[ccp4bb] suitabel buffer condition for protein
i dont know how people decide that this buffer ph ,salt concn,dtt,etc in perticular concm is suitable for protein to be in folded state,i am doing crystallisation of few proteins but i dont know how to select the parent buffer for the protein,is it CD or crysatllisation trials that tells abt this -Original Message- From: CCP4 bulletin board on behalf of James Stroud Sent: Mon 5/11/2009 8:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Manipulating electron density On May 10, 2009, at 12:21 PM, Phoebe Rice wrote: Of COURSE the map will look lovely if you carve it off at 1.5A from your atoms. And your gels will look lovely too if you just touch them up with with some white-out and a sharpie. Do the honest thing and show the whole truth, using the z-clipping to get a comprehensible slab. Maybe we should give Jason the benefit of the doubt that he wants to present an honest representation of his density. Clipping off symmetry related molecules is not the same as carving density from amorphous blobs. The former is more akin to showing only part of a lane of a gel, which is common practice even in prestigious journals. In fact, my other monitor presently displays such a carved gel picture from a recent Cell paper. James
Re: [ccp4bb] suitabel buffer condition for protein
Dear Atul, for crystallisation, I would say the most suitable buffer is water. If your protein concentrates well in pure water, why add anything else? Of course, many proteins will not concentrate well in pure water, so start adding buffer components until your protein is happy: - buffer (HEPES, Tris) at 10 mM. - a bit of salt - ions your protein may need for stability - etc. The thermofluor (Analytical Biochemistry, Volume 357, Issue 2, 15 October 2006, Pages 289-298) assay can be good for this. Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain researcherID: B-3678-2009 On 11 May 2009, at 07:48, atul kumar wrote: i dont know how people decide that this buffer ph ,salt concn,dtt,etc in perticular concm is suitable for protein to be in folded state,i am doing crystallisation of few proteins but i dont know how to select the parent buffer for the protein,is it CD or crysatllisation trials that tells abt this -Original Message- From: CCP4 bulletin board on behalf of James Stroud Sent: Mon 5/11/2009 8:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Manipulating electron density On May 10, 2009, at 12:21 PM, Phoebe Rice wrote: Of COURSE the map will look lovely if you carve it off at 1.5A from your atoms. And your gels will look lovely too if you just touch them up with with some white-out and a sharpie. Do the honest thing and show the whole truth, using the z-clipping to get a comprehensible slab. Maybe we should give Jason the benefit of the doubt that he wants to present an honest representation of his density. Clipping off symmetry related molecules is not the same as carving density from amorphous blobs. The former is more akin to showing only part of a lane of a gel, which is common practice even in prestigious journals. In fact, my other monitor presently displays such a carved gel picture from a recent Cell paper. James
Re: [ccp4bb] phasing with se-met at low resolution
Dear Engin I would also like to comment. I our recent structure determination of the sodium pump (3.5 A) (see morth JP et al 2007) we did not have experimental phasing to more than 6 A for the Ta6Br12 clusters and 7 A for the Pt sites. Both with extensive multicrystal averaging and phase combination it was possible to trace the structure. The data was what you could call lousy, but in the end I was able to identify the 3 Rubidium ions present in the data set based on the their anomalous scattering power. Not detectable in the reflection statistics of the native data set (see Schack VR et al 2008). So even though your selenium sites will not help the phasing initially, they will still guide the model building extensively (see Hunte C et al 2005 Nature, 3.5 A res structure with Se) with your improved model phases the selenium site positions will improve and at a later stage they will be valuable, when combined as additional phase information. The initial maps always look really bad, your 3 years of heavy atom derivatisation might not have been in vain, you can still use the initial Se phases to try to locate more heavy atoms sites (Fredslund F et al 2006 jmb) good luck Preben On 11/05/2009, at 05.24, Engin Ozkan wrote: Wow, I got quite a number of responses. Thanks everyone. Let's elaborate. Petr, I don't know my anomalous signal, because I haven't yet done the experiment. If I had, I would have definitely talked about chi2 values for I+/I- merged and unmerged, Anomalous Patterson maps, or other measures of anomalous signal (Dauter, Acta Cryst D, 2006 is a great paper to read on that, I suggest every grad student to present it in their Crystallography Journal Club). I was merely pondering today, but I plan to do the experiment very soon (crystals are waiting for synchrotron time). About your example, 2.9 A diffracting crystals (with 4 A anomalous signal) are in the doable range, as you suggested. The question is what would happen if your crystals diffract to 4 A, and anomalous signal dies at 6 A. The interesting bit of course is 1 Met per 200 residue, which should put to death the 1 in 50 or 1 in 100 Methionine myths: it depends on the quality of your data. Engin On 5/10/09 2:50 PM, Leiman Petr wrote: Dear Engin Ozkan, You have told us how bad your crystals are, but you did not mention how good your anomalous signal is: 1. To what resolution does your anomalous signal extend and what statistic is used for this estimate? 2. Do your dispersive and Bijvoet Pattersons look similar and what is the measure of similarity? This structure http://www.pdb.org/pdb/explore/explore.do?structureId=1K28 which contains ~1100 residues in the asymmetric unit (and ~3500 in the entire complex), was solved using a chimerical SeMet derivative, in which one protein was SeMet labeled (17 Se per a.u.) and the other was native. The Semet dataset had a detectable anomalous signal to 4 A resolution (at most). The diffraction extended to 2.9A resolution. Sincerely, Petr --- Petr Leiman Institut de physique des systèmes biologiques École Polytechnique Fédérale de Lausanne (EPFL) Cubotron/BSP-415 CH-1015 Lausanne Switzerland -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Engin Ozkan Sent: Sunday, May 10, 2009 11:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phasing with se-met at low resolution Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures only using phases from selenium in a realistic case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, whereI/ sigma in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick- Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them.
[ccp4bb] System virtual machine recommendation for crystallography?
Hello everyone, I would like to install a system virtual machine to run Ubuntu Linux as a guest OS on a 32-bit Vista laptop. The idea is to allow occasional use of crystallographic refinement programs while I'm away from lab. The laptop has an Intel Core 2 duo processor (2.0 GHz) and 3 GB RAM. There are popular software programs available (VMWare, Parallels, VirtualBox, etc.), but is anyone aware of any considerations that would make one better for the above purposes? For example, will one offer easy control over distributing hardware resources to prevent crippling Vista while running refinement within the guest Linux? My Google and CCP4bb searches have not turned up anything so far. Thanks in advance for any advice or reference material. I will post a summary e-mail as well. Best Regards, -Andy Torelli -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry and Chemical Biology Cornell University =
Re: [ccp4bb] phasing with se-met at low resolution
On Sun, May 10, 2009 at 08:24:34PM -0700, Engin Ozkan wrote: The question is what would happen if your crystals diffract to 4 A, and anomalous signal dies at 6 A. The interesting bit of course is 1 Met per 200 residue, which should put to death the 1 in 50 or 1 in 100 Methionine myths: it depends on the quality of your data. Have a look at 2jk4 ... which had the added 'fun' of being a membrane protein with very anisotropic diffraction (best direction to about 4A) and no NCS for averaging ... Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Manipulating electron density
Hi, I was not implying to cut-out the bad pieces and apologize if it appeared as such. In my short time in crystallography the lowest resolution I have worked with is 1.95 A. This was only an example to help-out, and appeared to be the best and complete answer to the question. Jason, I goofed in typing, you can leave out the parenthesis if you create an object. isomesh mesh1, your map, 1.0, chainA I removed carve :) Sincerely, Carlos Phoebe Rice pr...@uchicago.edu 05/10/09 2:22 PM Of COURSE the map will look lovely if you carve it off at 1.5A from your atoms. And your gels will look lovely too if you just touch them up with with some white-out and a sharpie. Do the honest thing and show the whole truth, using the z-clipping to get a comprehensible slab. Original message Date: Sat, 9 May 2009 18:53:35 -0500 From: Carlos Huerta carlos.hue...@utsouthwestern.edu Subject: Re: [ccp4bb] Manipulating electron density To: CCP4BB@JISCMAIL.AC.UK Hi Jason, If you already created a .map from CCP4 and changed the extension to .ccp4. Then, to create a map in PyMol for your protein only is the following. create chainA, (chain A pdb name) #I think you can leave out the pdb name isomesh mesh1, your map, 1.0, (chainA), carve=1.5 If the chain contains other atoms beside the protein atoms. create chainA, (chain A resi #-#) isomesh mesh1, your map, 1.0, (chainA), carve=1.5 Sincerely, Carlos Jason Porta jpo...@unmc.edu 05/09/09 3:19 PM Hi everybody, Sorry if this message was already asked (I could not find it in the archives). I am making a figure of a recently solved protein structure including the electron density. I would like the electron density to cover only the protein, and not the surrounding space where the symmetry-related atoms are. I remember this being a simple task with Xfit, but I cannot find a copy for Intel-based Macs. I have tried doing this using Coot and PyMol, but have had no luck as of yet. I have also tried using Mapmask in CCP4, which also did not work. Any suggestions would be greatly appreciated. Jason Porta Graduate Student Dept. Biochemistry Molecular Biology University of Nebraska Medical Center Omaha, NE 68198 Phoebe A. Rice Assoc. Prof., 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 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] System virtual machine recommendation for crystallography?
I'm using VirtualBox 2.2.2 to run a Ubuntu 9.04 on WinXP (32 bit) as a testbed for migrating my lab servers/workstations from Fedora 8. It runs surprisingly well with 512 Mbyte of assigned memory and 64 Mbyte of assigned graphics memory. I have even used it to test Wine to run some Win-only crystallography software within Ubuntu within WinXP (!) with very good performance on a dual core 2.4 GHz machine with 2 Gbyte total memory. The only thing you will not get in the virtual machine is accelerated graphics performance. If you are expecting to run Coot or Pymol in a virtual machine, forget it. (However, it does work, and I can get about 15 frames/sec with the non-native graphics. It's certainly usable, if a bit slow.) What you can do is set up share folders so that you can open files in Vista and get native graphics performance in your favorite graphical applications. VMWare is snappier, but VirtualBox is free. Cheers, Roger Rowlett Andy Torelli wrote: Hello everyone, I would like to install a system virtual machine to run Ubuntu Linux as a guest OS on a 32-bit Vista laptop. The idea is to allow occasional use of crystallographic refinement programs while I'm away from lab. The laptop has an Intel Core 2 duo processor (2.0 GHz) and 3 GB RAM. There are popular software programs available (VMWare, Parallels, VirtualBox, etc.), but is anyone aware of any considerations that would make one better for the above purposes? For example, will one offer easy control over distributing hardware resources to prevent crippling Vista while running refinement within the guest Linux? My Google and CCP4bb searches have not turned up anything so far. Thanks in advance for any advice or reference material. I will post a summary e-mail as well. Best Regards, -Andy Torelli -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu
Re: [ccp4bb] Manipulating electron density
Jason Porta jpo...@unmc.edu 05/09/09 3:19 PM Hi everybody, Sorry if this message was already asked (I could not find it in the archives). I am making a figure of a recently solved protein structure including the electron density. I would like the electron density to cover only the protein, and not where the symmetry-related atoms are. [...] I have tried doing this using Coot [...], but have had no luck as of yet. Jason, quick note: the reason this fails in Coot is because Coot assumes a match of the symmetry and cell in the model and map. To make this work in Coot you would need to create a map that does not have the same symmetry as the model (e.g. generate an extended map in P1). Perhaps this is possible with the SYMM card in mapmask. (You can then use the usual map masking extensions). Paul.
[ccp4bb] 2nd Announcement:Fragment screening library for protein crystallography
Many people responded with questions following our first announcement of a ready-to-use small fragment screening library designed for protein crystallographic screening. Given suitable crystals, the methodology is of interest to anyone wishing to probe ligand binding interactions and motifs in accessible sites on the protein. A document describing the library design philosophy, chemical property parameters, practical application in protein crystallography and provision of structure data is available from Leslie Hernandez (les...@zenobiatherapeutics.com). A SD file showing the chemical structures of compounds in the library (352 ring-containing compounds with mean MW 154Da) is also available on request. Zenobia Therapeutics http://www.zenobiatherapeutics.com/
Re: [ccp4bb] phasing with se-met at low resolution
If experience from intrinsic zinc is ok, I'll add my two cents. trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other Bert already mentioned collecting in wedges for SAS, so I'll add to the chorus there. For dispersive differences, wedges (20 degrees at inf, 20 at rmt) helps a great deal for some of our crystals as well. P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one. Last time I emailed the RCSB about this (a few years back), it wasn't possible to search by phasing method. You can try using advanced search - keyword search - advanced and doing a full text search, but this is somewhat less than ideal. To be fair though, I suspect relatively few people searching the PDB are concerned about the phasing method used. Pete
Re: [ccp4bb] CCP4 updates and the problem pages
I just did the same today. Those that are missing from the 4-03 patch (at least the src version) included: scala.f version.fh refmac5.tcl scalepack2mtz.com dtrek2mtz.com pname2.com import_scaled.script also there was a tcl++ fix William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On May 11, 2009, at 3:35 PM, Ben Eisenbraun wrote: If I download and apply a patch; e.g.: ftp://ftp.ccp4.ac.uk/ccp4/6.1.1/updates/ccp4-6.1.1-linux-i386-patch-04_03_09.tar.gz Can I assume that all the problems listed on the problem pages prior to April 3, 2009 will be in that patch? Or do I still need to go through the problem pages and update things by hand? Thanks. -ben -- | Ben Eisenbraun | Software Maintainer| | Structural Biology Grid | http:// sbgrid.org | | Harvard Medical School | http:// hms.harvard.edu |
