[ccp4bb] weak magnet of goniometer of MAR345dtb
Hi, We have recently installed a new home source diffraction system using MAR 345 dtb. It works much better than the rigaku because this new device has automatic motor control system. However, we have a lot of trouble to mount crystals. The magnet of MAR goniometer is significantly weak so the pins do not stick on. The old pins we used nicely in old rigaku are not working most of time. The new pins from hampton research particularly designed for ALS high-through put data collection are not easily working either. I wonder whether anyone has similar experience and it could be fixed by replacing with a stronger magnet base. Thanks for your suggestions in advance. Sincerely, Aidong
Re: [ccp4bb] Lipid Removal from Proteins
I dug around on the net and found this method to remove lipids from proteins: More precisely, from denatured proteins. That's what methanol/chloroform phase does for most proteins. Wessel Fluegge (1984), Anal. Biochem. 138:141-143. Itґs a methanol/ chloroform precipitation and gives you a pellet that is easily redissolved. The method was especially devised for removing lipids or detergents, so it should be perfect for you. -- http://www.bio.net/bionet/mm/methods/1996-December/052513.html By far the best method of concentration/desalting/de-lipidizing proteins for SDS gels. I've used it extensively over the years. Even then, the efficiency of precipitation drops off very significantly for most small proteins at low [protein]. Is this still the preferred way? I do not want to use reagents that are *themselves* likely to denature my protein. Has anyone tried cyclodextrins? Lots of people did. They work. So if you have protein that you can easily immobilize, washing the matrix extensively with b-cyclodextrin will do the trick. But immobilized cyclodextrins are not readily available for reasonable price. So for untagged protein your next bet would be various detergent removal sorbents available from Calbiochem, Pierce, Bio-Rad and likely many others. All of these WILL bind your protein to various extent, but usually not completely because they are also work as size exclusion. I'm specifically trying to strip sarcosyl. I want to do it completely. What's the definition of completely? If you are lucky and your protein binds to cation exchangers, simply washing the column with 20 CV of low salt buffer (even better with non-denaturing concentrations of alcohols or glycols) usually will decrease sarcosyl concentration by ~ 100X. Pretty much the same if your his-tagged protein is bound to IMAC sorbent. - Dima
Re: [ccp4bb] weak magnet of goniometer of MAR345dtb
Quoting aidong a...@xmu.edu.cn: Hi, We have recently installed a new home source diffraction system using MAR 345 dtb. It works much better than the rigaku because this new device has automatic motor control system. However, we have a lot of trouble to mount crystals. The magnet of MAR goniometer is significantly weak so the pins do not stick on. The old pins we used nicely in old rigaku are not working most of time. The new pins from hampton research particularly designed for ALS high-through put data collection are not easily working either. I wonder whether anyone has similar experience and it could be fixed by replacing with a stronger magnet base. Thanks for your suggestions in advance. Have you made sure that the electro-magnet for the goniometer is switched on and working. You can switch the magnet on and off on the control panel and a pin will not 'stick' without a green light here. This message was sent using IMP, the Internet Messaging Program.
Re: [ccp4bb] Is there Windows version of O ?
On Thu, 27 Aug 2009, lei feng wrote: maybe a little offtopic, but I really need anotehr model buiding software that can run on windows system ( I already have coot) May I ask why you need another modelling tool if you don't even know it? I found Alwyn Jones 's website, which has version 12 of the O software , but the win_ono.exe is a very small file. I am wondering is it only less than 3MB? I opened the file, it was not a typical windows interface software like win-coot. Am I missing something ? anyone used the windows version? You probably picked up the right file. The Linux version also only has 2.5MB. You need a lot a data-files, too, if you want to enjoy the program. You get what you need from the same ftp-server. But I suggest you find someone already familiar with O to start using it. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] Conference Membranes and Modules in Berlin
Dear crystallographers, we are organising a conference Membranes and Modules which takes place in Berlin from December 10 - 13, 2009. It covers a number of topics related to structural biology, macromolecular complexes, biophysics and biochemistry of membranes/membrane proteins and cellular dynamics, and might be of interest to many of you. We have an amazing speaker list, please have a look at our webpage (www.mam2009.de) and convince yourself. Hope to see you in Berlin in December (also for a Gluehwein on one of the christmas markets), Oli Daumke -- Dr. Oliver Daumke Max-Delbrück-Centrum for Molecular Medicine Crystallography Robert-Roessle-Strasse 10 13125 Berlin Tel.: 0049-309406-3425 Fax.: 0049-309406-3814
Re: [ccp4bb] Arpwarp not recognised by latest version of ccp4
Hi - There is no specific reason, to my knowledge, why ARP/wARP will not be recognized by the latest version of ccp4i. At worse, you can re-install the GUI from 'Install Task' from the ccp4i, which should still work. If you have any specific trouble/error messages, please do let me know. Best, Tassos On Aug 26, 2009, at 16:23, Sylvia Fanucchi wrote: Hi Does anyone know how to configure ArpWARP manually so that it will be recognized by the latest version of ccp4? Best regards Sylvia Fanucchi Ph.D Protein Structure-Function Research Unit East Campus, Gate House Room 416 School of Molecular and Cell Biology University of the Witwatersrand Johannesburg 2050 South Africa Tel: +27 (11) 717-6348 Fax: +27 (11) 717-6351 E-mail: sylvia.fanuc...@wits.ac.za This communication is intended for the addressee only. It is confidential. If you have received this communication in error, please notify us immediately and destroy the original message. You may not copy or disseminate this communication without the permission of the University. Only authorized signatories are competent to enter into agreements on behalf of the University and recipients are thus advised that the content of this message may not be legally binding on the University and may contain the personal views and opinions of the author, which are not necessarily the views and opinions of The University of the Witwatersrand, Johannesburg. All agreements between the University and outsiders are subject to South African Law unless the University agrees in writing to the contrary. P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
Re: [ccp4bb] Is there Windows version of O ?
Apropos to installing windows crystallography programs: Re: [ccp4bb] Computer hardware and OS survey Jon Wright Fri, 01 May 2009 10:24:28 -0700 Link,Todd M wrote: ... I did not find an equal web support page for Windows. It just isn't needed. If there is a windows version of a program you get to download, install, run and then get on with your life. Jon http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg10636.html But if for some reason everything is not manifestly self-evident, this page might help: http://alpha2.bmc.uu.se/~alwyn/O_to_Go/O_to_Go_frameset.html On Aug 28, 2009, at 1:16 AM, Tim Gruene wrote: On Thu, 27 Aug 2009, lei feng wrote: maybe a little offtopic, but I really need anotehr model buiding software that can run on windows system ( I already have coot) May I ask why you need another modelling tool if you don't even know it? I found Alwyn Jones 's website, which has version 12 of the O software , but the win_ono.exe is a very small file. I am wondering is it only less than 3MB? I opened the file, it was not a typical windows interface software like win-coot. Am I missing something ? anyone used the windows version? You probably picked up the right file. The Linux version also only has 2.5MB. You need a lot a data-files, too, if you want to enjoy the program. You get what you need from the same ftp-server. But I suggest you find someone already familiar with O to start using it. Tim
Re: [ccp4bb] Lipid Removal from Proteins
James, Dima is right. One of the protocols for protein-refolding with sarcosyl uses cyclodextrins to remove it. However, sarcosyl is an anionic detergent above pH 5.5 under most conditions, so any ion exchange platform should work well to markedly reduce its presence in a preparation. Also, it is extremely insoluble in the presence of divalent cations (as SDS is) and crystallizes readily. I probably wouldn't try removing it with an IMAC column as it will bind to some extent and lead to continued contamination (something we have seen). It is possible that hydroxyapatite column might be a good matrix for removing it. As sarcosyl is anionic, it is slower to dialyze across most membranes. However, we have had good results for detergent removal just using a standard spin-desalting column with G-25 Sephedex. It is not absolutely quantitative detergent removal, but 100x decreases are easily reached in less than 20 min. For sarcosyl, you could set up the column with G-25 DEAE-Sephedex at a pH where your protein doesn't bind and comes out in the flow-through. After 1-2 centrifugations, your protein should be free of sarcosyl. Regards, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: garav...@msu.edu On Aug 28, 2009, at 1:24 AM, James Stroud wrote: Hello All, I dug around on the net and found this method to remove lipids from proteins: Wessel Fluegge (1984), Anal. Biochem. 138:141-143. It´s a methanol/chloroform precipitation and gives you a pellet that is easily redissolved. The method was especially devised for removing lipids or detergents, so it should be perfect for you. -- http://www.bio.net/bionet/mm/methods/1996-December/052513.html Is this still the preferred way? I do not want to use reagents that are *themselves* likely to denature my protein. Has anyone tried cyclodextrins? I'm specifically trying to strip sarcosyl. I want to do it completely. James
[ccp4bb] Postdoctoral position, Karolinska Institute, Sweden
*Postdoctoral position in molecular and structural biology of protein complexes in nerve development and disease* Dept. of Medical Biochemistry and Biophysics, Karolinska Institute A postdoctoral position is available immediately in the Molecular Structural Biology group at the Karolinska Institute in Stockholm (http://phillips.mbb.ki.se/). The position is initially available for 2 years with the possibility of extension to three years. We are seeking a motivated scientist to join a new research project on structural and functional studies of protein-protein complexes involved in nerve cell development and disease. The project focuses on the characterization of the complexes and their function in the cell. The experiments will involve a broad range of techniques, such as protein expression/purification, site-directed mutagenesis, enzyme kinetics, and various methods of structure determination (X-ray crystallography, EM, SAXS). Applicants should have a background in biochemistry or molecular biology, and possess experience with cloning, protein expression/purification techniques. Biochemists and molecular biologists who would like to learn structural biology are encouraged to apply. Experience in mammalian cell culturing is a plus. Interested candidates should send a cover letter including CV and names and e-mail addresses of three references before the 15th September 2009 to: Dr. Bernhard Lohkamp Div. of Molecular Structural Biology Dept. of Medical Biochemistry and Biophysics Karolinska Institutet SE-17177 Stockholm Sweden e-mail: Bernhard.Lohkamp(at)ki.se, phone: +46 8 5248 7673, fax: +46 8 32 7626
[ccp4bb] Rfactor got stuck with a data having alternate strong and weak reflections.
Dear all: I am currently refining a structure solved by MAD and somehow the R factor got stuck around 30% with 2.2 resolution. There are four molecules in one ASU. Two had very good density map and the other two were not equally good. I tried using NCS during refinement but it did not help much. Then I checked my data. Actually I found that there are alternate layers of strong and weak reflections. THe crystal is in a thin-plate shape with orthorombic space group. Then I looked at my molecular replacement solution from Phaser using my native data. Actually phaser gave two sets of solutions, which showed slightly different positions. You can also see that there is a translation inside the same set of solution. SOLU SET RFZ=12.8 TFZ=21.4 PAK=0 LLG=452 RFZ=10.7 TFZ=47.9 PAK=0 LLG=1693 RFZ=13.0 TFZ=47.6 PAK=0 LLG=2791 RFZ=10.7 TFZ=46.1 PAK=0 LLG=4045 SOLU 6DIM ENSE ensemble1 EULER 184.0520.185 175.770 FRAC -0.49889 -0.00218 -0.0 SOLU 6DIM ENSE ensemble1 EULER 225.1160.167 134.696 FRAC -0.47056 0.49706 0.00051 SOLU 6DIM ENSE ensemble1 EULER 359.333 31.677 180.633 FRAC 0.75769 -0.71475 -0.14004 SOLU 6DIM ENSE ensemble1 EULER 359.373 31.969 180.711 FRAC 0.73074 -0.21423 -0.14108 SOLU SET RFZ=12.8 TFZ=21.4 PAK=0 LLG=452 RFZ=10.7 TFZ=47.9 PAK=0 LLG=1693 RFZ=13.0 TFZ=47.6 PAK=0 LLG=2791 RFZ=10.7 TFZ=47.3 PAK=0 LLG=4042 SOLU 6DIM ENSE ensemble1 EULER 213.1150.173 146.741 FRAC -0.49931 -0.00269 0.00045 SOLU 6DIM ENSE ensemble1 EULER 248.1730.254 111.665 FRAC -0.47091 0.49661 0.00101 SOLU 6DIM ENSE ensemble1 EULER 359.399 31.602 180.578 FRAC 0.75808 -0.71455 -0.13980 SOLU 6DIM ENSE ensemble1 EULER 359.378 31.255 180.361 FRAC 0.78370 -0.21555 -0.13830 I remember there is a discussion in CCP4bb about the same topic with the focus of pseudo-symmetry or translational pseudo-symmetry. Can anybody give some troubleshooting about my issue? Thanks a lot and have a nice weekend, Jerry McCully _ Windows Live: Keep your friends up to date with what you do online. http://windowslive.com/Campaign/SocialNetworking?ocid=PID23285::T:WLMTAGL:ON:WL:en-US:SI_SB_online:082009
[ccp4bb] Problem with Coot reading monomer library file.
Dear Crystallographers, I've been spending 10 hours trying (googling, manually editting cif files based on templates in Coot's library, asking around, rtfm and reading this bbs) to figure out why Coot the geometric restraints wouldn't load. The molecule I have is difluoroacetate (and some analogs). I drew it in sketcher and tried regularized it using both the refmac from sketcher as well as the 'normal' refmac. From what I can tell, Coot can read this cif file without errors, but when it keeps telling me that it doesn't have the library descriptions when I try to do real space refinement. My system and programs: -Windows Vista 32 bit Home -Coot 0.5 as well as 0.6 -CCP4i 6.1.1 Out of desperation, I also tested this on Coot v0.1 which has worked for me before... and I was surprised that I can do real space refinement without problem. Could there be some settings that I've missed? Any insights would be greatly appreciated. Peter _ Stay in the loop and chat with friends, right from your inbox! http://go.microsoft.com/?linkid=9671354
