Re: [ccp4bb] Off topic: How to identify a unknown ligand
Hi Xiaopeng To add to Artem's comments: Does the presumed gsh make a mixed disulfide in the active site?i.e. is it covalently bonded to the active site via a s-s bond? If yes then MS on your purified sample should easily give you the answer. If a mixed s-s is indeed the scenario then purifying the enzyme in the presence of reducing agents and playing with the pH a bit should yield a gsh free preparation. Best regards Savvas On 16 Mar 2011, at 02:19, Artem Evdokimov artem.evdoki...@gmail.com wrote: Tightly bound ligands commonly survive purification :) Several unexpected discoveries have been made this way! If you think your stuff is GSH, soak some 'real' GSH or co-crystallize with it, and see if density shape changes from what you had before. This is not guaranteed to work because sometimes the ligand may be bound so tightly/exchange slowly that the original ligand just won't budge. This was a significant issue with some of the kinase inhibitors and nuclear hormone receptor antagonists that I've had a chance to work with - the binding was almost 'one way' in real-time situation, and (partial) denaturation was required to get the ligands out. If your ligand is indeed GSH, in equimolar amount with the protein, then you could also try MS as detection technique. The ligand should come off when protein is subjected to the normal MS environment (typically 0.1% TFA or formic acid, in a mixture of water and acetonitrile, etc.). To detect it, don't forget to expand the mass window to get the mass, and set the ionization mode to positive - you should see a clear 308.3 Da peak, with a lovely isotope splitting pattern (assuming you have access to MS). In negative mode the mass will be 1/2 of the already low m.w. of 307, since GSH has two negative charges. Notably, GSH should also accept e.g. an iodoacetamide group on the -SH, meaning that you should be able to treat crystals with iodoacetamide and observe the addition of -CH2CONH2 to the sulfur. Naturally, the S atom should be pretty prominent in the density anyway. Ditto mercurials, but they may wreck the crystal. Since your enzyme may be a GST (assumption on my part here) it also may present GSH to be reactive with whatever substrates that yor GST is targeting, so you may be able to identify conjugation products. Artem 2011/3/14 Xiaopeng Hu huxp...@mail.sysu.edu.cn Dear all, Sorry for this off topic question. We are working on protein/inhibitor complex structure although we can not get our inhibitors in. However, we did find a strange density at the active site, it looks really like GSH, the natural co-enzyme of thiis protein.We tried to use very simple solution to get crystal then exclude the possiblity of buffer moleculors,but that density is aways there. I am wondering how this ligand (if it is GSH) can survive all purification steps and want to indentify it. Are there any methodes to do this work? Let's say to pick up a crystal and do some analysis? Many many thanks!!! Xiaopeng
[ccp4bb] Limited proteolysis affected by metal binding?
Dear all, I was working with a protein which is known to bind zinc. I tried to make a limited proteolysis (with trypsin) after purification (metal affinity, ion exchange and gel filtration; last step uses EDTA to remove bound metal ions) in the presence and absence of zinc ions and I was quite surprised that the proteolysis pattern is completely different although all parameters were the same during the proteolysis (except for the presence of zinc ions). Since the protein I'm using is His-tagged (and I did not remove the His-tag), I was wondering whether anybody of you knows if zinc 1.) affects trypsin in it's activity? 2.) zinc can bind to the His-tag and affects the result of the limited proteolysis? 3.) zinc does not have any effect on His-tagged proteins? Just another comment: I also tried the same proteolysis in the presence of magnesium and manganese, but the proteolysis pattern looks the same as the one without metal ions. Any comments are welcome, Greg
Re: [ccp4bb] Limited proteolysis affected by metal binding?
To add more information: The proteolysis buffer was 50 mM Tris / HCl pH 8.0, 150 mM NaCl, 0.5 mM ZnCl and 0.1 mM TCEP; protein concentration was ~ 25 µM. Proteolysis was carried out at 4°C over 2 hours. Thank you very much for the literature, Mark - I'll look into it. Greg 2011/3/16 Matthias Zebisch matth...@strubi.ox.ac.uk how much zinc would be essential to know... Am 16/03/2011 09:18, schrieb Greg Carter: Dear all, I was working with a protein which is known to bind zinc. I tried to make a limited proteolysis (with trypsin) after purification (metal affinity, ion exchange and gel filtration; last step uses EDTA to remove bound metal ions) in the presence and absence of zinc ions and I was quite surprised that the proteolysis pattern is completely different although all parameters were the same during the proteolysis (except for the presence of zinc ions). Since the protein I'm using is His-tagged (and I did not remove the His-tag), I was wondering whether anybody of you knows if zinc 1.) affects trypsin in it's activity? 2.) zinc can bind to the His-tag and affects the result of the limited proteolysis? 3.) zinc does not have any effect on His-tagged proteins? Just another comment: I also tried the same proteolysis in the presence of magnesium and manganese, but the proteolysis pattern looks the same as the one without metal ions. Any comments are welcome, Greg -- Dr. Matthias Zebisch The Division of Structural Biology The Henry Wellcome Building for Genomic Medicine Roosevelt Drive Oxford, OX3 7BN United Kingdom Phone : +44-1865-278549 (office) Mobile: +44-786-6841877 Fax : +44-1865-2785 email: matth...@strubi.ox.ac.uk
Re: [ccp4bb] Limited proteolysis affected by metal binding?
Hi Greg, I am not sure why you are so surprised! If the zinc is altering the conformation and/or folding of your protein, this might change the accessibility of trypsin cleavage sites, thus changing your limited proteolysis pattern. Eg: Metal-ion induced conformational changes in alkaline phosphatase from E. coli assessed by limited proteolysis V. Bučević-Popovića, M. Pavela-Vrančiča, , and R. Dieckmannb Biochimie Volume 86, Issue 6, June 2004, Pages 403-409 http://bit.ly/h2EtHj HTH, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 16 March 2011 09:52, Greg Carter greg.carter...@gmail.com wrote: To add more information: The proteolysis buffer was 50 mM Tris / HCl pH 8.0, 150 mM NaCl, 0.5 mM ZnCl and 0.1 mM TCEP; protein concentration was ~ 25 µM. Proteolysis was carried out at 4°C over 2 hours. Thank you very much for the literature, Mark - I'll look into it. Greg 2011/3/16 Matthias Zebisch matth...@strubi.ox.ac.uk how much zinc would be essential to know... Am 16/03/2011 09:18, schrieb Greg Carter: Dear all, I was working with a protein which is known to bind zinc. I tried to make a limited proteolysis (with trypsin) after purification (metal affinity, ion exchange and gel filtration; last step uses EDTA to remove bound metal ions) in the presence and absence of zinc ions and I was quite surprised that the proteolysis pattern is completely different although all parameters were the same during the proteolysis (except for the presence of zinc ions). Since the protein I'm using is His-tagged (and I did not remove the His-tag), I was wondering whether anybody of you knows if zinc 1.) affects trypsin in it's activity? 2.) zinc can bind to the His-tag and affects the result of the limited proteolysis? 3.) zinc does not have any effect on His-tagged proteins? Just another comment: I also tried the same proteolysis in the presence of magnesium and manganese, but the proteolysis pattern looks the same as the one without metal ions. Any comments are welcome, Greg -- Dr. Matthias Zebisch The Division of Structural Biology The Henry Wellcome Building for Genomic Medicine Roosevelt Drive Oxford, OX3 7BN United Kingdom Phone : +44-1865-278549 (office) Mobile: +44-786-6841877 Fax : +44-1865-2785 email: matth...@strubi.ox.ac.uk
Re: [ccp4bb] molrep NOSG=-1 (space group checking)
I guess the only real choice is P2 21 21 or P21 21 21 - the absences alng h00 could be a result of the pseudo-translation. I cant explain the score - maybe there is something in the documentation? But I am afraid after refinement in P212121 the resultant model is sure to give the best score in that SG. Eleanor PS - I believe in belt-and-braces. What SG did PHASER suggest with the original model? When two programs agree one feels more confident On 03/15/2011 05:30 PM, Francis E Reyes wrote: Hi all I'm checking all space groups under P222 for data that contains a pseudotranslation. The data integrates in P222 but a 26% PST peak (0.500, 0.000, 0.23) makes this look like a C2221 cell (see previous CCP4bb post subject: Let's talk pseudotranslational symmetry (or maybe it's bad data). I was able to get a solution (2 molecules per ASU related by PST) in P 21 21 21, and able to build into additional density and refine to R/Rfree of 0.274/0.317 for 3.5A data. There still a few uninterpretable blobs (a linker region of about 6 residues) left. I'm now trying to do the MR of the refined model in all combinations of P222 via MOLREP (NOSG=-1). Now I'm trying to interpret the output of molrep. --- Space Group Checking. --- I,Nsg,Scor,Cntr: 1 16 P 2 2 2 0.375 5.521 I,Nsg,Scor,Cntr: 2 17 P 2 2 21 0.415 12.143 I,Nsg,Scor,Cntr: 3 1017 P 21 2 2 0.337 1.722 I,Nsg,Scor,Cntr: 4 2017 P 2 21 2 0.327 14.237 I,Nsg,Scor,Cntr: 5 18 P 21 21 2 0.419 5.169 I,Nsg,Scor,Cntr: 6 2018 P 21 2 21 0.414 10.808 I,Nsg,Scor,Cntr: 7 3018 P 2 21 21 0.463 12.357 I,Nsg,Scor,Cntr: 8 19 P 21 21 21 0.621 18.538 Time: 11h 22m 8s Elapsed: 0h 11m 30s MOLREP(ccp4): Failure [1] Is there somewhere in the man page or documentation that explains how 'Scor' is computed? [2] Based on the results above, it seems that P 21 21 21 is the correct s.g.? [3] Why the failure? Thanks! F - 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
[ccp4bb] MAR IMAGE PLATE DETECTOR
MAR describe their latest Image Plate as a CCD. Their early Image Plate designs were described as barium halide phosphor doped with Eu2+. Does anyone know why they have kept the name Image Plate when everyone else calls it a CCD? Rex Palmer Birkbeck College
Re: [ccp4bb] MAR IMAGE PLATE DETECTOR
Maybe because a CCD detector still requires a phosphor layer to detect X-rays? Tim On Wed, Mar 16, 2011 at 12:59:14PM +, REX PALMER wrote: MAR describe their latest Image Plate as a CCD. Their early Image Plate designs were described as barium halide phosphor doped with Eu2+. Does anyone know why they have kept the name Image Plate when everyone else calls it a CCD? Rex Palmer Birkbeck College -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
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[ccp4bb] protein lost activity after size exclusion chromatography
Dear all, Recently, I came across an obstacle on the purification and acitivty measurement of my protein. My protein was expressed with an C terminal His tag in the HEK 293T cells and purified by nickel affinity, anion exchange and size exclucion chromatography. For every purification step, I preserved some sample to test the activty. Strikingly, the protein retains activity after nickel affinity column even for three days but lost almost all the activty immediately after Mono Q and SEC. Therefore, I speculated that something (metal ion or co-factor) binding to the protein was striped by the Mono Q column. Then I skipped this step and only use the SEC for further purification. However, the protein is still not active no matter what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel column is also in the PBS buffer and no additive was added. Buffer exchange in the concentrator doesn't affect the activity of the protein. Can anyone explain why anion exchange or size exclucion chromatography destroy the activity of the protein? Any comment or proposal is appreciated! Harvey
[ccp4bb] 4L Vapor shipper for sale--NEVER USED
Hi everyone, I recently purchased a 4L Vapor Shipper (Taylor-Wharton) from VWR. Turns out it's designed for samples kept in canes, I wanted one that could hold pucks for APS data collection. VWR won't take it back because I got rid of the packaging. Please see description on the website for further info (cat# 55709-226) https://www.vwrcanlab.com/catalog/product/index.cgi?partnumber=V-48sim_code=1.0catalog_number=55709-226inE=1highlight=55709-226reference_type=1 List price $2107.44 Purchased price $1400 (after academic discount) Asking price $1200 (will include cost of shipping) If you are interested, please contact me at r...@queensu.ca or 613-533-6392. Thanks, Natalie Roy
[ccp4bb] Fw: Re: [ccp4bb] Solidarity with Japan
I was very relieved to learn that my friend and colleague Hideaki Niwa who took his MSc and PhD with me at Birkbeck is safe and well in Japan. I believe that International the Red Cross is doing great work out there and need all the help they can get. You can donate by going to the link below: http://clicks.aweber.com/y/ct/?l=7vf_Vm=1adA9w4Zgg1yh1b=_szL3OnaO1I3NyRX06YTVA Rex Palmer Birkbeck College
Re: [ccp4bb] protein lost activity after size exclusion chromatography
I guess it depends on what your activity is. Can you divulge that? Could it be that Ni is necessary? Jacob On Wed, Mar 16, 2011 at 11:28 AM, Harvey Rodriguez h.rodriguez.x...@gmail.com wrote: Dear all, Recently, I came across an obstacle on the purification and acitivty measurement of my protein. My protein was expressed with an C terminal His tag in the HEK 293T cells and purified by nickel affinity, anion exchange and size exclucion chromatography. For every purification step, I preserved some sample to test the activty. Strikingly, the protein retains activity after nickel affinity column even for three days but lost almost all the activty immediately after Mono Q and SEC. Therefore, I speculated that something (metal ion or co-factor) binding to the protein was striped by the Mono Q column. Then I skipped this step and only use the SEC for further purification. However, the protein is still not active no matter what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel column is also in the PBS buffer and no additive was added. Buffer exchange in the concentrator doesn't affect the activity of the protein. Can anyone explain why anion exchange or size exclucion chromatography destroy the activity of the protein? Any comment or proposal is appreciated! Harvey -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] protein lost activity after size exclusion chromatography
Depending on what the expected activity is, its worth considering the highly-depressing possibility that the activity seen in the impure sample was due to impurities: for example, barely-visible-on-a-gel chaperones can give a nice ATP hydrolysis signal, and DNA ligases float about with an AMP covalently attached, and thus can do one round of ligation with no ATP/NAD added to the soup. Phoebe = Phoebe A. Rice 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 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 16 Mar 2011 12:16:49 -0500 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Jacob Keller j-kell...@fsm.northwestern.edu) Subject: Re: [ccp4bb] protein lost activity after size exclusion chromatography To: CCP4BB@JISCMAIL.AC.UK I guess it depends on what your activity is. Can you divulge that? Could it be that Ni is necessary? Jacob On Wed, Mar 16, 2011 at 11:28 AM, Harvey Rodriguez h.rodriguez.x...@gmail.com wrote: Dear all, Recently, I came across an obstacle on the purification and acitivty measurement of my protein. My protein was expressed with an C terminal His tag in the HEK 293T cells and purified by nickel affinity, anion exchange and size exclucion chromatography. For every purification step, I preserved some sample to test the activty. Strikingly, the protein retains activity after nickel affinity column even for three days but lost almost all the activty immediately after Mono Q and SEC. Therefore, I speculated that something (metal ion or co-factor) binding to the protein was striped by the Mono Q column. Then I skipped this step and only use the SEC for further purification. However, the protein is still not active no matter what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel column is also in the PBS buffer and no additive was added. Buffer exchange in the concentrator doesn't affect the activity of the protein. Can anyone explain why anion exchange or size exclucion chromatography destroy the activity of the protein? Any comment or proposal is appreciated! Harvey -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] protein lost activity after size exclusion chromatography
Hi Harvey, Well, knowing nothing about your protein, allow me to ruminate anyway... It sounds like you are exploring the possibility of a metal ion or other cofactor being lost. This is a reasonable first thing to check, but your buffer exchange steps should allow small cofactors (smaller than most proteins that is) to pass through your membrane and away from your protein. This suggests that your loss of activity is due to the loss of something the size of your protein. Four things come to mind right away. 1) The least exotic possibility I can think of is maybe your protein is inactive all along according to your assay (your assay could have a problem in it, I would suggest trouble shooting your assay as a first step). This could result in your relatively dirtier prep falsely reporting activity because of another protein component (i.e. an impurity) that is active according to your assay, and then lost later during your purification. 2) This next idea seems unlikely, but you asked so... Could there be another protein component missing that is necessary for activity that you don't know about? This protein would be lost during purification resulting in an inactive form or your protein. 3) Probably another red herring here... Maybe your protein is not stable without lots of other proteins around. I have personally seen proteins that go to pot at low concentrations, but are very stable at high concentrations, for which this sort of reasoning is invoked. You could try adding Arg or other amino acids to keep it folded. 4) Is your protein active in a cleaved form? I have seen kinases with competent kinase domains in the absence of regulatory domains. If you run an activity assay that included the cleaved form of your protein, and then lose this cleaved form later after purifying away the cleaved protein, it would appear that you have lost activity. The most important advice I can give you is to pay attention to what your assays are really telling you, not what you think they are telling you because of useful assumptions we all make, but what the data really reports. For example, your activity assay shows no activity, the problem could be your protein, or a component of the assay, it is a bad idea to assume the protein is the only place something could be wrong. A factual analysis will hopefully allow you to trace back what you really know and where things could be going wrong. Hope this doesn't give you too many gooses to chase, hopefully somewhere in here is a spark to help you reason yourself out of your problem. Cheers~ ~Justin Quoting Harvey Rodriguez h.rodriguez.x...@gmail.com: Dear all, Recently, I came across an obstacle on the purification and acitivty measurement of my protein. My protein was expressed with an C terminal His tag in the HEK 293T cells and purified by nickel affinity, anion exchange and size exclucion chromatography. For every purification step, I preserved some sample to test the activty. Strikingly, the protein retains activity after nickel affinity column even for three days but lost almost all the activty immediately after Mono Q and SEC. Therefore, I speculated that something (metal ion or co-factor) binding to the protein was striped by the Mono Q column. Then I skipped this step and only use the SEC for further purification. However, the protein is still not active no matter what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel column is also in the PBS buffer and no additive was added. Buffer exchange in the concentrator doesn't affect the activity of the protein. Can anyone explain why anion exchange or size exclucion chromatography destroy the activity of the protein? Any comment or proposal is appreciated! Harvey
Re: [ccp4bb] protein lost activity after size exclusion chromatography
Hi Harvey, Could it be that the activity you're measuring comes from a contaminant? Did you test the other fractions from SEC or IEX? Cheers, Alex 2011/3/16 Harvey Rodriguez h.rodriguez.x...@gmail.com Dear all, Recently, I came across an obstacle on the purification and acitivty measurement of my protein. My protein was expressed with an C terminal His tag in the HEK 293T cells and purified by nickel affinity, anion exchange and size exclucion chromatography. For every purification step, I preserved some sample to test the activty. Strikingly, the protein retains activity after nickel affinity column even for three days but lost almost all the activty immediately after Mono Q and SEC. Therefore, I speculated that something (metal ion or co-factor) binding to the protein was striped by the Mono Q column. Then I skipped this step and only use the SEC for further purification. However, the protein is still not active no matter what buffer I use, eg. Tris,Hepes or PBS. The protein I purified by nickel column is also in the PBS buffer and no additive was added. Buffer exchange in the concentrator doesn't affect the activity of the protein. Can anyone explain why anion exchange or size exclucion chromatography destroy the activity of the protein? Any comment or proposal is appreciated! Harvey
Re: [ccp4bb] Fw: Re: [ccp4bb] Solidarity with Japan
Le 16/03/2011 17:59, REX PALMER a écrit : Would it be possible to get information through the CCP4BB about colleagues who do not answer mails ? I'd like to have news about TAKENAKA Akio, Faculty of Pharmacy, Iwaki Meisei University, Tokyo Institute of Technology. Thank you if somebody can transmit the information. Philippe Dumas I was very relieved to learn that my friend and colleague Hideaki Niwa who took his MSc and PhD with me at Birkbeck is safe and well in Japan. I believe that International the Red Cross is doing great work out there and need all the help they can get. You can donate by going to the link below: http://clicks.aweber.com/y/ct/?l=7vf_Vm=1adA9w4Zgg1yh1b=_szL3OnaO1I3NyRX06YTVA http://clicks.aweber.com/y/ct/?l=7vf_Vm=1adA9w4Zgg1yh1b=_szL3OnaO1I3NyRX06YTVA Rex Palmer Birkbeck College attachment: p_dumas.vcf
Re: [ccp4bb] Fw: Re: [ccp4bb] Solidarity with Japan
http://japan.person-finder.appspot.com/?lang=en The latest numbers I read is that they have 140,000 records, unfortunately there is no information about TAKENAKA Akio available yet. Best wishes, Thomas On Wed, Mar 16, 2011 at 21:17, Philippe DUMAS p.du...@ibmc-cnrs.unistra.frwrote: Le 16/03/2011 17:59, REX PALMER a écrit : Would it be possible to get information through the CCP4BB about colleagues who do not answer mails ? I'd like to have news about TAKENAKA Akio, Faculty of Pharmacy, Iwaki Meisei University, Tokyo Institute of Technology. Thank you if somebody can transmit the information. Philippe Dumas I was very relieved to learn that my friend and colleague Hideaki Niwa who took his MSc and PhD with me at Birkbeck is safe and well in Japan. I believe that International the Red Cross is doing great work out there and need all the help they can get. You can donate by going to the link below: http://clicks.aweber.com/y/ct/?l=7vf_Vm=1adA9w4Zgg1yh1b=_szL3OnaO1I3NyRX06YTVA Rex Palmer Birkbeck College
Re: [ccp4bb] Fw: Re: [ccp4bb] Solidarity with Japan
Thanks for the concern. I have replied to Prof. Dumas some minutes ago, but should have done to CCP4BB (Thanks a lot for letting us to use CCP4BB in this way. I think this to be an exceptional use of CCP4BB under the catastrophic circumstances in Japan.). I heard from one of his ex-students, Dr Midori Kamimura, who told me that Prof. Akio Takenaka came back home yesterday by combining many taxi trips from Iwaki to Yokohama, more than 200 km. Soichi Wakatsuki (2011/03/17 7:04), Thomas Juettemann wrote: http://japan.person-finder.appspot.com/?lang=en The latest numbers I read is that they have 140,000 records, unfortunately there is no information about TAKENAKA Akio available yet. Best wishes, Thomas On Wed, Mar 16, 2011 at 21:17, Philippe DUMAS p.du...@ibmc-cnrs.unistra.fr mailto:p.du...@ibmc-cnrs.unistra.fr wrote: Le 16/03/2011 17:59, REX PALMER a écrit : Would it be possible to get information through the CCP4BB about colleagues who do not answer mails ? I'd like to have news about TAKENAKA Akio, Faculty of Pharmacy, Iwaki Meisei University, Tokyo Institute of Technology. Thank you if somebody can transmit the information. Philippe Dumas I was very relieved to learn that my friend and colleague Hideaki Niwa who took his MSc and PhD with me at Birkbeck is safe and well in Japan. I believe that International the Red Cross is doing great work out there and need all the help they can get. You can donate by going to the link below: http://clicks.aweber.com/y/ct/?l=7vf_Vm=1adA9w4Zgg1yh1b=_szL3OnaO1I3NyRX06YTVA http://clicks.aweber.com/y/ct/?l=7vf_Vm=1adA9w4Zgg1yh1b=_szL3OnaO1I3NyRX06YTVA Rex Palmer Birkbeck College
Re: [ccp4bb] PDB data mining
Thank you to everyone who replied. I went through all the suggestions and in the end used Jason's PyMOL script, using Thomas' cpv.distance suggestion (which did make it much faster for me) and a few more modifications to eliminate redundant pairing listings. Bellow is the modified script, saved as dist_set1.py, run in terminal using /Applications/PyMOLX11Hybrid.app/Contents/MacOS/MacPyMOL -cq dist_set1.py It works on all pdb files in the directory /Users/cale/pdb/set1 It works on gziped pdb files so that the folder pdb can be compressed to be smaller All pdb files must be in the same folder (set1) as it does not traverse subdirectories It is limited to at most ~30,000 files in one folder so I had to split the ~72,000 files mirrored off the PDB into three folders (set1, set2 and set3) and generate an output file for each individually. the files can then be merged together into one using cat. # begin script # import glob, os, pymol, sys from pymol import cmd from chempy import cpv the_pdb=/Users/cale/pdb/set1 files = glob.glob(the_pdb+os.sep+*.ent.gz) if not len(files): print Please set 'the_pdb' variable to a valid path containing PDB files. sys.exit(1) else: print Processing %d files. % len(files) s, outFile = resn HIS and name ND1, dist_set1.csv f = open(outFile, 'wb') # write the header f.write(PDB\tCHAIN\tRESI\tATOM-A\tCHAIN\tRESI\tATOM-B\tDISTANCE\n) # for each file in the mirror for x in files: cmd.load(x,finish=1) n = cmd.get_names()[0] m = cmd.get_model(s).atom # pairwise for each atom for aa in m: for bb in m: # avoid distances to self if aa==bb: continue # avoid duplicates if aabb: continue distance = cpv.distance(aa.coord, bb.coord) # don't list if distance is above 10 angstroms # if distance 10 : continue f.write( %s\t%s\t%s\t%s\t%s\t%s\t%d\t%f\n % (n, aa.chain, aa.resi, aa.index, bb.chain, bb.resi, bb.index, distance)) cmd.delete(n) f.close() print Processed %d files. Please see %s for results. % (len(files), outFile) # end script # Cheers, Cale
