Re: [ccp4bb] Optimizing xtals conditions
Dear Christine, Slow crystallization may also be due to some residual protease activity, which cuts away a flexible loop. You could check this with mass spec or running a gel with protein from a crystal. If it turns out that you need proteolysis to get crystals, you could try in situ proteolyis by adding a trace amount of protease e.g. Trypsin of Chymotrypsin to your crystallization trials. The link below gives a recent paper in this subject. Good luck! Herman http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0005094 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Harman, Christine Sent: Tuesday, August 14, 2012 7:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Optimizing xtals conditions Hi all, I need some advice on reproducing crystals that emerged in about 2 months from a screen. The background is I set up a 96-well hangdrop screen and checked the tray after 1 week and then every 2-3day for about 3 weeks. I did notice that this particular drop seemed very dynamic overtime. I stopped looking at the tray for about 2 weeks and then when I re-checked after that time I found ~5 beautiful single growing crystals. These crystals continued to grow with a few more emerging over the next week. I am not sure of the exact time the first crystals emerged, but between the time I set up the screen to when I saw the crystals it was not quite 2 months (~1 week short). I am in the process of reproducing this hit. I set up drops with the protein at the same concentration used in the screen (~5mg/mL) and at a higher concentration ~2.5X. This protein is a Fab that was complexed with peptide before setting up initial screen. The protein is in only 0.1M Tris pH8.0. The crystal condition is 13.4% PEG 8K, 2% MPD and 0.1M imidazole pH6.5. I have checked drops (~3 weeks old) and with the more concentrated protein I see some interesting crystalline like ppt similar to what I see in the drop in the screen; however, no crystals yet. The drops with the lower concentration of protein are still somewhat clear with some having crystalline like ppt. I varied the PEG 8K concentration 12.5-14% vs MPD at 2, 4, and 6%. What I need advice on is what else can I do to speed up the crystallization process. Does anyone have suggestions besides increasing the protein concentration. I have limited amounts of protein left for optimization so I was wondering if there were any additives that were better known to facilitate faster crystallization as opposed to testing everything under the sun. Any suggestions would be great :) Thanks, Christine
Re: [ccp4bb] phaser with MR------search ensemble
The ensemble should be a set of coordinates Eleanor On 15 Aug 2012, at 04:42, 李华 wrote: Dear ccp4er, I try to use Phaser MR to solve a structure. A mtz file from oasis was used as a ensemble. All of the parameters containing protein MW, nucleic acid MW, extent of X, Y, Z and center of X, Y, Z and RMS error were assigned. However, when I ran phaser mr, the program complain that NOT SET: search ensemble. Does anybody has any advice about this? Thanks very much! Hua
Re: [ccp4bb] CCP4 6.3.0 release
To anyone with problems with coot after installing 6.3.0 in Ubuntu Linux: I added the line export PATH=/path/to/ccp4-6.3.0/coot-0.6.2/bin:$PATH to my .bashrc after removing the referrals to my old ccp4 installation setup scripts. If you don't removed or comment them out I guess that your old coot installation will still be pathed correctly. You could also add the export line to the ccp4.setup-sh if you wish, I guess. Other than that I must say that this went extremely smoothly and it seems to be the right Coot version as well. Well done! Cheers, Morten On 18 July 2012 10:36, Marcin Wojdyr marcin.woj...@stfc.ac.uk wrote: Two Windows-specific problems have been reported and fixed yesterday: - cif_mmdic.lib is in %CCP4%\share\ccif, should be in %CCP4%\lib - rapper doesn't work (missing DLL) cif_mmdic.lib can be moved manually, so if you don't use rapper there is no need to update. Installer with fixes is available from ccp4 website and ftp: ftp://ftp.ccp4.ac.uk/ccp4/6.3.0/CCP4-6.3.0.1.msi Additionally ctruncate has been updated, some non-critical issues have been fixed. Marcin -- Scanned by iCritical. -- Morten K Grøftehauge, PhD Pohl Group Durham University
Re: [ccp4bb] phaser with MR------search ensemble
No, it's possible also to specify a model in the form of the molecular transform in an MTZ file, derived either from coordinates or a density map. I see that I failed to Reply all in my earlier reply to this email, which was the following: = Does the MTZ file from oasis contain electron density for another crystal form of the same protein (or a close relative)? If so, I presume you've made sure to cut out the density corresponding to one molecule and place it in a large unit cell. Assuming that's right, the error message you got arises when you haven't told the ccp4i GUI for Phaser which ensemble you want to search for. Even if you've only defined one ensemble, you have to specify that that's the one you want to search for. This is in a pulldown in the Search Parameters pane labelled Perform search using. I've just tested with the new CCP4 6.3.0 release, and this works for me. Good luck! Randy Read = On 15 Aug 2012, at 10:07, Eleanor Dodson wrote: The ensemble should be a set of coordinates Eleanor On 15 Aug 2012, at 04:42, 李华 wrote: Dear ccp4er, I try to use Phaser MR to solve a structure. A mtz file from oasis was used as a ensemble. All of the parameters containing protein MW, nucleic acid MW, extent of X, Y, Z and center of X, Y, Z and RMS error were assigned. However, when I ran phaser mr, the program complain that NOT SET: search ensemble. Does anybody has any advice about this? Thanks very much! Hua -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
[ccp4bb] 答复: [ccp4bb] phaser with MR------search ensemble
Hi Hua, I am so curious why you used an mtz file from OASIS as ensemble. OASIS has ability to complete the model, position and orientation of which have been determined correctly. And PHASER is a quite excellent program to fix MR model. So I think a normal procedure is PHASER - OASIS, but not OASIS - PHASER. Or do you have some special idea？ Zhang, Tao 发件人: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] 代表 李华 发送时间: 2012年8月15日 11:42 收件人: CCP4BB@JISCMAIL.AC.UK 主题: [ccp4bb] phaser with MR--search ensemble Dear ccp4er, I try to use Phaser MR to solve a structure. A mtz file from oasis was used as a ensemble. All of the parameters containing protein MW, nucleic acid MW, extent of X, Y, Z and center of X, Y, Z and RMS error were assigned. However, when I ran phaser mr, the program complain that NOT SET: search ensemble. Does anybody has any advice about this? Thanks very much! Hua
[ccp4bb] Fall BioSAXS beamtime available at MacCHESS
BioSAXS beamtime at CHESS is now available for Fall 2012! Dear BioSAXS users, Beamtime is still available during the Fall running period (Sept 12 - Nov 19) for BioSAXS. To apply for time, please fill out the short online express-mode form at express.chess.cornell.edu/EM_form.phphttp://express.chess.cornell.edu/EM_form.php Under Choice of experimental technique specify Other In the box provided for Special experimental and facility needs type standard BioSAXS. beamline = F2 Please also send me an email when you submit your express mode form: r...@cornell.edumailto:r...@cornell.edu To insure a successful visit, please carefully follow the sample preparation guidelines and other information given on our recently updated BioSAXS web page:www.macchess.cornell.edu/MacCHESS/bio_saxs.htmlhttp://www.macchess.cornell.edu/MacCHESS/bio_saxs.html News -- The F2 station is now dedicated to BioSAXS. You can read about our robotic sample loading system in our recent paper: High-throughput biological small-angle X-ray scattering with a robotically loaded capillary cell, Nielsen, S. S., Moller, M. Gillilan, R. E. (2012). J. Appl. Cryst. 45. We now have dual SAXS/WAXS Pilatus 100K detectors that give us automatic q-space coverage from 0.008 - 0.8 inverse Angstroms! Our AKTA Purifier size-exclusion chromatography (SEC) system is available for use at the F2 beamline. If your samples seem to aggregate rapidly after preparation, or you think you may have a mixture, you may want to investigate on-site SEC. Fractions can be collected directly into the same 96-well plates used by the BioSAXS robot. I you are interested, contact me (Richard Gillilan) for details. -- Hope you can join us this Fall! Best Richard Gillilan MacCHESS
[ccp4bb] Br-dU in Phenix refinement
Dear all: I tried to build 5’-Br-dU in my coordinate. I imported Br-dU from monomer lib (coot) and put it in the position, merged the molecules, renamed the Chain ID and renumber the residues, everything was ok. However, when I did refinement with Phenix (using elbow to generate cif file for Br-dU) afterward, in the resulting PDB, the Br-dU were always found disconnected with the adjacent nucleotide. In coot, I could manually fix this problem with real-space refinement, but it seems that the Phenix always fall to do that. In addition, when using real-space refinement for nucleotide in coot, the hydrogen always falls apart. Is there anyone encounter the similar problems or know how to deal with them? Many thanks! Cui sheng
[ccp4bb] Mr. Bump and adding FASTA to CCP4 6.3 installation?
Hello, Mr. Bump requires Fasta to be installed. Could someone tell me what needs to be done to install Fasta so that it is seen and used by CCP4 6.3 programs? Installers for previous versions, 6.1 and 6.2, also installed Fasta, for 6.3 we are supposed to download and install manually. OK, I downloaded and unpacked into C:\CCP4\fasta\ - now what? The download site, http://fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml , says not a word about installation in Windows, nor it is mentioned in the Readme file. Thanks! - Nate
