[ccp4bb] Industrial postdoc position at Biogen
Hi All, This is a job posting on behalf of a friend at Biogen, Cambridge, MA, US: We have a postdoctoral position open to study Wnt signaling and its role in neurodegeneration. A number of studies have shown the importance of the Wnt signaling pathway in the survival and maintenance of dopaminergic neurons. The candidate will conduct original and significant basic investigations in elucidating the mechanisms through which this pathway regulates these processes. The laboratory explores these pathways using a structural (x-ray crystallography), biophysical and biochemical approaches. The successful candidate will have opportunities to collaborate internally and externally to succeed in contributing towards research that will result in novel and important discoveries leading to understanding the pathway biology and also publications in top tier scientific journals. The requisition number for the job is: 30685BR. Please use the link below to apply for the position: https://jobs.biogen.com/job/Cambridge-Postdoctoral-Scientist%2C-Structural-Biology-MA-02138/403117100/ --- Matt Chu
[ccp4bb] scala - handling of anomalous data
Hi all, Can someone kindly explain what is that match pairs related by inversion of indices option doing for handling anomalous data? If I have collected the inverse beam data, does it mean I have to use this option to match the I+ and I- pairs in merging? But why does it lead to seriously incomplete data (compare to the merging data without the MATCH option ON)? Thanks, Matt -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Stanford School of Medicine
[ccp4bb] Fwd: 6th International Conference: Hybrid Methods (March 14-18)
This is a forwarded message. For inquires please contact Bill Weis ( bill.w...@stanford.edu). Dear Colleagues: We are delighted to announce that the* 6th International Conference on Structural Analysis of Supramolecular Assemblies by Hybrid Methods *will be held from *March 14-18, 2012 in Lake Tahoe, California at the Granlibakken Conference Center*. We invite you to visit our Symposium website at: http://www.hybridmethodsconference.com/ to register for the Symposium ($375 before January 31st), submit an abstract, view the current program, and to also publicize the attached meeting poster. Abstracts are due on February 28th and on March 5th, abstract-selected talks will be announced, though late abstracts will be accepted through March 11th. This Symposium builds on a series of very successful previous meetings on the same theme from 2004-2010. As in previous years, the overall *goal is to illustrate the power of combining state of the art methods to tackle important and challenging biological problems and to identify limitations and gaps in currently practiced hybrid methods*. The central premise is that gaining a comprehensive understanding of the highly sophisticated machines, complexes, and organelles of the cell requires the coordinated application of a number of complementary biophysical approaches (hybrid methods). New innovations at the 2012 meeting will include a special Vision talk on the future of Hybrid Structural Methods by *Michael Rossmann*, Purdue University, new characterization methods will be combined with advances within more traditional hybrid approaches such as the interfaces between electron microscopy, X-ray crystallography, and computational biology. Featured topics will also include other biophysical methods, single molecules in motion and cell biology. We are fortunate to have recruited two outstanding Keynote Speakers: *Wes Sundquist* (University of Utah), and *Susan S. Taylor* (UCSD), as well as a great program of platform speakers who utilize multiple approaches to investigate a variety of complex biological systems. Additional platform speakers will be selected from submitted abstracts. Sincerely yours, *The Organizing Committee:* Phoebe Stewart, Chair, Andrej Sali, Co-Chair, Taekjip Ha, Dorit Hanein, Ron Milligan, Felix Rey, Alasdair Steven, and Bill Weis. *Cami Thompson* Graduate Programs Department of Pharmacology Case Western Reserve University (216)368-4617 c...@case.edu -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Stanford School of Medicine
Re: [ccp4bb] Pymol questions
1. Yes, just Save Molecule (individually) after align 2. show stick, HETATM around 4 ( is your pdb name, 4 = 4 angstrom) On Wed, May 4, 2011 at 3:26 PM, jlliu liu jlliu20022...@gmail.com wrote: Hi All, I have two questions for Pymol. 1. Can you write out the PDB file after structural alignment? 2. How to only show structure that is several angstrom from the ligand? Thanks a lot in advance! Eric -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Stanford School of Medicine
Re: [ccp4bb] Question about movie making
Hey Mark, In Window PC, I used ImageReady, which comes with Photoshop. see this: http://sage.ucsc.edu/~wgscott/xtal/movie/making_movie.html Alternatively, using Mac version of Pymol, you can easily make a movie and save it in quick time format see this: www.chem.umass.edu/.../*BMS2006*Files/.../PymolMorphingMovie.pdf HTH, Matt On Mon, Mar 7, 2011 at 1:21 PM, mjvdwo...@netscape.net wrote: All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu
Re: [ccp4bb] Question about movie making
sorry, this is the right link for the mac-pymol movie: people.chem.umass.edu/jhardy/BMS2006Files/pymol%20morphing/PymolMorphingMovie.pdf On Mon, Mar 7, 2011 at 2:07 PM, Matthew Chu linghonmatt...@gmail.comwrote: Hey Mark, In Window PC, I used ImageReady, which comes with Photoshop. see this: http://sage.ucsc.edu/~wgscott/xtal/movie/making_movie.html Alternatively, using Mac version of Pymol, you can easily make a movie and save it in quick time format see this: www.chem.umass.edu/.../*BMS2006*Files/.../PymolMorphingMovie.pdf HTH, Matt On Mon, Mar 7, 2011 at 1:21 PM, mjvdwo...@netscape.net wrote: All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu
Re: [ccp4bb] AMP-PNP Hydrolysis
Is it possible that the phosphates are just disordered rather than being cleaved? It's always the case for inactive kinase-ATP or AMPPNP complexes that the phosphates are not stabilized by Mg2+ or the residues in the binding pocket and hence they become disordered and are not seen in the electron density. It may be worth to take a look at those phosphate-binding residues in your enzyme and see if they are positioned towards your AMPPNP's phosphates. HTH, Matt On Mon, Feb 14, 2011 at 5:30 AM, Soisson, Stephen M stephen_sois...@merck.com wrote: Hi there, Was recently looking at a structure of an enzyme with AMP-PNP added to the crystallization mix, and all I see is density for ADP. I was wondering if hydrolysis of AMP-PNP to ADP is relatively common - either as a result of extended time in crystallization or exposure of the resultant crystals to synchrotron radiation? I know that there can be up to 10% contamination of ADP in the purchased material, so it could just be that we have selected that form in the crystal, or that there was endogenous ADP bound that failed to substitute. Just curious if hydrolysis is a common observation. Thanks in advance- Steve Stephen M. Soisson, Ph.D. *Structural Chemistry Site Lead, WP* Merck Research Laboratories 770 Sumneytown Pike, WP14-1101 West Point, PA 19486 Phone: (215) 652-6185 Fax:(215) 652-9051 stephen_sois...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432
Re: [ccp4bb] List of commonly used cryoprotectants and buffer molecules
ISRDB Cryoprotectant database for protein crystals is a good resource, not only for the cryoprotection methods but the freezing methods used from literature. http://idb.exst.jaxa.jp/db_data/protein/200304E02478000.html It is under maintenance at the moment though... HTH, Matt On Thu, Sep 9, 2010 at 5:33 PM, Chris Weichenberger ch...@came.sbg.ac.atwrote: Dear All, I am trying to find out which molecules are frequently used by X-ray crystallographers serving as cryoprotectants or as buffer molecules. The idea behind this is to sort native ligands from molecules that appeared in the electron density just because they were used in the crystallization buffer or as a cryoprotectant. Can anybody point out literature, a web site, or simply provide a (subjective) list extending my collection of GOL, EDO, and different size PEGs? What about sugars? Thanks for your help, Chris -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu Facebook: http://www.facebook.com/MatthewLingHonChu Skype: matthew.lh.chu
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
Yea, Parveen has actually asked about this here a year ago and I found this discussion quite useful indeed: www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg11717.html and I think we all get tons of these crystals especially in conditions with PEGs. There are lots of things you can try and have been suggested, but I found this reference most helpful by far: A general protocol for the crystallization of membrane proteins for X-ray structural investigation. http://www.ncbi.nlm.nih.gov/pubmed/19360018 (this is actually the paper in the link that Eric posted: http://kb.psi-structuralgenomics.org/update/2009/06/full/th_psisgkb.2009.25.html) Best, Matt On Tue, Aug 31, 2010 at 12:53 PM, Parveen Goyal parveen...@gmail.comwrote: Hi, The picture looks like detergent crystals, specially DDM crystals. I have same crystals in many conditions. With regards, Parveen -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu
Re: [ccp4bb] ligand fitting, refinement?
Hi Rui, for some reasons, I also always encounter problems when building new ligand by CCP4 Sketcher. You can try PRODRUG ( http://davapc1.bioch.dundee.ac.uk/prodrg/index.html) to build your new ligand and get the cif from the server, it always works for me. HTH, Matt 2009/11/27 rui ruis...@gmail.com Hi, I found this old post in ccp4 and it's very useful. I used the same procedure Scott described to add a ligand into pdb file. What I did, is in coot, search for the ligand in the library and find the ligand and then merge. However, when I tried to refine in refmac, it has some problems, it complains about that New ligand has been encountered, stop now. I didn't create the cif file myself, it's been pulled from the library directory. Do anyone know what could be wrong? Thanks a lot for your help. Thanks. On Thu, Feb 5, 2009 at 10:55 AM, Scott Pegan pe...@uic.edu wrote: Andy, We do a lot of liganding fitting with CCP4. This is the general order of steps we take (post initial solution of the protein itself): 1) Build the potential ligand in CCP4 Sketcher a) Rename all the Hydrogens to H#, CCP4 Refmac has some issues with Hydrogens marked OH1, NH1, etc. To simplify things I normally just renumber all the Hydrogens starting from 1. Also makes for less hassle when using the definition file, as the labels in the definition file has to match the pdb of the ligand (this will be more important below). b) Use the regularize function with Refmac 2) Using Coot, load the protein and maps 3) Load the ligand and definition file (_mon_lib.cif) 4) Use the find ligand function in Coot (find it under other modeling tools) a) select the protein, map you want to search 5) If you find results you desire, merge those ligands with the main pdb 6) Run Refmac on the merged PDB with the library for the ligand in the library input space. Hope this helps, Scott On Thu, Feb 5, 2009 at 9:27 AM, ANDY DODDS andy.dod...@googlemail.comwrote: Hello, does anyone know of a tutorial which lays out some sort of pipeline, hopefully using CCP4 packages, to fit and refine a small molecule ligand please? cheers andy -- Scott D. Pegan, Ph.D. Senior Research Specialist Center for Pharmaceutical Biotechnology University of Illinois at Chicago -- Matthew L.H. Chu, PhD The University of Manchester, UK Hong Kong Tel: (852) 26729515 [home] / (852) 9169 2427 [mobile] Email: linghonmatt...@gmail.com Facebook: http://www.facebook.com/MatthewLingHonChu MSN: chuling_...@hotmail.com Skype: matthew.lh.chu
Re: [ccp4bb] MSD PISA
Hi Koustav, I had the same question before and here is the answer from Eugene Krissinel, the developer of PISA: Yes PISA has a database of compounds, which contains certain interaction parameters. If a compound is not found in the database, then PISA does not know how it interacts with other molecules and for this the compound is ignored. The only way to repair this is to process your compound, which cannot be done until it is deposited to our database. But even then, I am afraid, nothing can be done because I am no longer with the EBI and full knowledge of PISA was not digested by people left behind. Sorry for the crude truth. Good luck, Matt 2009/11/25 Koustav Maity mai...@gmail.com Hi All, I am using MSD PISA web server to analyze the protein ligand interface in my newly solved structure. The ligand in the structure is a new inhibitor (generated using PRODRG), so there is no entry for the inhibitor in the PDB. Therefore when I am uploading my pdb file to PISA server it is not recognizing the ligand molecule. Probably PISA reads the ligand identifier and matches with the database to process it as a ligand. Is there any way I can specify my inhibitor library file ? or is there some other way to resolve this issue? Koustav -- Matthew L.H. Chu, PhD The University of Manchester, UK
Re: [ccp4bb] generating a cif file using refmac
Hi Madhavi, try the prodrg server: http://davapc1.bioch.dundee.ac.uk/prodrg/index.html 2009/6/26 Nalam, Madhavi madhavi.na...@umassmed.edu Hello: I am using CCP4 version 6.1.1 now. I refine protein-small molecule complexes (the small molecules are new and hence I have to input the cif file). In the previous versions of CCP4, refmac used to fail and generate a cif file in the first run when I introduce the inhibitor into the pdb file. Now, in the new version, refmac doesn't run at all and asks me to input the cif file. I tried Sketcher but the problem is Sketcher is opening a proline molecule although I am trying to open my inhibitor molecule as a pdb file (again this problem is not there before) In my experience the cif files generated by refmac and sketcher are slightly different, at least for the molecules I am working with, and the ones generated by refmac are correct. Although there are other programs that generate cif files, I would like to generate them using refmac if possible. Any help would be greatly appreciated. Thanks, Madhavi -- Matthew LH Chu PhD School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] soaking problems
Hi Marek, I had a similar problem before, which the active site was partially occupied by a PEG molecule and my ligand couldn't get into the site. Sequential soaking-out can solve my problem; basically you need to slowly remove the PEGs from the crystal (in order to balance the osmotic shock): Stepwise transfer your crystal into three different drops of reservoir solution containing increasing concentrations of glycerol but decreasing PEG concentration in the presence of substrate for 1 min each, say your crystal grown in 20 % PEG: e.g. Solution (1) = 15 % PEG, 5 % glycerol + 0.5 mM substrate; (2) = 10 % PEG, 10 % glycerol + 0.5 mM substrate; (3) = 20 % glycerol only + 0.5 mM substrate. And of course you can try different combinations. It works for me nicely and is repeatable. HTH, Matt 2009/6/9 herman.schreu...@sanofi-aventis.com Dear Marek, A lot may happen: Your active site might be blocked by PEG or crystal contacts, or your sugar substrate gets converted into product and leaves the active site. In your case I would try the following: - soak or cocrystallize with a non-hydrolizable suger analog - soak with as high a substrate concentration as you can get (preferably saturated) - try crystallizing with larger pegs (20k) which may not enter the active site. Good luck! Herman -- *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Marek Frischerkase *Sent:* Tuesday, June 09, 2009 1:56 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] soaking problems Dear all, I tried to soak substrate (sugar) into my enzyme-crystals. After solving the structure I found PEG molecules instead of substrate in the active site. Does this mean that the active site is blocked and the substrate isn´t able to get into the enzyme-crsytals anymore? I used PEG 1000 in my crystallisation setups. Cocrystallisation showed no success so far. Can anyone give some suggestion what I should do? Thanks a lot, Marek -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] ARP/wARP Solvent
Thanks, Anastassis, Solvent is working now after installing the fixed file. Cheers, Matt 2009/1/8 Anastassis Perrakis a.perra...@nki.nl Hi - Its a bug in version 7.0.1 that went (almost) unnoticed ... there was one more complaint a year ago and I had fixed it but there was no release in between. Sorry. Apart from simply doing the solvent building from the REFMAC interface instead (either with arp_warp as since now or with Coot as since CCP4 6.1) I will mail you the fixed file (which might work). Anyone else interested ask me ; the fix will be in the imminent 7.1 release. A. On Jan 8, 2009, at 12:59, Matthew Chu wrote: Thanks Damian, but I have been using my library file for refmac refinement and it works fine. And I can't find the line Unrecognized atom type, but presumably, if it works in refmac refinement, why not in Arp/wArp? Yes Gerrit, the [ and ] should not be there.so auto_solvent.sh can recognize my mtz fp, etc. BUT I did check the box 'input a user-defined library file' in GUI and the command 'extralibrary' in the script, and again, it fails to read my library file or the one refmac just created, but obviously the extralibrary command works fine? Here is the log: Working directory/home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/ Job ID is set to 20090108_114504 X-ray data file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent//L1.mtz Protein file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent//L1.pdb Extralibrary file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent//refmac5_temp1.10491_lib.cif TLS input file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent//L1_TLS.tls Creating directory /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504 Output solvent file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504/L1_solvent.pdb mtz labels taken: F_New SIGF_New FreeR_flag Parameter file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504/arp_warp_solvent.par Job launched in /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504 The log file is /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504/L1_warp_solvent_details.log But ERROR: Important, Important, Important! Your coordinate file has a ligand which has either minimum or no description in the library A new ligand description has been added to /tmp/mchu/refmac5_temp1.11125_lib.cif Picture of the new ligand can be viewed using postscript file. See above Check description in this file and, if satisfied, use it as the input library Otherwise either edit bond orders manually or use CCP4i Sketcher to view and edit the ligand and create a library entry by running libcheck It is strongly recommended that dictionary entry should be checked carefully before using it If you are happy with the library description then use the keyword (MAKE CHECK NONE) I.e. do not check correctness of the coordinates === Error: New ligand has been encountered. Stopping now I really have no idea what is the problem? Any suggestion would be greatly appreciated! Kind regards, Matt 2009/1/8 Damian Ekiert dceki...@scripps.edu Matt, I had a similar sounding problem once, but it may or may the problem in your case. Do you have any ligands or glycans on your protein that you just added in? I was working on a protein with glycosylations and hadn't yet removed the oxygens from my sugars that would be lost after condensation (e.g., the oxygen that would be lost as water when the first residue was attached to Asn). My suggestion would be to look a little bit higher up in the log file and see if you see any lines saying something like Unrecognized atom type. I think when there is a discrepancy between the residue name and the names of the atoms in the residue, you can have this problem. Hope that helps. Best, Damian Ekiert 2009/1/8 Gerrit Langer ger...@embl-hamburg.de Dear Matt, have you tried the 'input a user-defined library file' check box under 'refmac parameters' in the gui? Else try the keyword 'extralibrary' when using the 'auto_solvent.sh' script from the command line. Both options define a string 'LIB_IN mylib.cif' that is passed on to refmac. When using the auto_solvent.sh script, please omit the '[' and ']' characters. Type e.g.: auto_solvent.sh datafile L1.mtz protein L1.pdb fp F_New sigfp SIGF_New extralibrary refmac5_templ.03957_lib.cif I hope this will help. Regards, Gerrit. Matthew Chu wrote: Dear all, I tried to use ARP/wARP 7.0.1 GUI for solvent building, however it couldn't recognize my ligand library file (.cif), which works fine in refmac refinement. Apparently, the error message is: === Error: New ligand has been encountered. Stopping now Refmac_5.2.0019: New ligand has been encountered. Stopping now Your coordinate file has a ligand which has either minimum or no description in the library A new ligand description has been added to /tmp/mchu
Re: [ccp4bb] ARP/wARP Solvent
Thanks Damian, but I have been using my library file for refmac refinement and it works fine. And I can't find the line Unrecognized atom type, but presumably, if it works in refmac refinement, why not in Arp/wArp? Yes Gerrit, the [ and ] should not be there.so auto_solvent.sh can recognize my mtz fp, etc. BUT I did check the box 'input a user-defined library file' in GUI and the command 'extralibrary' in the script, and again, it fails to read my library file or the one refmac just created, but obviously the extralibrary command works fine? Here is the log: Working directory/home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/ Job ID is set to 20090108_114504 X-ray data file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent//L1.mtz Protein file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent//L1.pdb Extralibrary file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent//refmac5_temp1.10491_lib.cif TLS input file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent//L1_TLS.tls Creating directory /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504 Output solvent file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504/L1_solvent.pdb mtz labels taken: F_New SIGF_New FreeR_flag Parameter file /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504/arp_warp_solvent.par Job launched in /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504 The log file is /home/mchu/ArpwARP/l1/ArpwARP_070109/solvent/20090108_114504/L1_warp_solvent_details.log But ERROR: Important, Important, Important! Your coordinate file has a ligand which has either minimum or no description in the library A new ligand description has been added to /tmp/mchu/refmac5_temp1.11125_lib.cif Picture of the new ligand can be viewed using postscript file. See above Check description in this file and, if satisfied, use it as the input library Otherwise either edit bond orders manually or use CCP4i Sketcher to view and edit the ligand and create a library entry by running libcheck It is strongly recommended that dictionary entry should be checked carefully before using it If you are happy with the library description then use the keyword (MAKE CHECK NONE) I.e. do not check correctness of the coordinates === Error: New ligand has been encountered. Stopping now I really have no idea what is the problem? Any suggestion would be greatly appreciated! Kind regards, Matt 2009/1/8 Damian Ekiert dceki...@scripps.edu Matt, I had a similar sounding problem once, but it may or may the problem in your case. Do you have any ligands or glycans on your protein that you just added in? I was working on a protein with glycosylations and hadn't yet removed the oxygens from my sugars that would be lost after condensation (e.g., the oxygen that would be lost as water when the first residue was attached to Asn). My suggestion would be to look a little bit higher up in the log file and see if you see any lines saying something like Unrecognized atom type. I think when there is a discrepancy between the residue name and the names of the atoms in the residue, you can have this problem. Hope that helps. Best, Damian Ekiert 2009/1/8 Gerrit Langer ger...@embl-hamburg.de Dear Matt, have you tried the 'input a user-defined library file' check box under 'refmac parameters' in the gui? Else try the keyword 'extralibrary' when using the 'auto_solvent.sh' script from the command line. Both options define a string 'LIB_IN mylib.cif' that is passed on to refmac. When using the auto_solvent.sh script, please omit the '[' and ']' characters. Type e.g.: auto_solvent.sh datafile L1.mtz protein L1.pdb fp F_New sigfp SIGF_New extralibrary refmac5_templ.03957_lib.cif I hope this will help. Regards, Gerrit. Matthew Chu wrote: Dear all, I tried to use ARP/wARP 7.0.1 GUI for solvent building, however it couldn't recognize my ligand library file (.cif), which works fine in refmac refinement. Apparently, the error message is: === Error: New ligand has been encountered. Stopping now Refmac_5.2.0019: New ligand has been encountered. Stopping now Your coordinate file has a ligand which has either minimum or no description in the library A new ligand description has been added to /tmp/mchu/refmac5_temp1.03957_lib.cif Even if I use the one refmac created after the error, it still can't recognize this new cif file... I also tried to run it from command line, another problem was raised. It couldn't recognize the FP label in my mtz when I used the keyword [fp F_New] [sigfp SIGF_New] [freer FreeR_flag] Error message: Label FP does not match the content of the datafile /home/mchu/ARP_wARP/solvent/L1.mtz Possible mtz labels are: F_New FC FWT DELFWT Does anyone have any idea why and how I can fix it? Thank you so much in advance! Kind regards, Matt -- Matthew LH Chu PhD Student School of Pharmacy
[ccp4bb] ARP/wARP Solvent
Dear all, I tried to use ARP/wARP 7.0.1 GUI for solvent building, however it couldn't recognize my ligand library file (.cif), which works fine in refmac refinement. Apparently, the error message is: === Error: New ligand has been encountered. Stopping now Refmac_5.2.0019: New ligand has been encountered. Stopping now Your coordinate file has a ligand which has either minimum or no description in the library A new ligand description has been added to /tmp/mchu/refmac5_temp1.03957_lib.cif Even if I use the one refmac created after the error, it still can't recognize this new cif file... I also tried to run it from command line, another problem was raised. It couldn't recognize the FP label in my mtz when I used the keyword [fp F_New] [sigfp SIGF_New] [freer FreeR_flag] Error message: Label FP does not match the content of the datafile /home/mchu/ARP_wARP/solvent/L1.mtz Possible mtz labels are: F_New FC FWT DELFWT Does anyone have any idea why and how I can fix it? Thank you so much in advance! Kind regards, Matt -- -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester --
Re: [ccp4bb] Sequence of crystallised protein fragment
N-terminal sequencing / MS for intact mass analysis are the only ways that I can think of. Matt 2008/7/1 Klaus Futterer [EMAIL PROTECTED]: We have a 150 kDa protein that reproducibly crystallises at one of the Hampton Screen conditions. However, we know from SDS gel analysis that the crystals contain only a 45 kDa fragment, that forms through proteolytic cleavage over time. We would like to determine the sequence boundaries of the fragment. We believe a combination of N-terminal sequencing plus MS analysis might give us the information we need, but I was wondering whether the ccp4bb community has other suggestions. Klaus - Klaus Fütterer, Ph.D. School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: [EMAIL PROTECTED] Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ - Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] pH gradient in Mono Q
Thank you for all your advices. I usually employ salt gradient in MonoQ for my proteins, but I just wonder if we can use pH gradient in the same column and see if it can improve the separation. I know the condition that my protein binds to Mono Q, so I will try to titrate toward its pI to reduce the affinity of the column. I am going to try Tris / phosphate buffer first, will let you guys know the results. Really appreciate for all your advices again. Matt 2008/6/25 John A. Newitt [EMAIL PROTECTED]: At 1:50 PM -0400 6/24/08, R.M. Garavito wrote: Matthew, You're not going to ruin your column, but you won't get great performance either. Elution by pH change is a very common method, but getting a really linear pH gradient is very hard. The Mono Q matrix is a strong anion exchanger, meaning that it is insensitive to pH changes, i.e., you can't titrate it smoothly with acid or base. DEAE resins, which are weak anion exchangers, have a nice pH titration curve and lend themselves better to elution by pH change. This is the reason chromatofocusing is not a commonly used method, and its expensive. There is a company the sells a proprietary buffer system and gradient programming calculator to create a stable pH gradient for separation on a MonoQ column or other strong ion exchanger. http://www.cryobiophysica.com/ My problem with pH gradient techniques is that they don't work very well unless your protein is happy in low ionic strength buffers, which is almost never the case with my projects. This company now claims that it can create the pH gradient with NaCl present, but I haven't tried this yet. - John -- http://xri.net/=john.newitt -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] pH gradient in Mono Q
Hi Nadir, No I didn't and I am not concerning CHROMATOFOCUSING, and you are right, I have been thinking of using both pH and salt gradients to refine the separation. Thanks! Matt 2008/6/25 Nadir T. Mrabet [EMAIL PROTECTED]: John, You can adjust your ionic strength not only with NaCl/KCl/etc but also by increasing the concentration of the components in your buffer mixture. If you do it right, I can assure you can get a linear pH gradient. And, as mentioned earlier by Tom, you can use both pH and salt gradients to refine your separation, even batchwise (no linear gradient required... if it works). The problem working with pH gradients is re-equilibrating the resin, since what you are actually doing is titrate a supposedly high-capacity buffer with another high-capacity buffer. Therefore, patience is required and sufficient volumes of buffer A. Again, I ask the question: Did Matthew ever mentioned before he intended to perform a CHROMATOFOCUSING experiment? Nadir -- Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] John A. Newitt wrote: At 1:50 PM -0400 6/24/08, R.M. Garavito wrote: Matthew, You're not going to ruin your column, but you won't get great performance either. Elution by pH change is a very common method, but getting a really linear pH gradient is very hard. The Mono Q matrix is a strong anion exchanger, meaning that it is insensitive to pH changes, i.e., you can't titrate it smoothly with acid or base. DEAE resins, which are weak anion exchangers, have a nice pH titration curve and lend themselves better to elution by pH change. This is the reason chromatofocusing is not a commonly used method, and its expensive. There is a company the sells a proprietary buffer system and gradient programming calculator to create a stable pH gradient for separation on a MonoQ column or other strong ion exchanger. http://www.cryobiophysica.com/ My problem with pH gradient techniques is that they don't work very well unless your protein is happy in low ionic strength buffers, which is almost never the case with my projects. This company now claims that it can create the pH gradient with NaCl present, but I haven't tried this yet. - John -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
[ccp4bb] pH gradient in Mono Q
Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] pH gradient in Mono Q
Thanks Guenter and Andreas, Yea, I have taken a look for the Mono P before, I thought the material they used in Mono P is basically the same as in Mono Q and I found the bookProtein Purification Protocols: Second Edition by Paul Cutler mentioned that phosphate buffer can also generate a continuous gradientthat's why I reckon we can also perform pH gradient in Mono Q. But I am worrying if it is a good idea to go for it, as both mono Q or mono P are quite expensive. Matt 2008/6/24 Andreas Förster [EMAIL PROTECTED]: Hey Matt, it seems to me that what you're asking for is chromatofocusing. See the official GE documentation: http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin(260949098-R350)http://www6.gelifesciences.com/aptrix/upp00919.nsf/Content/WD:Chromatofocusin%28260949098-R350%29 The proprietary buffers are a bit expensive, but as you found out, they're a bit complicated to make. I don't know if you'd ruin your Mono Q with a pH gradient. If in doubt, buy one of the dedicated Mono P column. Hope that helps. Andreas Matthew Chu wrote: Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] Refinement in half occupancy
Hi Bithika, Basically, I combined the coordinates of the two different conformations of the ligand (e.g. *ZZZ*), which modeled in Coot individually, in one pdb, and then put A/B label in front of the residue code and set the occupancy to 0.5, e.g. ATOM 4052 O2A*AZZZ* X1234 -42.055 -10.565 -13.483 *0.50*50.93 O ATOM 4053 O2A*BZZZ* X1234 -40.667 -10.029 -15.836 *0.50*59.20 O Kind regards, Matt 2008/6/10 Bithika Bera [EMAIL PROTECTED]: Hi Matt, Could you please pass me all advices regarding 'refine the ligand in half occupancy'. And which one is working for you. Thanks, bb *Matthew Chu [EMAIL PROTECTED]* wrote: Thank you for all the advices, I am now able to refine the ligand in half occupancy! Matt 2008/6/10 Guillaume Marassio [EMAIL PROTECTED]: I'am not sure it is your problem but you can try with A and B before the residue number. Hope it will be helpfull. Guillaume Marassio Matthew Chu a écrit : Dear All, Can anyone teach me how to refine a ligand in a protein structure with half occupancy in refmac? I have tried to combine the coordinates of the two different conformations of that particular ligand in one pdb, after modeling in Coot individually, and then changed the occupancy to 0.5 for each of the conformation. However, I couldn't manage to refine this pdb in Refmac. Thanks in advance! Best regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester -- Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
[ccp4bb] Refinement in half occupancy
Dear All, Can anyone teach me how to refine a ligand in a protein structure with half occupancy in refmac? I have tried to combine the coordinates of the two different conformations of that particular ligand in one pdb, after modeling in Coot individually, and then changed the occupancy to 0.5 for each of the conformation. However, I couldn't manage to refine this pdb in Refmac. Thanks in advance! Best regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
