[ccp4bb] summary: tricoordinate ion?
Dear all, thanks a lot for the many replies suggesting me what to put in my misterious density. Molecules that have been suggested are: acetate, Mg ion with waters, bicarbonate or nitrate, sulphate, borate, glycerol, tris, alternative conformation of the Arg side chain, waters. I have to say that, as you've noticed, the resolution is only 2.6, so it'll be hard to conclude something beyond reasonable doubts. What I'm doing is, as Roger Rowlett and David Borhani suggested, calculating the maps by omitting the waters, and deciding upon the shape of the density I see there, trying than to fit different molecules. In doubt, it'd seem reasonable to me to place one of the molecule of the crystallization conditions. At the moment, it seems that glycerol could fit nicely. I also have to apologize for the large attachment. I'm sorry I didn't notice it was so large (I have to admit, I just sent what Wincoot wrote out! ;-)). Next time I'll be surely posting the image to some website. Thanks again, all the best, S -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 574 303 310
[ccp4bb] Summary - crystals grown from high ammonium sulphate
Thanks a lot for all the helpful replies I got! Here is a summary: - increase drop size to 4+4ul if protein is not the limiting factor - limits skin formation - add 10-20ul well solution directly to drop - freeze straight from the drop - microseeding into less conc. AmSO4 to get more crystals - add AmSO4 free motherliquor to well solution to equilibrate drop to lower AmSO4 - add cryo solution in 1/4 of drop volume steps to drop and freeze directly from drop - glucose more gentle than glycerol - http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Cryo - reduce evaporation from drop by mounting and freezing crystals in the cold room - try mineral oils - either add small amount directly to drop or move crystal to drop with oil first - test diffraction of crystal at RT - crystals could be grown in a plastic tube, which can be put directly in the cryo stream (Yevgeniy Kalinin and Robert Thorne, Acta Cryst. (2005). D61, 1528–1532.) - be quick Sabine Sabine Schneider wrote: Hi everyone, We got crystals that grew in ~3.2M ammonium sulphate and some tris-buffer at 18dgC. Unfortunately the crystals take a while to grow (~4-5 weeks) and so far we only have 4-5 xtals. I tried to freeze the crystals, but as soon as I broke though the skin of the drop the ammonium sulphate started to crystallise. I got the crystals out, froze them using sort of an artifical mother liquor with sodium malonate as cryo and tested the diffraction. The freezing seems to be OK and it is definitely a protein crystal. The crystal suffered when the ammonium sulphate in the drop started to crystallise, but didn't seem to deteriorate anymore in the cryo. Well the corners had already more or less disappeared by the time I got them out of the drop... Since we only have a few xtals at the moment and I can't try out a lot of things, I was wondering if anyone has experienced and solved a similar problem? My freezing attempt so far was in an airconditioned room with 18dgC. I thought about higher humidity and temperature in the room, and/or adding the cryo directly to the drop Any ideas are very much appreciated!
[ccp4bb] summary of cartoon problem and a question
Dear all, Thank you for the suggestions regarding cartoon problem that I posted on bulletin board. The problem was due to the separate segID put out after the phenix refinement. According to the PDB format, if I am not wrong, column number 73 should be blank which is followed by element name. But phenix puts segID in that column. During the refinement it has assigned the segID in column 22 througout where as in other column there were missing in some cases. Because of that I had seen a break in column in pymol. The suggestion given by Ingo P. Korndoerfer to assign the segID to those missing ones helped to close the gap. And also suggestion by Warren DeLano set retain_order and rebuild did the trick. Presence of segID in column 73 appears to be off the PDB format. But I would like to know why then phenix.refine adds that additional column in the pdb file? Thanking you Madoori pramod
[ccp4bb] Phylogenetic analysis??
Hi Everyone, Could someone please tell me how to display the evolutionary/ phylogenetic tree of the homologs of my protein of interest. When I perform a PSI-BLAST search for my protein, I receive about 130 top hits for homologs. The NCBI or EBI tools that I've laid my hands on seem to only display a 'phylogenetic' tree based on the distance relationships between the protein sequences and that is not what I am after. I'd like to find a way to resort the results and redisplay a tree that say progresses from, say 'yeast to human'. What I have now is the organisms shown in some random order, say, rat followed by C. elegans followed by human. I am going bonkers trying to find a simple way to do what I want. Thanks and sorry for this Bioinformatics101-type question. Raji
[ccp4bb] User proposal submission for Collaborative Crystallography at BCSB
Dear Users, Through the Collaborative Crystallography Pilot Program (CC) at the Advanced Light Source, scientists can send protein crystals to Berkeley Center for Structural biology (BCSB) staff researchers for data collection and analysis. The CC Pilot Program can provide an number of benefits to researchers: * Obtain high quality data and analysis through collaborating with expert beamline researchers; * Rapid turn around on projects; * Reduced travel costs. Thumbnail Description: == The Collaborative Crystallography program will be piloted on beamlines 5.0.1 and 5.0.2 for one year; if successful it will be implemented on a permanent basis. CC proposals will go through the regular ALS General User proposal review process for beamtime allocation. Proposals will be reviewed and ranked by the Proposal Study Panel, and beamtime will be allocated accordingly. BCSB staff will schedule the projects to fit into the available resources. Only non-proprietary projects will be accepted. As a condition of participation, BCSB staff researchers who participate in data collection and/or analysis must be appropriately acknowledged - typically being included as authors on publications and in PDB depositions. Please consult the website for additional information at: http://bcsb.als.lbl.gov/wiki/index.php/Collaborative_Crystallography = How To Apply: To submit a proposal, go to: http://www-als.lbl.gov/als/quickguide/independinvest.html Click on protein crystallography to begin proposal. For question 3 select Collaborative PX 501/502. For question 9, please describe a specific research project with a clear end point. In order to request CC time for January/February 2009 allocation period, proposals must be submitted by November 15, 2008. The deadline for CC proposals for the time period March/April 2009 will be January 15, 2009. Regards, Banumathi Sankaran
