Re: [ccp4bb] New Version of the Protein Geometry Database Now Available
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 The Protein Geometry Database looks at proteins as collections of bond lengths, angles, and torsion angles. It is not the place to go when you want to know how a protein part is related in space to some other (covalently) distant part. Andy tells me that Jacque Fetrow, who was at Wake Forest University, has a database that might answer your query. There is a paper at J Mol Biol. 2003 Nov 28;334(3):387-401. Structure-based active site profiles for genome analysis and functional family subclassification. Neither one of us has used it. Hope that helps, Dale Tronrud On 6/27/2014 1:49 PM, Keller, Jacob wrote: I have wanted for some time to search for catalytic-triad-like configurations by defining three Ca-Cb bonds from known catalytic triads, then searching the pdb for matches, but have not thought of a quick and/or easy way to do this--can your software do this sort of thing, or is there some other software which could be used for this? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale Tronrud Sent: Friday, June 27, 2014 4:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] New Version of the Protein Geometry Database Now Available Protein Geometry Database Server V 1.0 http://pgd.science.oregonstate.edu/ Developed by Andy Karplus' laboratory at Oregon State University We are pleased to announce the availability of an enhanced version of the Protein Geometry Database (PGD) web service, originally announced in Berkholz et al (2010) Nucleic Acids Research 38, D320-5. This server allows you to explore the many backbone and side chain conformations that exist in the PDB as well as the protein geometry (lengths and angles) that occur in those conformations. This service is ideal for finding instances of particular conformations or peculiar bond lengths or angles. It is also quite adept at identifying sets of fragments that can then be examined for systematic variation in ideal geometry. The expanded PGD now includes all conformational and covalent geometry information not just for the backbone but also for the sidechains. There are three basic operations available: selecting a set of fragments via a delimited search, analyzing the geometry of those fragments, and dumping the results to your computer for more specialized analysis. To control bias in statistical analyses due to the variable number of entries with the same or similar sequence, the database contains only the highest quality model in each sequence cluster as identified by the Pisces server from Roland Dunbrack's lab. Two settings, 90% and 25% sequence identity, are available. Currently, at the 90% sequence identity level there are 16,000 chains and at the 25% level this drops to about 11,000 chains. You can filter a search based on the quality of the model as indicated by resolution and R values. A search can also be filtered based on DSSP secondary structure, amino acid type, the phi/psi/omega angles and bond lengths, angles, and chi angles. For example, you can find all cysteine residues in the center of three-residue peptide fragments (i.e. not at a peptide terminus), in beta sheet, with both peptide bonds trans, and CB-SG length greater than 1.85 A from models with resolution better than 1.5 A. By the way, in the no more than 25% sequence identity category there are 25 of them. Once you have a set of results, you can create 2D plots showing the relationships of up to three features (i.e. bond lengths, bond angles, or conformational angles). For instance, you can look at how a given feature varies with phi and psi using a phi(i)/psi(i) plot. Or, you can just as easily look at the variation with psi(i)/phi(i+1), or even the relationships between any selected bond angles. As one example, it is instructive to perform a default search and plot NCaCb vs NCaC colored based on CbCaC. As this search is restricted to just the highest resolution models, you can see the justification for chiral volume restraints. Finally, all of your results can be downloaded for your own analysis. Development of the PGD continues. If you have worked with the site and have any ideas and suggestions for how to improvement it, please drop us a line. The publication describing the PGD is: Berkholz, D.S., Krenesky, P.B., Davidson, J.R., Karplus, P.A. (2010) Protein Geometry Database: A flexible engine to explore backbone conformations and their relationships with covalent geometry. Nucleic Acids Res. 38, D320-5. Also, some examples of published analyses enabled by earlier versions of the PGD are listed here:. Berkholz, D.S., Shapovalov, M.V., Dunbrack, R.L.J. Karplus, P.A. (2009). Conformation dependence of backbone geometry in proteins. Structure 17, 1316-1325. Hollingsworth, S.A., Berkholz, D.S. Karplus, P.A. (2009). On the occurrence of
[ccp4bb] AW: [ccp4bb] Solvent channels
Dear Bernard, we once worked with a series of protease inhibitors which turned out to be slow substrates, e.g. an acyl intermediate was formed that was subsequently hydrolyzed. Here we had to reduce the soaking times to below 30 minutes, otherwise we would see nothing, e.g. the large excess of added inhibitor was completely turned over. The precipitant was 25% PEG4000, which I consider a typical PEG condition. The inhibitors were the usual bunch of a few (aromatic) rings linked (unfortunately) by an amide linker. I agree with the others who reacted to your post that soaking times of 10 hrs are atypical and more likely caused by a slow Kon than by slow diffusion. Cheers, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Bernhard Rupp Gesendet: Samstag, 28. Juni 2014 10:46 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Solvent channels Here you are starting to mix equilibrium arguments with the previous kinetic arguments. I don't think I am mixing them; both are relevant. If it cannot diffuse there, forget the kinetics - necessary but not sufficient requirement. Nonetheless, the fact that in high concentrations you can force even weak non-native binders into binding sites (but I reiterate, never in 100% occupancy, at best asymptotically approaching it) is the reason for the many buffer 'ligands' observed in structures (also basis for fragment screening.) Your movie doesn't include any details of concentration of your dye, nor what its binding constant is to any sites in a protein nor any mention of kon or koff. The movie does not claim to be a study of any specific ligand binding, it simply illustrates soaking. Graphs of concentration vs achievable equilibrium occupancy at different Kds are in separate figures eg 3-40. Cheers, BR Dale Tronrud -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Friday, June 27, 2014 3:07 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels And yet halides--even iodide--permeate those same lysozyme crystals and others entirely in 30--60 sec. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp Sent: Friday, June 27, 2014 9:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels Just a remark: diffusion is a slow and random-walk process. Particularly large molecules in viscous media (PEG anybody?) move (diffuse) slowly in solution. To simply extrapolate from the fact that the ligand is smaller than the solvent channels to the odds of the presence of a ligand is a risky proposition. Positive omit difference density after 'shoot first' as Boaz indicated is a much better indication. And shoot you probably will a lot. The little movie below shows how slowly even a small aromatic dye molecule soaks into a crystal. Total time 10 hrs. http://www.ruppweb.org/cryscam/lysozyme_dye_small.wmv The literally hundreds of empty ligand structures collected in Twilight attest to that fact. http://journals.iucr.org/d/issues/2013/02/00/issconts.html Best, BR Science is a way of trying not to fool yourself: The first principle is that you must not fool yourself - and you are the easiest person to fool. R. Feynman, 1974 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Boaz Shaanan Sent: Friday, June 27, 2014 2:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels Hi, I'm not aware of a program with an option to display channels in crystals but if you use any of the currently available molecular display program and ask to display symmetry-related molecules + adjacent unit cells, it should give you a good enough idea of the spaces between molecules. Using programs for calculation of intermolecular distances would also be helpful here. Independently of the calculation, I would try soaking first and consult the calculations later (in the spirit of Rossmann's American method: shoot first ask later). Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat [rkha...@ccny.cuny.edu] Sent: Friday, June 27, 2014 2:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Solvent channels Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the
[ccp4bb] a warning message from XDS (IDXREF)
Hi all, I use XDS to process a data set. When I checked the log files I found a warning message at the end of IDXREF (as below). What should I do next? How to judge which solution is correct? !!! WARNING !!! SOLUTION MAY NOT BE UNIQUE. OTHER POSSIBLE SOLUTIONS CAN BE TRIED IN THE CORRECT STEP BY SPECIFYING ALTERNATIVE SETS OF UNIT CELL BASIS VECTORS: UNIT_CELL_A-AXIS= -25.024 -66.429 -28.634 UNIT_CELL_B-AXIS= 20.919 22.359 -70.153 UNIT_CELL_C-AXIS= 157.798 -70.095 24.714 Thanks so much! Yamei Yu
[ccp4bb] AW: [ccp4bb] a warning message from XDS (IDXREF)
Nothing to get worried about. It means that other, equally valid choices of axes may be possible (e.g. with polar space groups). Only if you want to merge data from different crystals or if you want to scale native and derivative data, you have to make sure that the same choice of axes is being made for all crystals. If you provide XDS with a reference data set, XDS will figure this out for you. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Yamei Yu Gesendet: Montag, 30. Juni 2014 09:33 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] a warning message from XDS (IDXREF) Hi all, I use XDS to process a data set. When I checked the log files I found a warning message at the end of IDXREF (as below). What should I do next? How to judge which solution is correct? !!! WARNING !!! SOLUTION MAY NOT BE UNIQUE. OTHER POSSIBLE SOLUTIONS CAN BE TRIED IN THE CORRECT STEP BY SPECIFYING ALTERNATIVE SETS OF UNIT CELL BASIS VECTORS: UNIT_CELL_A-AXIS= -25.024 -66.429 -28.634 UNIT_CELL_B-AXIS= 20.919 22.359 -70.153 UNIT_CELL_C-AXIS= 157.798 -70.095 24.714 Thanks so much! Yamei Yu
[ccp4bb] FR591 and Osmic VariMax HF
For free: After 16 years of service our home source is retiring. It is an FR591 Rotating anode generator. The anode motor needs replacement (estimated cost 4-5000 €) but otherwise the generator is functional. The generator can be picked up for free. Not for free: In 2005 the generator was fitted with a set of Osmic VariMax HF Confocal Multilayer Mirrors, which still may have some residual value. The mirrors have been kept in a He-atmosphere during operation. If interested, please get in touch to discuss the price. Best wishes Søren Thirup Søren Skou Thirup Asc. Professor, Ph.D Centre for Structural Biology Dept. of Molecular Biology and Genetics, Aarhus University Gustav Wieds Vej 10C, DK8000 Aarhus C, Denmark s...@mb.au.dkmailto:s...@mb.au.dk Tel: +45 8715 5464 Mob: +45 2058 5981
[ccp4bb] BM14 Postdoctoral Fellowship
Dear all Please note that a Postdoctoral Fellowship is immediately available to join the BM14 MAD MX beamline team at the ESRF, Grenoble, France (www.bm14.eu). Context: EMBL Grenoble operates the ESRF BM14 beamline under a Memorandum of Understanding with the Department of Biotechnology (DBT) of India and thus provides access to both European and Indian MX communities. We are seeking to recruit a motivated postdoctoral fellow to support BM14 users. The candidate should have proven experience in structure determination using synchrotron crystallography especially data collection, processing and de novo phasing (MAD/SAD methods). This is a unique opportunity to develop your crystallography knowledge and expertise! On the science side, the candidate will be offered to join the structural studies of Toxoplasma gondii epigenetic machinery that we conduct in close collaboration with Dr Hakimi, (Grenoble Medical University). We are in particular interested in in silico methods to screen small compound libraries and perform docking studies. In this context, knowledge of the Schrödinger program suit would be an advantage. For additional information please contact me (belrhali at embl.fr) Application Instructions: Please apply online through www.embl.org/jobs Regards Hassan
[ccp4bb] WAXSiS: New web server for SAXS/WAXS calculations based on explicit-solvent molecular dynamics
Dear SAXS and crystallography communities, it is our pleasure to announce a new web server for the calculation and fitting of SAXS/WAXS curves of biomolecules in solution. WAXSiS (WAXS in Solvent) computes SAXS/WAXS curves based on explicit-solvent all-atom molecular dynamics simulations. The key differences to established web services are: * No fitting parameters associated with the solvation shell or excluded solvent are required. * Solvation shell and excluded solvent are described at atomic detail, allowing accurate scattering predictions also at wide angles. * Thermal fluctuations of solvent and biomolecule are naturally included. Please find WAXSiS at: http://waxsis.uni-goettingen.de/ WAXSiS is up and running, and it has passed a few hundred test calculations. We would be pleased to receive feed-back, comments, and functionality requests from the SAXS community. With best wishes, The WAXSiS team at the University of Göttingen
Re: [ccp4bb] New Version of the Protein Geometry Database Now Available
Jacob Keller There is a service that will hopefully do the calculation you want, search for a template of residues (eg. catalytic-triads). This is just a through space superposition of atoms. It is part of a tool set of fragments found by knowledge based analysis (pdbetemplate) http://www.ebi.ac.uk/pdbe-as/pdbetemplate/ and there is a tool called DB search accessible from this page , or directly from here... http://www.ebi.ac.uk/pdbe-as/pdbesearch/PDBeSearch.jsp You can upload a small template of residues (up to 6) in PDB format using the [upload and analyse] button. The search is weighted based on occupancy - where weights for each atom a defined by the occupancy value. You can specify to just the CA atoms, the side chain (based on occupancy), and use fuzzy matching (slow) where ASP looks like GLU etc. Here is an example PDB file fragment you can use to try out the search - it is a catalytic quartet of residues - a superset of the catalytic triad. Regards Tom Oldfield, PDBe. HEADERHYDROLASE (SERINE PROTEINASE) 22-JAN-85 5CHA 5CHA 3 COMPNDALPHA CHYMOTRYPSIN A (E.C.3.4.21.1) 5CHA 4 SOURCECOW (BOS $TAURUS) 5CHA 5 AUTHOR R.A.BLEVINS,A.TULINSKY 5CHA 6 SITE 1 CTA 3 HIS A 57 ASP A 102 SER A 195 5CHA 160 ATOM403 N HIS A 57 28.981 28.453 20.681 1.00 10.49 5CHA 550 ATOM404 CA HIS A 57 28.691 28.510 22.151 1.00 10.51 5CHA 551 ATOM405 C HIS A 57 29.897 28.096 22.990 1.00 11.71 5CHA 552 ATOM406 O HIS A 57 29.926 28.441 24.252 1.00 11.27 5CHA 553 ATOM407 CB HIS A 57 27.441 27.724 22.518 1.00 10.23 5CHA 554 ATOM408 CG HIS A 57 27.711 26.268 22.692 1.00 11.48 5CHA 555 ATOM409 ND1 HIS A 57 27.449 25.299 21.720 1.00 11.56 5CHA 556 ATOM410 CD2 HIS A 57 28.206 25.605 23.753 1.00 10.62 5CHA 557 ATOM411 CE1 HIS A 57 27.787 24.143 22.229 1.00 12.29 5CHA 558 ATOM412 NE2 HIS A 57 28.219 24.290 23.459 1.00 12.00 5CHA 559 ATOM729 N ASN A 100 21.139 29.092 17.682 1.00 15.17 5CHA 876 ATOM730 CA ASN A 100 21.091 28.846 16.199 1.00 14.91 5CHA 877 ATOM731 C ASN A 100 22.361 29.384 15.589 1.00 14.15 5CHA 878 ATOM732 O ASN A 100 22.993 30.366 16.007 1.00 14.62 5CHA 879 ATOM733 CB ASN A 100 19.865 29.473 15.542 1.00 14.99 5CHA 880 ATOM734 CG ASN A 100 19.459 28.894 14.195 1.00 15.90 5CHA 881 ATOM735 OD1 ASN A 100 19.768 27.721 13.910 1.00 15.64 5CHA 882 ATOM736 ND2 ASN A 100 18.776 29.679 13.303 1.00 15.91 5CHA 883 ATOM745 N ASP A 102 25.217 27.828 15.245 1.00 8.90 5CHA 892 ATOM746 CA ASP A 102 26.263 27.454 16.230 1.00 8.03 5CHA 893 ATOM747 C ASP A 102 27.602 26.986 15.681 1.00 7.56 5CHA 894 ATOM748 O ASP A 102 28.030 25.875 15.937 1.00 6.97 5CHA 895 ATOM749 CB ASP A 102 25.612 26.547 17.282 1.00 7.61 5CHA 896 ATOM750 CG ASP A 102 26.373 26.259 18.576 1.00 7.61 5CHA 897 ATOM751 OD1 ASP A 102 26.215 25.289 19.348 1.00 6.70 5CHA 898 ATOM752 OD2 ASP A 102 27.250 27.077 18.886 1.00 7.66 5CHA 899 ATOM 1404 N SER A 195 30.503 18.936 23.287 1.00 10.84 5CHA1551 ATOM 1405 CA SER A 195 30.268 20.144 22.534 1.00 11.39 5CHA1552 ATOM 1406 C SER A 195 31.398 20.500 21.546 1.00 11.49 5CHA1553 ATOM 1407 O SER A 195 32.602 20.510 21.836 1.00 10.92 5CHA1554 ATOM 1408 CB SER A 195 30.081 21.398 23.463 1.00 11.44 5CHA1555 ATOM 1409 OG SER A 195 28.708 21.332 23.826 1.00 13.00 5CHA1556 On 27/06/14 21:49, Keller, Jacob wrote: I have wanted for some time to search for catalytic-triad-like configurations by defining three Ca-Cb bonds from known catalytic triads, then searching the pdb for matches, but have not thought of a quick and/or easy way to do this--can your software do this sort of thing, or is there some other software which could be used for this? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale Tronrud Sent: Friday, June 27, 2014 4:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] New Version of the Protein Geometry Database Now Available -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Protein Geometry Database Server V 1.0 http://pgd.science.oregonstate.edu/ Developed by Andy Karplus' laboratory at Oregon State University We are pleased to announce the availability of an enhanced version of the Protein Geometry Database (PGD) web service, originally
Re: [ccp4bb] Thermofluor assay
I've tried to post my reply to this quite a few times but as best I can tell (and my friends can tell from their CCP4bb subscriptions) I have been foiled by jiscmail every time. Hopefully this will go through. And hopefully I'm not reposting stuff I already sent. Here we go: Okay, the consensus here seems to be that you don't have a thermal shift assay up and running with your membrane protein or membrane proteins in general. Like a couple of people mentioned the Alexandrov 2008 is the standard reference for membrane proteins (CPM reacts with a thiol). You have options though: Thiol-reaction activated fluorophore (e.g. CPM) High-throughput static light scattering (Harbinger Stargazer, Avacta Optim) High-throughput intrinsic fluorescence (Avacta Optim) High-throughput fluorescence lifetime (NovaFluor PR Fluorescence Lifetime Plate Reader) Western blotting (CETSA, FASTpp) Environmental rigidity sensitive dyes (e.g. DCVJ) Environmental dielecticity / hydrophobicity sensitive dyes (e.g. SYPRO Orange, bis-ANS) Possibly differential scanning calorimetry (DSC) Thiol-reaction activated fluorophore Cysteine side chains are typically buried in the core of a protein. When the protein is denatured they become solvent accessible. A dye like CPM can then react with it and only the thiol-reacted compound is fluorescent. CPM has maximum excitation/emission of ~385/470 nm which is a slight problem. Most qPCR machines have excitation filters that begin at ~450 nm and emission filters that begin at ~500 nm. I have however seen somewhere in the literature someone excite CPM with about ~405 nm and reading fluorescence above 500 nm. I haven't tried it but I have tried bis-ANS where I excited at 455-485 nm but without getting a useful signal (bis-ANS has the same excitation maximum as CPM). Some qPCR machines can be fitted with excitation filters that start at 350 nm (Stratagene MX3000/3005, Qiagen Rotor Gene, possibly more). More fluorescence plate readers have these wavelengths but then often don't have temperature control or the temperature is capped at 42 or 65 C. 65 is probably fine for membrane proteins but I would want to start with a test protein and I can't think of any that would melt at that temperature. But actually, you could just add Gu-HCl to ß-lactoglobulin - ß-LG melts in the 70-80 degrees interval but with enough guanidinium it should be fine. Without temp. control you could incubate outside the plate reader but you should be quick about it. I've been told not to incubate with CPM since it does degrade. CPM is of course incompatible with ß-mercaptoethanol, DTT, and tris (tris because of the primary amine unless you are at pH where it is well protonated and has no buffering capacity). pH is restricted to ~neutral. http://www.ncbi.nlm.nih.gov/pubmed/18334210 I tried what Artem suggested with having a friendly chemist cook me up one of those Korean / Chinese compounds that work like CPM but fluoresces in the visible spectrum. It didn't work for me but perhaps I didn't try hard enough or it wasn't pure enough or something. There are a lot of them but keep in mind that a lot of them are designed to react with both the thiol and the amine of cysteine. In a protein that amine is usually an amide and therefore no go. Update: this is the one Artem used http://www.ncbi.nlm.nih.gov/pubmed/19343759 There's a paper where they used CPM with membrane protein in LCP. They had to centrifuge it after each heating step though because the LCP became cloudy. High-throughput static light scattering Using a specialised machine to read the aggregation state of many wells in parallel using static light scattering while heating. I know of two machines, Harbinger Stargazer and Avacta Optim, but unless you can find one to borrow this might be a bit over budget. High-throughput intrinsic fluorescence The Avacta Optim also reads intrinsic fluorescence at the same time. I don't know if this adds any useful information not already supplied by the light scattering, I haven't tried it. High-throughput intrinsic fluorescence lifetime The lifetime of tryptophan fluorescence differs between folded and unfolded protein and by measuring the lifetime of UV-excited fluorescence at temperature intervals you can get a measurement of the melting temperature of your protein. NovaFluor PR Fluorescence Lifetime Plate Reader is the only machine for this that I know of. Western blotting This is pretty interesting. As far as I can see CETSA is a simplified version of FASTpp but with broader applicability. FASTpp is Fast Parallel Protealysis. Crude lysate + thermolysin. Heat but take out aliquots at intervals. Thermolysin is specific for bulky hydrophobic residues and since most of them are buried the digestion is greatly accelerated when proteins denature (this is true of all proteases though, regardless of their specificity). The aliquots are run on an SDS gel and Western blotting is performed to determine at what temperature the protein of
[ccp4bb] Acorn, CRANK
Hi everyone, I have two questions: 1- I was trying to run the program Acorn, on a SAD dataset (Se derivative) that was scaled in scalepack/HKL2000. converted to .mtz using scalepack2mtz, then edited in REVISE and Ecalc. But when I ran Acorn it gives me the error message Segmentation fault. Does anyone know what is going on? 2- The same dataset I ran on CRANK in an attempt to calculate a substructure and the program stopped with an error message: not enough clear peaks in the patterson function eventhough xtriage suggests I have a an anomalous signal until 5.1A. Does this mean the anomalous signal is so weak for CRANK to detect? Thank you, Maher
