Re: [ccp4bb] hello

[Herman . Schreuder]

Dear Afshan,
 
I have been struggling with the same irrating "feature" (I would call it
a bug), which was for me one of the reasons to abandon refmac. For some
reason and against PDB standards, refmacs wants linked amino acids to be
consecutively numbered and otherwise decides that they are not linked
and creates a funny gap record. However, I do not know whether this
"feature" is still present in the latest refmac version.
 
My advice would be:
1) Make sure you have the most recent version of refmac.
2) check in coot that the affected residues have not been pushed away
from each other and delete the gap records from your pdb file.
3) run a round of refinement and see what happens. If the problem
persists you have two or three options:
a) try a different refinement program
b) renumber your pdb, so linked amino acids have consecutive residue
numbers
c) add explicit link records to link the affected residues. If I recall
correctly you have to specify the atoms (N and C). The link is probably
called "trans"
 
Good luck!
Herman




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Afshan Begum
Sent: Thursday, July 21, 2011 5:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] hello


Dear all,


I have facing one problem during the refinement of my protein .
Actually in my protein  there are some modified amino acids are present
like Cystein is modified into CME which i can get easily from monomer
libraray in coot . but after refinement in Pdb text file  indicated some
gaps while in the structures there are no gap in between these amino
acids so if any one suggest me what to do. I would appreciate your kind
suggestions.



LINKRGLU A 142 LEU A 144
gap 
LINKRSER A 328 GLY A 330
gap 
LINKRLEU A 138 GLU A 140
gap 
LINKRGLU A 126 ASP A 130
gap 
LINKRSER A 246 GLY A 248
gap 


Many thanks for your time

Best regards

Afshan

Re: [ccp4bb] hello

[Garib N Murshudov]

Dear AfshanMake sure that group name for CME is peptide (or L-peptide).  In the new version CME is peptide. I am not sure it was the case in older versions. I attach CME as a peptide. You can add this into your dictionary.Then CME can become part of a peptde. It would also be good to remove all the link records in the beginning of refmac. RegardsGarib

CME.cif
Description: Binary data
On 21 Jul 2011, at 16:13, Afshan Begum wrote:Dear all,I have facing one problem during the refinement of my protein . Actually in my protein  there are some modified amino acids are present  like Cystein is modified into CME which i can get easily from monomer libraray in coot . but after refinement in Pdb text file  indicated some gaps while in the structures there are no gap in between these amino acids so if any one suggest me what to do. I would appreciate your kind suggestions.LINKR    GLU A 142 LEU A
 144    gap LINKR    SER A 328 GLY A 330    gap LINKR    LEU A 138 GLU A 140    gap LINKR    GLU A
 126 ASP A 130    gap LINKR    SER A 246 GLY A 248    gap  Many thanks for your timeBest regardsAfshan
Garib N Murshudov Structural Studies DivisionMRC Laboratory of Molecular BiologyHills Road Cambridge CB2 0QH UKEmail: garib@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk

Re: [ccp4bb] Concentrating a protein solution - subbu

[M T]

A solution could be to control the solubility of your protein in different
pH and salts (you can also add some additives) by using the Thermal Shift
Assay. You may find a better buffer in which to concentrate your protein.

If you think that the reason of the low diffraction is the quality of your
crystals try the suggestion of Eric, but if you think that is due to the
small size of the crystals, you can also try to make drops of 3µl (2µl of
protein and 1µl of the well) or to feed your crystals during the growing
(i.e. adding 0.5µl of protein solution to your drop after when you see that
the crystals stop to grow), or to do seeding like Eric suggests, but in big
drops (5µl in hanging or more in sitting drop) and with serial dilutions of
the seeds.

Good luck...

Michel.

Dear All:
> We have been trying to crystallize a protein which is large - > 100 kDa.
> This is soluble but the best we can get is about 1 mg/mL.
> It did crystallize but did not diffract well. Efforts to increase the
> concentration has been unsuccessful. I am wondering whether there are
> methods that others use to increase the concentration other that using
> amicon columns.
> Any help will be appreciated.
> Thanks
> Subbu
>

Re: [ccp4bb] Phaser and Molrep gave different solutions

[ccp4]

The self rotation isnt the correct input to the translation function -
just run the Auto-Molrep option..
Eleanor

On Thu, 21 Jul 2011 20:57:02 +0800, Hubing Lou 
wrote:
> I also processed with Imosflm and ran Pointless, it was P21. Also it was
> indicated by the "Intensity systematic absences" in HKL2000 scale.log
file.
> 
> Best,
> Hubing
> 
> On Thu, Jul 21, 2011 at 8:44 PM,
> wrote:
> 
>> **
>>  Dear Hubing,
>>
>> One maybe stupid question: Your are sure the space group is P21 and not
>> P2
>> or even something else? Did you test other possible space groups?
>> Choosing
>> the wrong space group could exactly lead to the results you observe.
>>
>> Best,
>> Herman
>>
>>
>>  --
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
Of
>> *Hubing
>> Lou
>> *Sent:* Thursday, July 21, 2011 6:46 AM
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] Phaser and Molrep gave different solutions
>>
>> Dear all,
>>
>> I am stuck in a molecular replacement case and looking for advices.
>> I have been working on a protein-DNA complex structure.
>> Data was processed by HKL2000 to 2.6Ang and some of the data statistics
>> are
>> shown below:
>>
>> Space group: P21,
>> Unit Cell: 54.73, 104.91, 78.40, 90, 100.2, 90
>> Redundancy: 2.8 (2.7)
>> Completeness: 94.8 (93.1)
>> Linear R-fac: 0.051 (0.442)
>>
>> Data quality was checked by Phenix.xtriage and there's no problem. I
then
>> prepared a model by Chainsaw. Our protein shares only 30% of sequence
>> similarity with the model, but structurally they are in the same group
>> and
>> almost identical in apo form. Matthrews Coeff indaced two monomers in
>> AU. I
>> then ran Phaser in "automated search" mode and there's a solution with
>> RFZ
>> score 4.8, TFZ score 3.8. The electron density map was not bad with DNA
>> double helix clearly seen. However Refmac5 couldn't get Rfree lower
than
>> 50%.
>>
>> I then changed to MolRep, ran "self rotation function" first then used
>> the
>> first 10 peaks for translation search. Again there's a solution but it
is
>> different from that from Phaser. I attached a picture here. Checking in
>> coot, the packing is the same. But, the refinement couldn't get Rfree
>> lower
>> than 50%.
>>
>> I have tried to include NCS, TLS refinement in Refmac, both not
working.
>> Hope someone out there can help.
>> Thanks very much for your time.
>>
>> Hubing
>>
>>

Re: [ccp4bb] Concentrating a protein solution - subbu

[Stephen Weeks]

Subbu,
Don't forget you crystallization screen can also be used to find a
condition that your protein may be happier in ! As you already have setup a
 screen of your protein, have a look at  your drops again and see if there
are any conditions that are clear. If so, is there a consistent theme in the
conditions i.e pH or additive ? I've worked with one protein that behaved
similarly and we saw that in drops at pH 5 or below it remained clear. Upon
changing the purification procedure to this lower pH we could concentrate
the protein up to 40 mg/ml (and got nice crystals) instead of the borderline
2mg/ml we were getting at pH 7.4

Goodluck

Stephen


On 21 July 2011 17:53, Narayanan Ramasubbu  wrote:

> Dear All:
> We have been trying to crystallize a protein which is large - > 100 kDa.
> This is soluble but the best we can get is about 1 mg/mL.
> It did crystallize but did not diffract well. Efforts to increase the
> concentration has been unsuccessful. I am wondering whether there are
> methods that others use to increase the concentration other that using
> amicon columns.
> Any help will be appreciated.
> Thanks
> Subbu
>

[ccp4bb] Lion kills moleman (and all other USF programs on the Mac)

[Gerard DVD Kleywegt]

Hi all,

Just a heads-up - if you upgrade to OSX Lion, your trusty old USF executables 
will no longer work.


Help is on its way - in the near future the indefatigable and intrepid 
Lion-taming heroes of the USF will release a distribution kit that includes 
source code. However, for now you may want to postpone upgrading to Lion if 
your will to live would be compromised by not being able to run Moleman, 
Mapman, Lsqman, etc.


--Mr Anchovy (Proud owner of a lion taming hat. A hat with 'lion tamer' 
written on it. And it lights up saying 'lion tamer' in big red neon letters, 
so you can tame them after dark when they're less stroppy.)


PS: http://www.youtube.com/watch?v=XMOmB1q8W4Y

**
   Gerard J.  Kleywegt

http://xray.bmc.uu.se/gerard/  mailto:ger...@xray.bmc.uu.se
**
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
**

Re: [ccp4bb] Lion kills moleman (and all other USF programs on the Mac)

[Xiaoguang Xue]

Can I use the Linux version under Xterm (from Macports)? I noticed that the
Fink repository will not run on the Lion system, but it looks like Macports
is OK, because they have a new branch for Lion. And they also have a dmg
install file for Lion.

On Fri, Jul 22, 2011 at 12:59 PM, Gerard DVD Kleywegt  wrote:

> Hi all,
>
> Just a heads-up - if you upgrade to OSX Lion, your trusty old USF
> executables will no longer work.
>
> Help is on its way - in the near future the indefatigable and intrepid
> Lion-taming heroes of the USF will release a distribution kit that includes
> source code. However, for now you may want to postpone upgrading to Lion if
> your will to live would be compromised by not being able to run Moleman,
> Mapman, Lsqman, etc.
>
> --Mr Anchovy (Proud owner of a lion taming hat. A hat with 'lion tamer'
> written on it. And it lights up saying 'lion tamer' in big red neon letters,
> so you can tame them after dark when they're less stroppy.)
>
> PS: 
> http://www.youtube.com/watch?**v=XMOmB1q8W4Y
>
> **
>   Gerard J.  Kleywegt
>
>http://xray.bmc.uu.se/gerard/  mailto:ger...@xray.bmc.uu.se
> **
>   The opinions in this message are fictional.  Any similarity
>   to actual opinions, living or dead, is purely coincidental.
> **
>



-- 
Xiaoguang Xue, PhD student
Utrecht University
Crystal & Structural Chemistry
Padualaan 8. Room N807
3584 CH Utrecht
The Netherlands
Tel. +31-30-253-2383

Re: [ccp4bb] Lion kills moleman (and all other USF programs on the Mac)

[Andreas Förster]

As Lion seems to be, above all, an wonky iPad emulator, you're probably 
better off keeping the Snow Leopard alive until Apple obsolesces it in a 
few months.



Andreas



On 22/07/2011 11:59, Gerard DVD Kleywegt wrote:

Hi all,

Just a heads-up - if you upgrade to OSX Lion, your trusty old USF
executables will no longer work.

Help is on its way - in the near future the indefatigable and intrepid
Lion-taming heroes of the USF will release a distribution kit that
includes source code. However, for now you may want to postpone
upgrading to Lion if your will to live would be compromised by not being
able to run Moleman, Mapman, Lsqman, etc.

--Mr Anchovy (Proud owner of a lion taming hat. A hat with 'lion tamer'
written on it. And it lights up saying 'lion tamer' in big red neon
letters, so you can tame them after dark when they're less stroppy.)

PS: http://www.youtube.com/watch?v=XMOmB1q8W4Y

**
Gerard J. Kleywegt

http://xray.bmc.uu.se/gerard/ mailto:ger...@xray.bmc.uu.se
**
The opinions in this message are fictional. Any similarity
to actual opinions, living or dead, is purely coincidental.
**



--
Andreas Förster, Research Associate
Paul Freemont & Xiaodong Zhang Labs
Department of Biochemistry, Imperial College London
http://www.msf.bio.ic.ac.uk

Re: [ccp4bb] hello

[Ed Pozharski]

I recall using CME some time ago (within last couple of years) without
problems.  Also, an alternative approach is to incorporate BME as a
separate residue instead - the disulfide bond will be correctly
identified by refmac.  I would think ideologically this is more
appropriate - using a different residue type implies that this is the
way your protein is, as if somehow the modified residue was incorporated
during protein synthesis (as is the case with seleno-methionines).  With
post-translational modifications perhaps the better way is to represent
additional entities as separate monomers that are bound to the protein
via covalent bonds.

Cheers,

Ed.

On Fri, 2011-07-22 at 09:19 +0100, Garib N Murshudov wrote:
> Dear Afshan
> 
> 
> Make sure that group name for CME is peptide (or L-peptide).  In the
> new version CME is peptide. I am not sure it was the case in older
> versions. I attach CME as a peptide. You can add this into your
> dictionary.
> 
> 
> 
> 
> Then CME can become part of a peptde. It would also be good to remove
> all the link records in the beginning of refmac. 
> 
> 
> 
> 
> Regards
> Garib
> 
> 
> 
> 
> 
> 
> On 21 Jul 2011, at 16:13, Afshan Begum wrote:
> 
> > Dear all,
> > 
> > 
> > I have facing one problem during the refinement of my protein .
> > Actually in my protein  there are some modified amino acids are
> > present  like Cystein is modified into CME which i can get easily
> > from monomer libraray in coot . but after refinement in Pdb text
> > file  indicated some gaps while in the structures there are no gap
> > in between these amino acids so if any one suggest me what to do. I
> > would appreciate your kind suggestions.
> > 
> > 
> > 
> > LINKRGLU A 142 LEU A 144
> > gap 
> > LINKRSER A 328 GLY A 330
> > gap 
> > LINKRLEU A 138 GLU A 140
> > gap 
> > LINKRGLU A 126 ASP A 130
> > gap 
> > LINKRSER A 246 GLY A 248
> > gap 
> >  
> > 
> > Many thanks for your time
> > 
> > Best regards
> > 
> > Afshan
> > 
> > 
> > 
> > 
> 
> Garib N Murshudov 
> Structural Studies Division
> 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
> 
> 
> 
> 
> 
> 

-- 
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

[ccp4bb] CCP4 6.2.0 Released

[ronan . keegan]

Dear All,

We're very pleased to announce the release of the latest version of the CCP4 
software suite. Version 6.2.0 (Giggleswick) is now available from the CCP4 
download website:

http://www.ccp4.ac.uk/download.php

The release is available for Linux, Mac OSX and Windows platforms. Updates 
include the latest versions of Refmac (version 5.6.0117), Phaser (2.3.0), 
Molrep (11.0.0), Pointless (1.5.22), iMosflm (1.0.5)/Mosflm (7.0.7), Xia2 
(0.3.3.1) and Crank (1.4) along with many more. This version of the suite also 
includes several new programs including Cprodrg for generating ligand 
restraints, Jligand, a graphical interface for generating ligands and linking 
ligands, Csloop for loop building and "Multicomb" for multivariate phase 
combination. More information is available from:

http://www.ccp4.ac.uk/html/CHANGESinV6_2.html

We'd like to thank all of the developers who have contributed to CCP4 6.2.0 and 
all of those who have helped in testing it. See you in Madrid!

Kind regards,

Ronan



Ronan Keegan
CCP4 Group

[ccp4bb] VWR Symphony Incubator?

[Elena Menichelli]

Dear all,

we're in urgent need to replace an old Precision Scientific Low Temperature 815 
Incubator. We could quickly get a VWR Symphony Low-Temperature BOD Incubator 
but before purchasing I was wondering if anyone has any experience with this 
incubator for protein crystallization.
I'd appreciate any advice,

Thanks a lot in advance,
Elena

https://ca.vwr.com/store/catalog/product.jsp;jsessionid=2C90FEACA101B330B9392417C3203FFB?product_id=6789996

Re: [ccp4bb] Lion kills moleman (and all other USF programs on the Mac)

[Bosch, Juergen]

According to your link it's not worth it right ? I mean lion taming ?
Jürgen

On Jul 22, 2011, at 6:59 AM, Gerard DVD Kleywegt wrote:

Hi all,

Just a heads-up - if you upgrade to OSX Lion, your trusty old USF executables
will no longer work.

Help is on its way - in the near future the indefatigable and intrepid
Lion-taming heroes of the USF will release a distribution kit that includes
source code. However, for now you may want to postpone upgrading to Lion if
your will to live would be compromised by not being able to run Moleman,
Mapman, Lsqman, etc.

--Mr Anchovy (Proud owner of a lion taming hat. A hat with 'lion tamer'
written on it. And it lights up saying 'lion tamer' in big red neon letters,
so you can tame them after dark when they're less stroppy.)

PS: http://www.youtube.com/watch?v=XMOmB1q8W4Y

**
   Gerard J.  Kleywegt

http://xray.bmc.uu.se/gerard/  mailto:ger...@xray.bmc.uu.se
**
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
**

..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/