Re: [ccp4bb] Phaser and Molrep gave different solutions

2011-07-21 Thread Vellieux Frederic

Hi,

It's not a bad idea to read the Phaser manual for molecular replacement; 
see http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement


Soon after the start, in a table on the right hand side, there is: TFZ 
score  5, have I solved it ? No.


Hence with a TFZ score of 3.8 you do not have a solution using Phaser.

Fred.

Hubing Lou wrote:

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] Influence of symmetry related molecules on a protomer conformation in crystal structures

2011-07-21 Thread Thomas Holder

this is of course possible with PyMOL, try this:

pdbid, chain = '1a00', 'A'
cmd.fetch(pdbid, async=0)
cmd.symexp('__neighbors', pdbid, pdbid + ' and chain ' + chain, 5.0)
print 'Number:', len(cmd.get_object_list('(__neighbors*)'))
cmd.delete('__neighbors*')
cmd.delete(pdbid)

Cheers,
  Thomas

On 07/14/2011 01:48 PM, sukanta mondal wrote:

I know, in PyMOL using 'symexp' possible to generate symmetry related
molecules for a given crystal structure. But I'm looking for some
program/software (for batch) by which I can find out the number
of symmetry related molecules (distance cutoff= 5A) interacting with
a given chain in a crystal structure.

Thanking you,
suku
NIBIO, Osaka



--
Thomas Holder
MPI for Developmental Biology
Spemannstr. 35
D-72076 Tübingen


Re: [ccp4bb] Phaser and Molrep gave different solutions

2011-07-21 Thread Antony Oliver
Have you tried using the DNA as your search model? - I have had success this 
way round - certainly more phasing power than your protein model, I guess.  
Also, refine with your DNA in place, and your phases/ map should improve - 
hopefully allowing you to place your protein molecules with ease.

Tony.

Sent from my iPhone

On 21 Jul 2011, at 12:40, Hubing Lou 
louhub...@gmail.commailto:louhub...@gmail.com wrote:

I was worried as well with the low TFZ score. Usually successful cases with 
score 8. I am still puzzled why Phaser and Molrep gave different solutions. 
Does this mean molecular replacement do not work out in this case so more 
crystals have to be prepared?

A little more information might be helpful to dissolve the problem here. The 
model I used is a protein-DNA complex. The protein was Chainsaw editted but the 
DNA sequence was directly borrowed from the original model.

Best,
Hubing

On Thu, Jul 21, 2011 at 3:30 PM, Vellieux Frederic 
mailto:frederic.velli...@ibs.frfrederic.velli...@ibs.frmailto:frederic.velli...@ibs.fr
 wrote:
Hi,

It's not a bad idea to read the Phaser manual for molecular replacement; see 
http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement 
http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement

Soon after the start, in a table on the right hand side, there is: TFZ score  
5, have I solved it ? No.

Hence with a TFZ score of 3.8 you do not have a solution using Phaser.

Fred.

Hubing Lou wrote:
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









[ccp4bb] SLOW CCP4 Interface

2011-07-21 Thread Jan Dohnalek
Dear all,
my ccp4 interface 6.1.3 became REALLY slow in response on a new i7 PC with
Fedora core 15
2.6.38.8-35.fc15.x86_64 .

Any ideas where to go from here?


Jan Dohnalek


-- 
Jan Dohnalek, Ph.D
Institute of Macromolecular Chemistry
Academy of Sciences of the Czech Republic
Heyrovskeho nam. 2
16206 Praha 6
Czech Republic

Tel: +420 296 809 390
Fax: +420 296 809 410


[ccp4bb] Post-doc position available

2011-07-21 Thread Karen Fulan Discola
A 2-year postdoctoral position is available in the laboratory of Dr. A.
Dessen at the Institut de Biologie Structurale in Grenoble, France, to study
structure and function of new bacterial virulence factors.

The laboratory has state-of-the-art equipment for biochemistry and
crystallography studies, and the postdoctoral fellowship will involve
protein purification, crystallization, data collection at the synchrotron,
and structure solution and refinement.

The IBS is a member of the Partnership for Structural Biology and thus has
preferential access to equipment and techniques available on the PSB site,
which also includes the EMBL, the ILL, and the ESRF.

Any inquiries should be sent to andrea.des...@ibs.fr.


Re: [ccp4bb] Phaser and Molrep gave different solutions

2011-07-21 Thread Herman . Schreuder
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] Phaser and Molrep gave different solutions

2011-07-21 Thread Hubing Lou
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, herman.schreu...@sanofi-aventis.comwrote:

 **
  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] SLOW CCP4 Interface

2011-07-21 Thread Jan Dohnalek
Was a SELinux issue.

Jan Dohnalek


On Thu, Jul 21, 2011 at 1:44 PM, Jan Dohnalek dohnalek...@gmail.com wrote:

 Dear all,
 my ccp4 interface 6.1.3 became REALLY slow in response on a new i7 PC with
 Fedora core 15
 2.6.38.8-35.fc15.x86_64 .

 Any ideas where to go from here?


 Jan Dohnalek


 --
 Jan Dohnalek, Ph.D
 Institute of Macromolecular Chemistry
 Academy of Sciences of the Czech Republic
 Heyrovskeho nam. 2
 16206 Praha 6
 Czech Republic

 Tel: +420 296 809 390
 Fax: +420 296 809 410




-- 
Jan Dohnalek, Ph.D
Institute of Macromolecular Chemistry
Academy of Sciences of the Czech Republic
Heyrovskeho nam. 2
16206 Praha 6
Czech Republic

Tel: +420 296 809 390
Fax: +420 296 809 410


Re: [ccp4bb] Denzo/HKL2000 for Pilatus 6M detector

2011-07-21 Thread zhang yu
We could use HKL2000 for Pilatus 6M detector a few months ago in our lab. We
upgraded our HKL2000 to Version 0.98.701 and asked a new license file to
include Pilatus 6M detector.

Yu

2011/7/20 Petr Leiman petr.lei...@epfl.ch

 Dear all,

 What is the status of Denzo/HKL2000 availability/support for the Pilatus 6M
 detector?

 Thank you,

 Petr




-- 
Yu Zhang
HHMI associate
Waksman Institute, Rutgers University
190 Frelinghuysen Rd.
Piscataway, NJ, 08904


[ccp4bb] hello

2011-07-21 Thread Afshan Begum
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 time


Best regards

Afshan

Re: [ccp4bb] hello

2011-07-21 Thread Nicolas Foos

Hello Afshan,

Maybe the programm that you use for refinement need specific entry with 
restraint for your modified amino acids.

More precisely, i think about the *.cif file for exemple.

HTH.

Nicolas

Le 21/07/11 17:13, Afshan Begum a écrit :

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



Many thanks for your time

Best regards

Afshan


--
This message has been scanned for viruses and
dangerous content by *MailScanner* http://www.mailscanner.info/, and is
believed to be clean. 


--
This message has been scanned for viruses and
dangerous content by MailScanner, and is
believed to be clean.



[ccp4bb] Concentrating a protein solution - subbu

2011-07-21 Thread Narayanan Ramasubbu

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] Concentrating a protein solution - subbu

2011-07-21 Thread Antony Oliver
You could try loading a small 1ml HiTrap (or similar) Q or S column with your 
protein - and knocking it off it in one go with high salt, alternatively 
micro-dialysis against a solution containing PEGs can also work well. 

Tony

Sent from my iPhone

On 21 Jul 2011, at 17:54, Narayanan Ramasubbu ramas...@umdnj.edu 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


Re: [ccp4bb] Phaser and Molrep gave different solutions

2011-07-21 Thread Randy Read
Hi,

Although it's pretty much true that if TFZ8, the solution is correct, there 
are hard cases where a correct solution has a significantly lower TFZ score.  
Also, we've been finding that TFZ scores are generally lower for monoclinic 
space groups like P21, at least in the search for the first molecule.  (Among 
other things, the search for the first molecule in a polar space group only 
covers a plane, so there are fewer independent trials.)  Rob Oeffner has been 
doing some detailed statistics, which we're analysing so that we can give 
better rules of thumb.  However, a TFZ of 3.8 for the second molecule (where 
the search is 3-dimensional) is a bad sign.

One of the earlier posts suggested looking just for the DNA, which sounds like 
a good idea.  It's possible that the relative orientation of the protein and 
DNA is different enough that the complex model doesn't work, so it might well 
be worth trying the DNA and protein components separately.

Good luck!

-
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 21 Jul 2011, at 11:39, Hubing Lou wrote:

 I was worried as well with the low TFZ score. Usually successful cases with 
 score 8. I am still puzzled why Phaser and Molrep gave different solutions. 
 Does this mean molecular replacement do not work out in this case so more 
 crystals have to be prepared?
  
 A little more information might be helpful to dissolve the problem here. The 
 model I used is a protein-DNA complex. The protein was Chainsaw editted but 
 the DNA sequence was directly borrowed from the original model.
  
 Best,
 Hubing
 
 On Thu, Jul 21, 2011 at 3:30 PM, Vellieux Frederic frederic.velli...@ibs.fr 
 wrote:
 Hi,
 
 It's not a bad idea to read the Phaser manual for molecular replacement; see 
 http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement
 
 Soon after the start, in a table on the right hand side, there is: TFZ score 
  5, have I solved it ? No.
 
 Hence with a TFZ score of 3.8 you do not have a solution using Phaser.
 
 Fred.
 
 Hubing Lou wrote:
 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
 
 
 
 
 
 
 


[ccp4bb] Protein expression, purification and crystallization

2011-07-21 Thread Hena Dutta
Dear Members of CCP4BB and PHENIXBB,

Can you suggest a reliable website where I can get basic and advanced
information on protein expression, purification and crystallization. I like
to read on the monitor.
Thanking you in advance...
Hena


Re: [ccp4bb] Concentrating a protein solution - subbu

2011-07-21 Thread Eric Larson

Hi Subbu,

You got crystals at 1mg/ml so you probably don't need to concentrate your 
protein any higher, especially since you suggest that concentrating beyond that 
is problematic.  Instead, you may want to try to optimize the crystallization 
condition you have already identified.  Some possible things to try: additive 
screen, include specific ligands, different temperature, different ratios of 
protein solution to crystallization solution, seeding, different 
crystallization methods (hanging vs. sitting drop, batch, diffusion,...), ...

good luck,

Eric


Eric T. Larson, PhD
Biomolecular Structure Center
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195

email: larso...@u.washington.edu


On Thu, 21 Jul 2011, 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] PDBe introduces UniPDB - a UniProt-PDB sequence-coverage widget

2011-07-21 Thread Gerard DVD Kleywegt

Hi all,

As part of its recent summer update, the Protein Data Bank in Europe (PDBe; 
http://pdbe.org) introduced UniPDB (http://pdbe.org/unipdb), a widget for 
graphical display of the coverage in the PDB of any UniProt entry (e.g., 
http://pdbe.org/unipdb?uniprot=P19909). The widget can be used on the PDBe 
website or included in your own web pages.


For a quick overview of the functionality of the widget, see this 
illustration:


   http://www.ebi.ac.uk/pdbe-apps/widgets/html/unipdbsnap.png

Note how the PDBlogos instantly reveal which are X-ray or NMR entries, which 
entries contain DNA or ligands, etc. Pressing the button Related PDB 
sequences (to the left in the top bar of the widget) will launch a FASTA 
search of the PDB using the UniProt sequence. The results will be presented 
(and can be analysed) in the PDBeXplore browser (http://pdbe.org/fasta).


A simple way to make a link or bookmark to the UniPDB widget for your 
favourite protein is to use a URL in the following format: 
http://pdbe.org/unipdb?uniprot=P29373 (replacing P29373 by the UniProt code 
of your chosen protein, either its UniProt name, e.g. NGF_MOUSE, or its 
accession number, e.g. P01139). Incorporating the UniPDB widget in your own 
web pages is so easy even I can do it:


   http://xray.bmc.uu.se/gerard/unipdb_pdbportfolio.html

-

Why UniPDB?
---

UniProt (http://www.uniprot.org/) is the primary resource for information 
about protein sequence and function and the PDB is the single global archive 
of 3D structures of biomacromolecules and their complexes. Many PDB entries 
contain proteins that are also archived in UniProt. As a biologist interested 
in a particular protein, you may want to find out which entries in the PDB (if 
any) contain structures for (parts of) your favourite protein, and how these 
structures map to the UniProt sequence.


Structural biologists often work on partial sequences (e.g., stably folded 
domains) and sometimes have to modify the natural sequence to facilitate 
expression or crystallisation or to allow investigation of the effect of a 
mutation on the behaviour of the protein (such as catalytic activity or 
ligand-binding specificity). In addition, the same structure can be determined 
many times, e.g. in different laboratories, with different techniques, under 
different conditions, with different ligands, etc. For these reasons, it is 
not always easy to do sequence-based searches of the PDB and synthesise the 
results into an overview of what structural information is available for which 
parts of your favourite protein. This problem is addressed by the UniPDB 
widget. It uses the up-to-date mappings between UniProt sequences and PDB 
entries that are provided by the SIFTS resource (a collaboration between the 
UniProt and PDBe teams at the EBI; http://pdbe.org/sifts). SIFTS provides 
mappings from PDB entries to other bioinformatics resources as well, including 
Pfam (sequence-based protein domains), CATH and SCOP (both of these are 
structural fold classifications).


For more information about UniPDB (including instructions on how to include it 
in your own web pages), see: http://pdbe.org/unipdb


-

We hope that you will find the UniPDB tool useful. As always, we welcome 
constructive criticism, comments, suggestions, bug reports, etc. through the 
feedback button at the top of any PDBe web page.


--Gerard

---
Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK
ger...@ebi.ac.uk . pdbe.org
Secretary: Pauline Haslam  pdbe_ad...@ebi.ac.uk


Re: [ccp4bb] Protein expression, purification and crystallization

2011-07-21 Thread Bernhard Rupp (Hofkristallrat a.D.)
If you are eligible, I'd highly recommend the EMBO courses in Europe or the
CSHL courses in the US on those subjects. Reading is a good start but will
not be enough if you want to do real work.for strategy considerations, there
is always Ch 3 and 4 in le livre.  

 

Best, BR

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hena
Dutta
Sent: Thursday, July 21, 2011 10:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein expression, purification and crystallization

 

Dear Members of CCP4BB and PHENIXBB,

Can you suggest a reliable website where I can get basic and advanced
information on protein expression, purification and crystallization. I like
to read on the monitor.
Thanking you in advance...
Hena



Re: [ccp4bb] Concentrating a protein solution - subbu

2011-07-21 Thread Eric Larson
See below.  I believe Ray intended this to be sent to the entire bb and 
especially to Subbu!

And in reading Ray's message, I am reminded that I forgot to mention what he 
may be hinting at.  You should also try to optimize by creating a fine grid 
screen around your identified condition varying in particular pH and 
precipitant concentration.

best,
Eric 


On Thu, 21 Jul 2011, ray-br...@att.net wrote:

What is the pI? Perhaps the protein will be more soluble close to this pH?  
You could increase the salt and/or add some glycerol or a
cofactor?
 
Normally crystallization is most affected by the pH, 2 - 10% glycerol or 
temperature 4 - 25 degrees C.
Cheers
 
Ray Brown

_
From: Eric Larson larso...@u.washington.edu
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thu, July 21, 2011 1:40:51 PM
Subject: Re: [ccp4bb] Concentrating a protein solution - subbu

Hi Subbu,

You got crystals at 1mg/ml so you probably don't need to concentrate your 
protein any higher, especially since you suggest that
concentrating beyond that is problematic.  Instead, you may want to try to 
optimize the crystallization condition you have already
identified.  Some possible things to try: additive screen, include specific 
ligands, different temperature, different ratios of
protein solution to crystallization solution, seeding, different 
crystallization methods (hanging vs. sitting drop, batch,
diffusion,...), ...

good luck,

Eric


Eric T. Larson, PhD
Biomolecular Structure Center
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195

email: larso...@u.washington.edu


On Thu, 21 Jul 2011, 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] JLigand: Handedness related issue

2011-07-21 Thread Vineet Gaur
Dear all,
I am trying to generate a cif file for a new ligand (a sugar derivative)
using JLigand. The ligand needs to be in D- configuration. However, after
I input the coordinates for this ligand into J ligand and carry out geometry
regularisation, the program automatically converts D configuration to L. Had
anyone encountered similar kind of problem while working with JLigand and
how can I tackle this issue?

I am using JLigand since I have to define a covalent link between the ligand
and the mocromolecule later during the refinement. Is there any other
program other than Jligand (or Prodrg server) which I can use for generating
the cif file and defining the link?

Thanks,
Vineet Gaur


[ccp4bb] JLigand: Handedness related issue

2011-07-21 Thread Vineet Gaur
Dear all,

I am trying to generate a cif file for a new ligand (a sugar derivative)
using JLigand. The ligand needs to be in D- configuration. However, after
I input the coordinates for this ligand into J ligand and carry out geometry
regularisation, the program automatically converts D configuration to L. Had
anyone encountered similar kind of problem while working with JLigand and
how can I tackle this issue?

I am using JLigand since I have to define a covalent link between the ligand
and the mocromolecule later during the refinement. Is there any other
program other than Jligand (or Prodrg server) which I can use for generating
the cif file and defining the link?

Thanks,
Vineet Gaur


[ccp4bb] A new historical structural achievement

2011-07-21 Thread Justin Hall

Dear CCP4BB,

Forgive me for soap boxing, but yesterday the first structure of a  
GPCR/Gprotein complex was released (PDB:  
http://www.rcsb.org/pdb/explore/explore.do?structureId=3SN6, article:  
http://www.nature.com/nature/journal/vnfv/ncurrent/full/nature10361.html).


Recently a student preparing for their dissertation asked this board  
for its opinion about the most significant recent structural  
accomplishment, and among many things the progress on GPCRs was  
mentioned (albeit as being cute, I think).


Reading this work from Kobilka and Sunahara's two groups, I am floored  
by what it must have taken to achieve this - particularly if you know  
how hard and how long people have tried (both past and present) to get  
a GPCR/Gprotein complex structure. It is my opinion that this  
structure was something of a holy grail for the GPCR community.


So, even if you don't usually follow the developments of membrane  
crystallography, I wanted to invite your attention to this historical  
achievement in the GPCR field; and I hope you will join me in  
congratulating the scientist involved in this work.


Cheers~

~Justin


Re: [ccp4bb] JLigand: Handedness related issue

2011-07-21 Thread Christian Roth
Hi Vineet,

I did not used JLIGAND so far, so I cannot explain what happend, but maybe the 
quickest solution would be a manual intervention in the cif-files. Type at at 
the chirality remark positiv or negativ or whatever nomenclature is used and 
save it again. I do it sometimes for my ligands when I  genereated the wrong 
stereoisomer or the description for the racemate. 

Good Luck

Christian  

Am Donnerstag 21 Juli 2011 23:10:01 schrieb Vineet Gaur:
 Dear all,
 
 I am trying to generate a cif file for a new ligand (a sugar derivative)
 using JLigand. The ligand needs to be in D- configuration. However, after
 I input the coordinates for this ligand into J ligand and carry out
  geometry regularisation, the program automatically converts D
  configuration to L. Had anyone encountered similar kind of problem while
  working with JLigand and how can I tackle this issue?
 
 I am using JLigand since I have to define a covalent link between the
  ligand and the mocromolecule later during the refinement. Is there any
  other program other than Jligand (or Prodrg server) which I can use for
  generating the cif file and defining the link?
 
 Thanks,
 Vineet Gaur
 


[ccp4bb] Intensity-Weighted Reciprocal Lattice

2011-07-21 Thread Michael Thompson
Hello ccp4  phenix BB members,

I would like to view the intensity-weighted reciprocal lattice for several data 
sets that I have collected. (The data have been indexed, integrated and scaled 
with Denzo and Scalepack.) I was wondering if anyone could offer some advice on 
what might be the best and/or most practical way to do this?

I know that there are several programs out there that can generate sections 
(i.e. 0,k,l) of the reciprocal lattice, such as LABELIT and xrayplot. Are there 
any other options for doing this, perhaps within ccp4 and/or phenix? I once saw 
someone give a presentation and they had a little video that showed a three 
dimensional section of the reciprocal lattice rocking back and forth, which was 
really cool. I liked this because I felt like it gave a much more holistic 
representation as opposed to viewing a bunch of individual sections. I don't 
know if there is an easy way to do this, or if this person somehow managed to 
create this 3D depiction from a series of sections.

Any tips or recommendations would be appreciated.

Thanks,

Mike




-- 
Michael C. Thompson

Graduate Student

Biochemistry  Molecular Biology Division

Department of Chemistry  Biochemistry

University of California, Los Angeles

mi...@chem.ucla.edu