220.87816 40.52480
> >
> >AVER DOMAIN 4 REFINE
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 4 REFINE
> >ROTA MATRIX -0.34404 0.07038 0.93631 -0.03070 -0.99750 0.06370 0.93845
> >-0.00683 0.34534
> >TRAN -79.74300 222.43433 43.52758
> >
> >AVER DOMAIN 5 REFINE
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 5 REFINE
> >ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309
> >-0.01068 0.35948
> >TRAN -70.14525 224.81303 43.77335
> >
> >AVER DOMAIN 6 REFINE
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 6 REFINE
> >ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309
> >-0.01068 0.35948
> >TRAN -70.14525 224.81303 43.77335
> >
> >AVER DOMAIN 7 REFINE
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 7 REFINE
> >ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309
> >-0.01068 0.35948
> >TRAN -70.14525 224.81303 43.77335
> >
> >AVER DOMAIN 8 REFINE
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 8 REFINE
> >ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309
> >-0.01068 0.35948
> >TRAN -70.14525 224.81303 43.77335
> >
> >AVER DOMAIN 9 REFINE
> >ROTATE EULER   0.0000.0000.000
> >TRANSLATION0.0000.0000.000
> >
> >AVER DOMAIN 9 REFINE
> >ROTA MATRIX -0.35957 -0.00781 0.93309 -0.00716 -0.1 -0.01113 0.93309
> >-0.01068 0.35948
> >TRAN -70.14525 224.81303 43.77

Re: [ccp4bb] Off topic about program for multiple protein sequence alignment

[Bart Hazes]

mafft and muscle are both faster than clustalw and on average more 
accurate. You can also use different options for mafft to push for speed 
or accuracy depending on your needs and patience. Tcoffee has a flavour 
that includes structural information if available to assist alignment. 
Another flavour, MCoffee, runs a set of different alignment programs, 
including clustalw, mafft ..., and assembles the most consistent 
alignment based on all of them. If you are dealing with a non-trivial 
alignment and its accuracy matters to you then you should always take a 
look at it because even the best programs sometimes make obvious 
mistakes. It can also pay off to use two alignment programs based on 
different methodologies. But don't waste too much time staring at poorly 
defined regions as they may simply not be structurally or evolutionary 
equivalent.


Bart

On 12-05-15 03:50 AM, martyn.w...@stfc.ac.uk wrote:

Certainly different programs and different scoring matrices will give different 
answers. There is not necessarily a correct answer either, just different 
educated guesses. With high sequence identity, the answers should be fairly 
consistent. As you reduce the sequence identity (i.e. as it gets more 
interesting) then the answers will vary more.

I think clustalw is generally considered to be one of the poorer multiple 
alignment programs these days (though I am sure opinions will vary here). By 
poor, I mean it struggles in the twilight zone of 25 - 30% seq identity, but is 
perfectly adequate for routine use. There are many other programs to choose 
from: probcons, mafft, TCoffee, Muscle, etc etc. In some tests of MrBUMP, we 
found some marginal cases that relied on using an alignment from probcons or 
mafft, rather than clustalw.

HTH
Martyn



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Tim Gruene
Sent: 15 May 2012 08:20
To: ccp4bb
Subject: Re: [ccp4bb] Off topic about program for multiple protein
sequence alignment

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Donghui,

even within one program (clustalw) you would get different results by
picking different weighting schemes (clustalx: Aligment->Alignment
Parameter ->  Multiple Alignment Parameter: BLOSUM, PAM, Gonnet...).

As with any software I would assume the developers know best what they
are doing and recommend to stick to the defaults unless you know what
you are doing.

Regards,
Tim

On 05/15/12 08:02, wu donghui wrote:

Dear all,

I want to know your suggestions about current protein sequence
alignment programs. It seems that different programs give different
alignment results such as from analysis of Clustal W and MULTALIN.
Thanks for any input or comments.

Best regards,

Donghui


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

GPG Key ID = A46BEE1A

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Re: [ccp4bb] Multiple structure alignment and citing CCP4bb

[Ed Pozharski]

On Tue, 2012-05-15 at 07:27 +0100, Naveed A Nadvi wrote:
> I was wondering if there is any software out there that can be used
> for multiple structure superimposition and output some graphical plot
> of residues based on their deviation from the reference molecule.

And why exactly you cannot accomplish this using lsqkab?

-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

[ccp4bb] Opportunities to develop new MX integration software

[Gwyndaf Evans]

Dear All,

Can I draw your attention to the following vacancies at Diamond Light Source 
for scientific programmers to work on the development of new integration 
software for MX. The work forms part of a European collaboration under the 
BioStruct-X (www.biostruct-x.org) banner. Diamond 
are working closely with other European sites as well as CCP4 and the MRC on 
the development of new software for diffraction data analysis.

Please see http://www.diamond.ac.uk/Home/Jobs/Current/DIA0735_CG.html for 
details or contact me at 
gwyndaf.ev...@diamond.ac.uk for more 
information.

All the best
Gwyndaf


Dr Gwyndaf Evans
Principal Beamline Scientist for I24 Microfocus MX
Macromolecular Crystallography Village Coordinator
Diamond Light Source
Harwell Science and Innovation Campus
Didcot OX11 0DE
United Kingdom

Tel: +44-1235-778164Fax: +44-1235-778448   Email: 
gwyndaf.ev...@diamond.ac.uk

Re: [ccp4bb] how to ignore spot overlap in imosflm?

[Loes Kroon-Batenburg]

Dear Xinghua,

If you want to get the most out of your data, despite the fact that your data 
collection strategy was far from ideal, you may want to use EVAL15 
(www.crystal.chem.uu.nl/distr/eval). It it pretty good in modelling the complete 
profile, including overlap, and therefore is good at deconvoluting reflections 
that may overlap by as much as 60-70%.


Regards,
Loes Kroon-Batenburg

On 05/14/12 04:22, Xinghua Qin wrote:

Dear CCP4ers,
We collected a diffraction dataset with high percentage of spot overlaps, It
would be so kind to tell me how to ignore spot overlap in imosflm and explain
the hazard of high percentage of spot overlaps.
Thanks in advance.
Best wishes
Xinghua Qin
--
Xinghua Qin
State Key Laboratory of Plant Physiology and biochemistry
College of Biological Sciences
China Agricultural University
No.2, Yuan Ming Yuan West Road
Haidian District, Beijing, China 100193
Tel: +86-10-62732672
E-mail:/xinghua...@126.com/ 
// 





--
__

Dr. Loes Kroon-Batenburg
Dept. of Crystal and Structural Chemistry
Bijvoet Center for Biomolecular Research
Utrecht University
Padualaan 8, 3584 CH Utrecht
The Netherlands

E-mail : l.m.j.kroon-batenb...@uu.nl
phone  : +31-30-2532865
fax: +31-30-2533940
__

Re: [ccp4bb] Off topic about program for multiple protein sequence alignment

[martyn . winn]

Certainly different programs and different scoring matrices will give different 
answers. There is not necessarily a correct answer either, just different 
educated guesses. With high sequence identity, the answers should be fairly 
consistent. As you reduce the sequence identity (i.e. as it gets more 
interesting) then the answers will vary more.

I think clustalw is generally considered to be one of the poorer multiple 
alignment programs these days (though I am sure opinions will vary here). By 
poor, I mean it struggles in the twilight zone of 25 - 30% seq identity, but is 
perfectly adequate for routine use. There are many other programs to choose 
from: probcons, mafft, TCoffee, Muscle, etc etc. In some tests of MrBUMP, we 
found some marginal cases that relied on using an alignment from probcons or 
mafft, rather than clustalw.

HTH
Martyn


> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Tim Gruene
> Sent: 15 May 2012 08:20
> To: ccp4bb
> Subject: Re: [ccp4bb] Off topic about program for multiple protein
> sequence alignment
> 
> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Dear Donghui,
> 
> even within one program (clustalw) you would get different results by
> picking different weighting schemes (clustalx: Aligment->Alignment
> Parameter -> Multiple Alignment Parameter: BLOSUM, PAM, Gonnet...).
> 
> As with any software I would assume the developers know best what they
> are doing and recommend to stick to the defaults unless you know what
> you are doing.
> 
> Regards,
> Tim
> 
> On 05/15/12 08:02, wu donghui wrote:
> > Dear all,
> >
> > I want to know your suggestions about current protein sequence
> > alignment programs. It seems that different programs give different
> > alignment results such as from analysis of Clustal W and MULTALIN.
> > Thanks for any input or comments.
> >
> > Best regards,
> >
> > Donghui
> >
> 
> - --
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
> 
> GPG Key ID = A46BEE1A
> 
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.12 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
> 
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> VfD3ly3bmqycO0mX888oYfE=
> =7v6r
> -END PGP SIGNATURE-

Re: [ccp4bb] Off topic about program for multiple protein sequence alignment

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Donghui,

even within one program (clustalw) you would get different results by
picking different weighting schemes (clustalx: Aligment->Alignment
Parameter -> Multiple Alignment Parameter: BLOSUM, PAM, Gonnet...).

As with any software I would assume the developers know best what they
are doing and recommend to stick to the defaults unless you know what
you are doing.

Regards,
Tim

On 05/15/12 08:02, wu donghui wrote:
> Dear all,
> 
> I want to know your suggestions about current protein sequence
> alignment programs. It seems that different programs give different
> alignment results such as from analysis of Clustal W and MULTALIN.
> Thanks for any input or comments.
> 
> Best regards,
> 
> Donghui
> 

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

GPG Key ID = A46BEE1A

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