[ccp4bb] SHELX reference
Since SHELX is frequently cited incorrectly, after some arm-twisting by the Acta Cryst. editors I have written a paper: "A short history of SHELX". Sheldrick, G.M. (2008). Acta Cryst. A64, 112-122 which may be safely cited whenever any program with a name beginning with "SHELX..." is used. I do not expect anyone to actually read it, but for those who wish to do so, it is available as "Open Access" at: http://journals.iucr.org/a/issues/2008/01/00/issconts.html George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-2582
Re: [ccp4bb] Twinned crystals to single crystal
You can try e.g. - micro seeding - change MPD concentration (lower MPD especially with seeding) - scan the pH range - change protein concentration (lower protein conc. especially with seeding) - try different temperatures (4 degrees, 20 degrees, higher, if your protein supports it) - add another purification step - collect data and check whether you can solve the structure with the twinned data - carry out restricted proteolysis or slightly modify the protein (expression system, purification tags ...) For your second question, you can use small volume concentrators to check the effect of - different salts - different salt concentrations - different buffers - different pH - additives, especially reducing agents like DTT, beta-ME - addition of, say, 5% glycerol I am sure there are a lot more possibilities. If you explain a little more what you have already done, we might give even more concise answers. Cheers, Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Fri, 21 Dec 2007, shivesh kumar wrote: Dear all, I have crystallized a protein in 63% of MPD at pH 3.8-4.6.The crystals are twinned but with sharp edges.I welcome all the suggestions to get single crystals.Also,I have needles of other mutant protein,the problem is during concentration,it gets precipitated even in the centricon.What should I do to avoid this precipitation to have a concentrated protein.Thanx in advance for the suggestions. Shivesh
[ccp4bb] Twinned crystals to single crystal
Dear all, I have crystallized a protein in 63% of MPD at pH 3.8-4.6.The crystals are twinned but with sharp edges.I welcome all the suggestions to get single crystals.Also,I have needles of other mutant protein,the problem is during concentration,it gets precipitated even in the centricon.What should I do to avoid this precipitation to have a concentrated protein.Thanx in advance for the suggestions. Shivesh
[ccp4bb] Postdoc position at Genentech
Dear all, A position is available for a Postdoctoral researcher to join the crystallography group at Genentech, Inc. The group explores structural and functional aspects of proteins with scientific and/or therapeutic interest. The successful candidate will be involved in all aspects of crystallography, including the cloning and expression and purification of proteins, crystallization, data collection, structure solution and the analysis of protein structures. Job Qualifications: Highly motivated researchers, capable of independent work in a collaborative environment are sought for this position. The applicant must be familiar with the principles of protein crystallography and structure determination, in addition experience in molecular biology and protein chemistry is required. If you are interested in joining our team at Genentech, please submit a CV and cover letter referring to position 120834 at www.gene.com/careers. For additional information please contact Amber Welch [EMAIL PROTECTED] Christian Wiesmann, Ph.D. Senior Scientist Department of Protein Engineering Genentech, Inc. 1 DNA Way South San Francisco, CA 94080 E-mail: [EMAIL PROTECTED] Phone:(650)225-7484 Fax: (650)225-3734
[ccp4bb] detergent concentration used for membrane protein purification
Dear All sorry for a non-ccp4 related question. what is the typical concentration of detergents used in membrane protein purification? Is it usually within 1-10 times of CMC? My collaborator was using 10% DDM in purifying a membrane protein. I suggested to reduce it and now he is working with 2% DDM. He said lower than that the protein is not soluble. so another question is, can we purify the membrane protein first at high concentration of one detergent (say, 2% DDM), and then screen different detergents later to find a better one? Or it is better to screen the detergents first before purification? any suggestions will be appreciated. Have a nice holiday! Rongjin Guan
Re: [ccp4bb] detergent concentration used for membrane protein purification
Hi Rongjin, During extraction from the membrane, typically 10xCMC (e.g. FC12) to 100xCMC (e.g. DDM) may be used, depending on target under study. During purification, it is recommended to bring this down to ~2xCMC. Theoretically, 1xCMC should be ok to keep the protein soluble. In practice, it is difficult to get exactly 1xCMC in solution as the CMC value will fluctuate depending on salt in the buffer, solution temperature, inaccuracies in mass measurement when making the detergent solution, etc. Hence it is preferable to stay ~2xCMC. If you then use a 100K concentrator post purification, you should be fine. I have had a couple of cases when the protein seemed unstable at what we thought as 1xCMC but was ok at 2xCMC. Yes, you can purify the protein first at a high conc of one detergent, and then try detergent exchange during a SEC step into different detergents prior to crystallization. The problem with this is that detergent exchange is dependent on CMC value. If CMC value is very low, it is difficult to do a detergent exchange since micelles will form even at very low concentrations which may be difficult to get rid of. So, if you want to go this route, better to keep in mind the CMC value and proceed accordingly. It would also be ok to screen detergents at the extraction step and find the one that solubilizes/stabilizes best and then keep the same detergent throughout until crystallization. Since so much remains unknown about membrane proteins and best detergents, it is probably better to try both approaches and see what works. Strategy will depend only on available resources since most of the detergents are very expensive. Since a lot more detergent is required during extraction, it may be more economical to extract with an inexpensive detergent and then exchange into others during purification. On the other hand, since doing a test to see best or optimal detergents for extraction will require only lysing small amounts of cell paste, that will cut down on expense of screening during extraction, so that may work out fine as well. As long as you have a nice efficient protocol for screening worked out, you can go for both. A couple of years ago, I was involved in setting up a fast screening protocol for doing this, which we presented in a poster at an NIH PSI Protein Production Workshop. It contains a flowchart which can get you going. It can help to screen a variety of detergents in about 72 hours. Let me know if you want a copy, I'll have to look for it, but can send it to you. Regards, Debanu. -- Debanu Das, SSRL. From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Rongjin Guan Sent: Friday, December 21, 2007 11:09 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] detergent concentration used for membrane protein purification Dear All sorry for a non-ccp4 related question. what is the typical concentration of detergents used in membrane protein purification? Is it usually within 1-10 times of CMC? My collaborator was using 10% DDM in purifying a membrane protein. I suggested to reduce it and now he is working with 2% DDM. He said lower than that the protein is not soluble. so another question is, can we purify the membrane protein first at high concentration of one detergent (say, 2% DDM), and then screen different detergents later to find a better one? Or it is better to screen the detergents first before purification? any suggestions will be appreciated. Have a nice holiday! Rongjin Guan
Re: [ccp4bb] detergent concentration used for membrane protein purification
Hi Rongjin, the concentration (and total amount) of detergent during purification depends a lot on what stage of purification you're at. For membrane extraction (1st step), people typically use anything from 1-5%. This depends on your budget, on the cmc of the detergent, and of course on the amount of cells you have. b-OG, having a high cmc (~0.7%) is not very effective at 1%, so you have to use a higher concentration. 1% is often enough for things like DDM and LDAO. My lab uses typically 100 ml of 1% detergent to extract membrane proteins from 12 l Ecoli culture (so 1 g detergent total). Later on in purification, when the amounts of stabilizing endogenous lipids decrease, it makes sense to lower the detergent concentration as too high concentrations are often denaturing. I would not go lower than ~ 2-fold cmc (it is hard to know what the actual cmc is in your buffer). In the case of DDM, we do our 1st purification step in 0.2% DDM (Ni-column) and subsequent steps in 0.03-0.05% DDM (approx. 2x cmc). 2% DDM for purification seems excessive and will very likely not allow crystallization (if you're attempting that). Purification in one detergent followed by exchange later is perfectly fine. However, it seems your protein is not very happy in DDM, so I think it would make sense to start off (extraction) from a different detergent. Cheers, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm From: CCP4 bulletin board on behalf of Rongjin Guan Sent: Fri 12/21/2007 2:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] detergent concentration used for membrane protein purification Dear All sorry for a non-ccp4 related question. what is the typical concentration of detergents used in membrane protein purification? Is it usually within 1-10 times of CMC? My collaborator was using 10% DDM in purifying a membrane protein. I suggested to reduce it and now he is working with 2% DDM. He said lower than that the protein is not soluble. so another question is, can we purify the membrane protein first at high concentration of one detergent (say, 2% DDM), and then screen different detergents later to find a better one? Or it is better to screen the detergents first before purification? any suggestions will be appreciated. Have a nice holiday! Rongjin Guan
[ccp4bb] Visualization system for a Mosquito Robot
Hi all, sorry for the off topic post but we are in the market for a visualization system compatible with the Mosquito crystallization robot. We were just wondering what system, if any, do other crystallographers use as well as any experiences you may have had with it. Thanks for the input and the help! Adam
[ccp4bb] Problem again with mosflm
Sorry folks, but I have been very frustrated to get mosflm and imosflm to work. I have get around with X11, but I got another problem, >> CCP4 library signal ccp4_general:Cannot open environ.def (Success) raised in ccp4fyp << ipmosflm_universal: Cannot open environ.def ipmosflm_universal: Cannot open environ.def Times: User: 0.0s System:0.0s Elapsed: 0:00 I do have put environ.def into the folder where my mosflm resides,see below, drwxr-xr-x16 mnelson mnelson 544 Dec 21 21:59 . drwxr-xr-x14 mnelson mnelson 476 Dec 21 21:44 .. -rw-r--r-- 1 mnelson mnelson 6148 Dec 21 21:59 .DS_Store -rwsrwsrwt 1 mnelson mnelson 0 Dec 21 14:19 SUMMARY drwsrwsrwt55 mnelson mnelson 1870 Dec 20 23:19 bitmaps drwsrwsrwt11 mnelson mnelson 374 Aug 20 11:14 c -rwxr-xr-x 1 mnelson mnelson 3140 Dec 16 2004 default.def -rwxr-xr-x 1 mnelson mnelson 7586 Dec 16 2004 environ.def -rwsrwsrwt 1 mnelson mnelson 5407684 Dec 20 17:10 ipmosflm -rwsrwsrwt 1 mnelson mnelson 7141312 Dec 20 17:10 ipmosflm_osx_intel -rwsrwsrwt 1 mnelson mnelson 13948864 Dec 20 17:24 ipmosflm_universal drwsrwsrwt11 mnelson mnelson 374 Dec 21 21:59 lib -rwsrwsrwt 1 mnelson mnelson 690 Dec 21 14:19 mosflm.lp -rwsrwsrwt 1 mnelson mnelson 13947260 Dec 20 16:00 mosflm_osx_universal drwsrwsrwt 109 mnelson mnelson 3706 Dec 21 14:20 src -rwsrwsrwt 1 mnelson mnelson323544 Dec 6 2005 syminfo.lib I have been screwed up by this for a whole afternoon. Can anybody save me out of this heck. By the way, my machine is an intel mac with mac os 10.4.11 Thanks! Mike Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. http://mobile.yahoo.com/;_ylt=Ahu06i62sR8HDtDypao8Wcj9tAcJ
Re: [ccp4bb] Problem again with mosflm
Are your environment variables like $CINCL and $CCP4 defined? These usually get set up by sourcing the $CCP4/include/ccp4-setup.X file appropriate for your shell. michael nelson wrote: > Sorry folks, but I have been very frustrated to get > mosflm and imosflm to work. > I have get around with X11, but I got another problem, >>> CCP4 library signal ccp4_general:Cannot open > environ.def (Success) > raised in ccp4fyp << > ipmosflm_universal: Cannot open environ.def > ipmosflm_universal: Cannot open environ.def > Times: User: 0.0s System:0.0s Elapsed: > 0:00 > > I do have put environ.def into the folder where my > mosflm resides,see below, > drwxr-xr-x16 mnelson mnelson 544 Dec 21 > 21:59 . > drwxr-xr-x14 mnelson mnelson 476 Dec 21 > 21:44 .. > -rw-r--r-- 1 mnelson mnelson 6148 Dec 21 > 21:59 .DS_Store > -rwsrwsrwt 1 mnelson mnelson 0 Dec 21 > 14:19 SUMMARY > drwsrwsrwt55 mnelson mnelson 1870 Dec 20 > 23:19 bitmaps > drwsrwsrwt11 mnelson mnelson 374 Aug 20 > 11:14 c > -rwxr-xr-x 1 mnelson mnelson 3140 Dec 16 > 2004 default.def > -rwxr-xr-x 1 mnelson mnelson 7586 Dec 16 > 2004 environ.def > -rwsrwsrwt 1 mnelson mnelson 5407684 Dec 20 > 17:10 ipmosflm > -rwsrwsrwt 1 mnelson mnelson 7141312 Dec 20 > 17:10 ipmosflm_osx_intel > -rwsrwsrwt 1 mnelson mnelson 13948864 Dec 20 > 17:24 ipmosflm_universal > drwsrwsrwt11 mnelson mnelson 374 Dec 21 > 21:59 lib > -rwsrwsrwt 1 mnelson mnelson 690 Dec 21 > 14:19 mosflm.lp > -rwsrwsrwt 1 mnelson mnelson 13947260 Dec 20 > 16:00 mosflm_osx_universal > drwsrwsrwt 109 mnelson mnelson 3706 Dec 21 > 14:20 src > -rwsrwsrwt 1 mnelson mnelson323544 Dec 6 > 2005 syminfo.lib > > > I have been screwed up by this for a whole afternoon. > Can anybody save me out of this heck. > By the way, my machine is an intel mac with mac os > 10.4.11 > Thanks! > Mike > > > > > Be a better friend, newshound, and > know-it-all with Yahoo! Mobile. Try it now. > http://mobile.yahoo.com/;_ylt=Ahu06i62sR8HDtDypao8Wcj9tAcJ >
