Re: [ccp4bb] finicky protein
Try refolding before purification. On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: Hi all sorry, for offtopic query... I am trying to purify my protein by Ni-NTA affinity chromatography. After sonication as i centrifuge bacterial lysate, soon after 10 min whole lysates get precipitated during loading on the column and some time it remain soluble too. if i get purified through the column without precipitation, it gets precipitated during dialysis. I have tried lot, by chnaging buffers, increasing salt or deacreasing salt or no salt at are helpless. I do purifiaction in cold room. can any one suggest some solution? Thanks in advance. NSH -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
[ccp4bb] REMINDER: Job opportunities- Helmholtz Department of Structural Biology, DESY Hamburg.
Dear colleagues, we are writing to remind you that 6 March 2008 is the closing date for applications for the following job opportunities. The German Helmholtz Association, together with the University of Hamburg, is creating a new department of Structural Biology at the German Electron Synchotron (DESY) in Hamburg and invite applications for a professorship in Structural Biology (Head of the Helmholtz Department of Structural Biology) plus a Junior Professorship. For a full specification of these appointments, please see the advertisements at http://www.nature.com/naturejobs/science/jobs/42039 and http://www.nature.com/naturejobs/science/jobs/42040. You can also send your applications by email to [EMAIL PROTECTED] Please do not hesitate to contact us in case of further questions. We will be glad to provide any further information that you may need. Yours sincerely, Dirk Heinz Head of Division of Structural Biology Helmholtz Centre for Infection Research Braunschweig, Germany +49 (0)531 6181 7000 [EMAIL PROTECTED] -- Christine Bentz Project assistant BioPharma-initiative Division of Structural Biology Helmholtz Centre for Infection Research Inhoffenstrasse 7 38124 Braunschweig / Germany. Phone (0)531.6181.7002 Fax (0)531.6181.7099 [EMAIL PROTECTED]
Re: [ccp4bb] finicky protein
Did you filter your lysate through .45 then .22 filters? cheers b Quoting James Stroud [EMAIL PROTECTED]: Try refolding before purification. On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: Hi all sorry, for offtopic query... I am trying to purify my protein by Ni-NTA affinity chromatography. After sonication as i centrifuge bacterial lysate, soon after 10 min whole lysates get precipitated during loading on the column and some time it remain soluble too. if i get purified through the column without precipitation, it gets precipitated during dialysis. I have tried lot, by chnaging buffers, increasing salt or deacreasing salt or no salt at are helpless. I do purifiaction in cold room. can any one suggest some solution? Thanks in advance. NSH -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
[ccp4bb] new versions of Mosflm (7.0.3) and iMosflm (0.6.1)
Hi folks We are pleased to announce the release of Mosflm version 7.0.3 and iMosflm 0.6.1. These are bug-fix releases for versions 7.0.2 and 0.6.0, which affect the following detectors and circumstances only. If your detector is not listed, it isn't affected, so you shouldn't need to update your copies of Mosflm and/or iMosflm - but you may want to anyway. Many thanks to those people who brought these issues to our attention. Fixes to Mosflm: * Image display for R-Axis detectors was reversed in iMosflm (the new interface), not in the old interface. * The presence of extremely large pixel values (262128) for Mar IP scanners caused Mosflm to shut down. * Direct beam co-ordinates read from Mar CCD image headers not sited at ESRF had X and Y swapped. * Direct beam co-ordinates were not used from Ed Westbrook NOIR images by iMosflm (although they were used by Mosflm). * Reversephi check box in iMosflm did not function as intended. Fix to iMosflm: * Bruker .sfrm suffix included in list in image browser. Special thanks to Jan for reminding me that I should include the download sites when announcing new releases! Downloads are available through the normal web-pages and ftp sites - Everything Mosflm - http://www.mrc-lmb.cam.ac.uk/harry/mosflm specific places - Mosflm v.7.0.3 - http://www.mrc-lmb.cam.ac.uk/harry/mosflm/ver703 iMosflm v. 0.6.1 - http://www.mrc-lmb.cam.ac.uk/harry/imosflm/ Harry Luke -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH
Re: [ccp4bb] finicky protein / cell disruption
I will second this recommendation to use alternate cell disruption methods instead of sonication. Particularly with multi-protein complexes, where the preservation of the complex from disruption though crystallization is important, sonication can be quite deleterious. Even with the French press (or a cell emulsifier), disruption at low pressures (e.g. three passes at low pressure) increases the level integrity of full complexes, while giving good yield. My recommendation is to throw your sonicator away and use methods that apply less heat and energy to your cells and to your biomolecules. - Th Thomas Earnest, Ph.D. Senior Scientist and Group Leader Structural Proteomics Development Group Physical Biosciences Division MS64R0121 Lawrence Berkeley National Laboratory Berkeley CA 94720 [EMAIL PROTECTED] 510 486 4603 Mads Gabrielsen wrote: I am not a big fan of sonication. Try changing your way of disrupting the cells. I have compared sonication vs mechanical stress on several unrelated proteins, and for me a good old french press wins every time. If you want to get all modern and fancy, a cell disruptor gives similar results. Cheers, Mads Gabrielsen [Hide Quoted Text] On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: Hi all sorry, for offtopic query... I am trying to purify my protein by Ni-NTA affinity chromatography. After sonication as i centrifuge bacterial lysate, soon after 10 min whole lysates get precipitated during loading on the column and some time it remain soluble too. if i get purified through the column without precipitation, it gets precipitated during dialysis. I have tried lot, by chnaging buffers, increasing salt or deacreasing salt or no salt at are helpless. I do purifiaction in cold room. can any one suggest some solution? Thanks in advance. NSH
[ccp4bb] protein degraded when setting xtal?
Dear CCP4 experts, I'm working on a kind of cystein peptidase. It shows a little degradation after 2 days storing at 4 degree even I tried many kinds of protease inhibitors, or glycerol and DTT. So I decided to work fast (1 day) then set xtal. After one week I pick up some condition and check degradation by SDS-PAGE. It shows heavy degradation then I think it's impossible to obtain the crystal if I don't find out how to stop the degradation first. Do you have any idea about my situation? Is it true that I need to stop the degradation and how to deal with it? Thank you for your advices, My best regards, TriNgo
[ccp4bb] radiation damage question
Hi all, I had an interesting experience, and wonder if others have seen similar things. I was collecting data from a crystal that contains an iodinated macromolecule. After 2 days on a copper rotating anode, with the crystal at 100 K, we experienced a detector problem, so I put the crystal back into the dewar; it was diffracting nicely when I took it off. For various reasons, I didn't get back to this crystal until about 3 weeks later. When I put it back on the goniostat, the mother liquor was milky white in appearance. There were no ice rings, but alas the crystal only gave a few anemic spots around the beamstop. Annealing didn't help, and I noticed that when I blocked the cold stream, the milky white appearance didn't go away when the sample thawed. I finally took the crystal off and looked at it under a microscope, at which point I discovered that the milky white appearance was due to the presence of bubbles in the mother liquor. I seem to recall some talks on radiation damage in which people mention the evolution of a gas (H2?). So: Does this seem like a radiation damage phenomenon? And have others seen this kind of delay in the manifestation of damage during storage at liquid N2 temperatures? Thanks, Pat --- Patrick J. Loll, Ph. D. (215) 762-7706 Professor FAX: (215) 762-4452 Department of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA [EMAIL PROTECTED]
Re: [ccp4bb] protein degraded when setting xtal?
Ngo Duc Tri wrote: Dear CCP4 experts, I'm working on a kind of cystein peptidase. It shows a little degradation after 2 days storing at 4 degree even I tried many kinds of protease inhibitors, or glycerol and DTT. So I decided to work fast (1 day) then set xtal. After one week I pick up some condition and check degradation by SDS-PAGE. It shows heavy degradation then I think it's impossible to obtain the crystal if I don't find out how to stop the degradation first. Do you have any idea about my situation? Is it true that I need to stop the degradation and how to deal with it? Thank you for your advices, My best regards, TriNgo If I understand the problem properly, you are attempting to crystallize a peptidase that autolyzes in solution. If it is not possible to completely inhibit the protein with an analog or small molecule under crystallization conditions (and be able to crystallize the complex), then the most obvious solution to the problem is to prepare an inactive variant, e.g., with the mutation of the catalytically essential Cys to Ser. With any luck, the variant protein should still provide valuable structural and mechanistic context while avoiding pesky autolysis. Cheers, -- 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: [EMAIL PROTECTED]
Re: [ccp4bb] protein degraded when setting xtal?
If your protein is autolyzing, you may need to crystallize it in the presence of an inhibitor. In the case of trypsin, I have crystallized in the presence of benzamidine, then removed the inhibitor by dialysis after crystallization. kmj Ngo Duc Tri wrote: Dear CCP4 experts, I'm working on a kind of cystein peptidase. It shows a little degradation after 2 days storing at 4 degree even I tried many kinds of protease inhibitors, or glycerol and DTT. So I decided to work fast (1 day) then set xtal. After one week I pick up some condition and check degradation by SDS-PAGE. It shows heavy degradation then I think it's impossible to obtain the crystal if I don't find out how to stop the degradation first. Do you have any idea about my situation? Is it true that I need to stop the degradation and how to deal with it? Thank you for your advices, My best regards, TriNgo
Re: [ccp4bb] radiation damage question
Yes indeed ! H2 is one product of H2O radiolysis by recombination of two H radicals. Whether or not the cleavage of iodine (quite efficient under X-rays) is a factor increasing H2 production, I don't know. Philippe Dumas IBMC-CNRS, UPR9002 15, rue Rene Descartes 67084 Strasbourg cedex tel: +33 (0)3 88 41 70 02 [EMAIL PROTECTED] -Message d'origine- De : CCP4 bulletin board [mailto:[EMAIL PROTECTED] la part de Patrick Loll Envoye : Monday, March 03, 2008 6:00 PM A : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] radiation damage question Hi all, I had an interesting experience, and wonder if others have seen similar things. I was collecting data from a crystal that contains an iodinated macromolecule. After 2 days on a copper rotating anode, with the crystal at 100 K, we experienced a detector problem, so I put the crystal back into the dewar; it was diffracting nicely when I took it off. For various reasons, I didn't get back to this crystal until about 3 weeks later. When I put it back on the goniostat, the mother liquor was milky white in appearance. There were no ice rings, but alas the crystal only gave a few anemic spots around the beamstop. Annealing didn't help, and I noticed that when I blocked the cold stream, the milky white appearance didn't go away when the sample thawed. I finally took the crystal off and looked at it under a microscope, at which point I discovered that the milky white appearance was due to the presence of bubbles in the mother liquor. I seem to recall some talks on radiation damage in which people mention the evolution of a gas (H2?). So: Does this seem like a radiation damage phenomenon? And have others seen this kind of delay in the manifestation of damage during storage at liquid N2 temperatures? Thanks, Pat -- - Patrick J. Loll, Ph. D. (215) 762-7706 Professor FAX: (215) 762-4452 Department of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA [EMAIL PROTECTED]
[ccp4bb] Positions available in Australia
RESEARCH POSITION IN THE ROSSJOHN LABORATORY http://www.med.monash.edu.au/biochem/staff/rossjohn-lab.html The protein crystallography unit at Monash University, headed by Prof Jamie Rossjohn, ARC Federation Fellow, seeks two talented scientists to join his group. One position to work in collaboration with the ARC Centre of Excellence in Functional Microbial Genomics, http://www.microbialgenomics.net/, in the infection area of the laboratories’ research. The second position is to join a group looking into the structural basis of immune responses central to adaptive and innate immunity. Projects cover investigations of proteins involved in infectious diseases, and techniques of protein crystallography and molecular biology are used. The Research Fellows should have a PhD and knowledge of structural biology and protein crystallography. Appointments will be made at the level appropriate to the candidates’ qualifications and experience and in accordance with classification standards. Salary range: Level A $47472 - $64427. Level B $67818-$80535 Closing date 4th April, 2008. Inquiries to Margaret Bills, [EMAIL PROTECTED] Applications to: Reenu Johnson, [EMAIL PROTECTED] -- /Margaret Bills/ /Laboratory Manager/ /Protein Crystallography Unit/ /Department of Biochemistry and Molecular Biology/ /Monash University/ /Clayton/ /Victoria/ /Phone: 03 99058022/ [EMAIL PROTECTED]
Re: [ccp4bb] Crosslinking reagents
I used glutaraldehyde vapor by placing crystal drop above a bridge with 15% glutaradehyde for various amount of time. It worked for me for many crystals, but not for the one that I am trying now. The crystal didn't diffract any more. I intended to make the crystal tougher for heavy metal soaking. I am just wondering what will happen if I use some other crosslinking chemicals. Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 On Sun, Mar 2, 2008 at 12:28 AM, Nian Huang [EMAIL PROTECTED] wrote: Dear all, I know most people use glutaraldehyde as the crosslinking reagent for their crystals. But there are a lot of other crosslinking chemicals available both in protein chemistry and histology, such as simple formaldehyde. Did anybody have experience with other chemicals and how did they compared with glutaradehyde? I have a crystal very sensitive to glutaradehyde but I really wants to try the crosslinking method. Thanks, Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390
[ccp4bb] HIC-Update: Release 12.1 @ 2008-03-03 == 7,870 compounds
Dear structural(-ly interested) biologist ! HIC-Up, the Hetero-compound Information Centre - Uppsala, has been updated and now contains information on 7,870 hetero-entities. This is the first HIC-Up version after the release of the remediated PDB. A few changes had to be made to the underlying scripts and programs, but hopefully everything has been ironed out now. The compound pages now also contain links to ChEBI and [EMAIL PROTECTED] as well as to a new MSDmotif service that gives a PDB file for every instance of a ligand in the PDB along with all residues within 4.25A (plus a residue one each side; these PDB files are collected into one tar file for download). Links to PDBsum, ProCognate, PDBREPORT and ReLiBase have been updated. Please note that ReLiBase requires registration nowadays (free for academics, though). HIC-Up is available for local mirroring (follow the instructions in http://xray.bmc.uu.se/hicup/mirror.html). You can also download a single PDB file (ftp://xray.bmc.uu.se/pub/gerard/extras/hetero/hetero.pdb) that contains experimental coordinates for all the compounds. The URL for HIC-Up is: http://xray.bmc.uu.se/hicup For an example of the new HIC-Up pages, see the page for camphor, CAM, at URL http://xray.bmc.uu.se/hicup/CAM (or your own pet ligand's page) If you want to cite HIC-Up, you can use: Kleywegt, G.J. (2007). Crystallographic refinement of ligand complexes. Acta Cryst D63, 94-100. To request a reprint: http://xray.bmc.uu.se/cgi-bin/gerard/reprint_mailer.pl?pref=82 --dvd ** Gerard J. Kleywegt [Research Fellow of the Royal Swedish Academy of Sciences] Dept. of Cell Molecular Biology University of Uppsala Biomedical Centre Box 596 SE-751 24 Uppsala SWEDEN http://xray.bmc.uu.se/gerard/ mailto:[EMAIL PROTECTED] ** The opinions in this message are fictional. Any similarity to actual opinions, living or dead, is purely coincidental. ** ___ o-info mailing list (moderated) [EMAIL PROTECTED] http://o-info.bioxray.dk/mailman/listinfo/o-info
[ccp4bb] Copper Staining
Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
One down side is that copper sulfate is toxic to fish and plants. Please be good to the environment and do not pour copper sulfate solutions down the drain. We normally precipitate with NaOH, filter, and put the precipitated copper in an appropriate waste. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Tuesday, March 04, 2008 12:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Copper Staining Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
There's an even faster reagent for staining - takes abut 1 minute overall and the results look just like dear old Coomassie. This reagent works wonderfully for us. Yes, the recipe is proprietary - but if you add up the cost of reagents for normal Coomassie and compare - this one isn't much more expensive. http://www.novexin.com/products/InstantBlue.html Artem P.S. I am not affiliated with Novexin in any way shape or form, but their gel stain does kick butt. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Monday, March 03, 2008 6:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Copper Staining Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
Touche' Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Artem Evdokimov [EMAIL PROTECTED] To: 'Jacob Keller' [EMAIL PROTECTED]; CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 03, 2008 6:31 PM Subject: RE: [ccp4bb] Copper Staining There's an even faster reagent for staining - takes abut 1 minute overall and the results look just like dear old Coomassie. This reagent works wonderfully for us. Yes, the recipe is proprietary - but if you add up the cost of reagents for normal Coomassie and compare - this one isn't much more expensive. http://www.novexin.com/products/InstantBlue.html Artem P.S. I am not affiliated with Novexin in any way shape or form, but their gel stain does kick butt. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Monday, March 03, 2008 6:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Copper Staining Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
There's an even faster reagent for staining - takes abut 1 minute overall and the results look just like dear old Coomassie. This reagent works wonderfully for us. Yes, the recipe is proprietary - but if you add up the cost of reagents for normal Coomassie and compare - this one isn't much more expensive. http://www.novexin.com/products/InstantBlue.html Sounds like a good old Bradford reagent AKA colloidal Coomassie G-250. 60 mg G-250 dissolved in 50 ml ethanol and diluted into 1 L of 1/10 dilution of concentrated phosphoric acid (~8.5% final). Solubilizing agents is a smoke screen, for G-250 is fully soluble in ethanol and for the colloid particles to not penetrate the gel pores inclusion of any solubilizing agents will only be detrimental. Betcha it's a browish solution that forms blue pellet when centrifuged hard. If so, the claimed 5-10 ng sensitivity can only be achieved after 1) at least 15 min staining (30 min better; no one abolished laws of physics that requre diffusion into the gel for the interaction to take place), 2) some destaining to get rid of slight brown-blue background. Dima
[ccp4bb] superimpose two maps
Hello, I did the replacement soaking and it was successful. Now I want to superimpose the two maps to show (I hate to show off but I have to) the map diffrence of the ligand region to demonstrate that the replacement soaking was a success. Is there a way to superimpose the two maps? The two maps are from two different crystals with same space group but slightly different cell parameters. I know coot can superimpose NCS related maps and mapman can work on masks. I also know there are many 'map experts' out there in this list. Please help me. Thank you very much Yanming
Re: [ccp4bb] superimpose two maps
Yanming Zhang wrote: Hello, I did the replacement soaking and it was successful. Now I want to superimpose the two maps to show (I hate to show off but I have to) the map diffrence of the ligand region to demonstrate that the replacement soaking was a success. Is there a way to superimpose the two maps? The two maps are from two different crystals with same space group but slightly different cell parameters. If you want to use Coot, transform-map or transform-map-using-lsq-matrix are the functions you'd want to use. http://www.ysbl.york.ac.uk/~emsley/coot/doc/chapters/user-manual_6.html#SEC165 Paul.
