Re: [ccp4bb] Getting a copy of ESCET
Hi Mark - I am sure that Thomas will answer you soon, but: In the meantime I would like to take the opportunity to remind people to please be considerate and NOT post email addresses of yourselves, or most importantly of other people, to the bb, as you did today with Thomas. These feed all the spam engines. Some people (like Thomas) go to great lengths to not have their address on the web but only as an image, exactly for that reason, and would not be pleased to see it posted in public. Tassos
[ccp4bb] First Announcement: 7th International NCCR Symposium on New Trends in Structural Biology
Dear colleagues, it is our pleasure to announce the 7th International NCCR Symposium taking place this September. First Announcement: 7th INTERNATIONAL NCCR SYMPOSIUM ON NEW TRENDS IN STRUCTURAL BIOLOGY September 7 + 8, 2009 Lecture Hall HG F7, Swiss Federal Institute of Technology (ETH) Zurich, Switzerland This symposium brings together renowned scientists in structural biology from all over the world to exchange their knowledge and current research ideas in an interactive way. The symposium addresses latest developments and perspectives in the areas of membrane proteins, supramolecular assemblies interactions, as well as in the fields of related technologies. www.structuralbiology.uzh.ch/symposium2009 The registration slot opens end of March. Online registration will be possible directly from the above mentioned web site. We do hope that this conference is of interest to you and would be pleased to welcome you in Zurich this fall. With best regards, Patrick Sticher _ Visit the NCCR on the Internet www.structuralbiology.uzh.ch Dr. Patrick Sticher Moser NCCR Scientific Officer Institute of Biochemistry University of Zürich Winterthurerstrasse 190 CH - 8057 Zürich
Re: [ccp4bb] SUMMARY: Poor diffraction of eukaryotic membrane protein crystals
On request, this summary (slightly amended) has been posted to the CPP4 wiki Crystal Growth page. You can find it here: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality Regards, Damon. Damon Colbert schrieb: Thanks to everyone who responded with most helpful advice and suggestions. I have provided a summary of the suggestions (and clarifications to questions asked of me in return). Perma-Link to original question: _https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=CCP4BB;AJMLIg;20090205 170801%2B1300_ __ * Concentrate protein with a higher molecular weight cutoff (e.g. 50-100 kDa). * Protein is known to form a tetramer, and by approximation from gel filtration elution, exists as a 126 kDa species (~114 kDa tetramer and ~22 kDa OG micelle). It usually elutes as a single, well-resolved peak (unless, for example, I am using it to exchange detergent). DLS has shown monodispersity in samples, but I don't use it routinely. * Dialyse protein overnight (routinely or after centrifugal concentration) to reduce and define the detergent concentration. * This can get expensive, using relatively large volumes detergent to make the dialysis buffer. Nonetheless the most recent crystals were obtained from dialysed protein. * Trial extraction, purification, and crystallisation with different detergents (using desalting or Q-sepharose columns). Poor diffraction could be indicative of detergent-mediated crystal contacts (rather than protein-protein). * Use of shorter detergents (e.g. Cymal-3 to -6) or mixed detergent micelles * Reconstruct sparse matrix screens with each different detergent * See Lemieux /et al/. (2003), Protein Science. * Identify membrane lipids associated with protein (in-house by TLC or otherwise). Retaining some native lipid or adding it back in at crystallization may improve crystal quality. Conversely total delipidation may also help. * Need to correlate successful crystallisation with presence/absence of lipid * Could try using lipid-like detergents (FC or DHPC) * Deglycosylation is checked on SDS-PAGE, and confirmed by the loss of higher molecular weight smears (which are present before deglycosylation reaction). * Alternatively protein could be digested with Endolgycosidase H, which leaves one GlcNac residue on each glycosylation site. This could improve crystal contacts. See Chang, V.T. /et al/. (2007) Glycoprotein structural genomics: solving the glycosylation problem. Structure 15(3):267-73 * Chemical modification of surface residues may improve crystal contacts, for example lysine methylation. * See Walter /et al/. (2006) Lysine methylation as a routine rescue strategy for protein crystallization. Structure 14(11):1617-22 * Mutagenesis is another alternative, but we have not yet been successful producing a recombinant protein. * Adding salt (or PEG) to reservoir solution may promote crystal growth in the aqueous phase, rather than the 'oil/gel' phase. * Conditions producing the crystals grown in this 'gel' had PEG 1K or 2K as precipitant, and low [NaCl] present. (Is the suggestion 'to increase the concentration beyond that of the reservoir solution?'). * Test crystallisation conditions at low temperature (e.g. 4°C) * Test oils (paraffin or paraton-N) as cryoprotectants. Alternatively maintain detergent concentration in cryoprotectant. * Paratone oil (softened with some mineral oil) was used with poorly diffracting native crystals, and showed no improvement in diffraction. It has not been attempted with more recent protein crystals grown in presence of ligand. * Attempt to collect a 10Ang dataset and try MR with a close homolog. Many thanks. Regards, Damon.
Re: [ccp4bb] Issue on MOSFLM 7.0.4 with image collected on NOIR detector
hi folks I've had a look at Quentin's images, and have found out that some of the information that we are using in Mosflm to determine the pixel size and the beam centre is missing. For the record, if anyone else wants to process NOIR images with Mosflm and notices this problem, you should (for the present) tell Mosflm that the pixel size is 0.07767mm (same in both X Y) and also convert the beam centre from the image header from pixels to mm - while noting that the beam co-ordinates in the header are swapped in X Y compared to the Mosflm definition. Sorry for any inconvenience - we'll get this fixed for the next release! On 12 Feb 2009, at 21:08, Quentin Vicens wrote: Dear All, I used MOSFLM 7.0.4 to open some images originally collected on a NOIR-1 detector at the HHMI beamline at ALS. Problem #1: the resolution is not displayed accurately, even tough the reader seems to be read correctly (instead of being 1.28 in the attached example, it should be around 8 angstroems). Problem #2: autoindexing fails, which I now think may be related to problem #1. Thanks if you have any input! Quentin -- Quentin Vicens, Ph.D. University of Colorado Department of Chemistry and Biochemistry UCB 215 Boulder, CO 80309 USA tel: + 1 (303) 735-6338 fax: + 1 (303) 492-6194 e-mail: quentin.vic...@colorado.edu screen.tiff Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH
[ccp4bb] native gel
Dear all, I am wondering if anyone is working on Blue Natve PAGE or other alternate methods. I need some help to troubleshoot my Native PAGE experiments. It will be great if anyone can help me in this regard. I am working on membrane proteins which have pI around less than 7. i need to run Native PAGE for my samples which are purified with nonionic detergents. When i tried to run inhouse 4-16% gradient Native gel at running buffer pH of 8.9, the samples are getting stuck at the wells itself. I just found out that invitrogen Blue Natvie PAGE may be a good option but due to the cost if anyone can suggest me some other alternate methods, I will really appreciate it. Thanks in advance, regards, Jothi,
Re: [ccp4bb] native gel
Hi Jothi, You can make the blue native gel by yourself. It is quite cheap and easy to make. Other than the standard native PAGE supplies and some buffering reagents (if you want pH~7, imidazole will do), what you need in addition is commassie G-250. Please note, that commassie R-250, which you may find in your lab's protein gel stains, will not work for blue native gel (at lease didn't work for me). The reason probably is that R-250 has ethyl groups on the dye, making it more prone to irreversibly aggregate proteins.G-250 has methyl groups at the same places, so its complex with proteins are not so bad. There are some protocols on internet, also nature protocols has an article for blue native PAGE: http://www.nature.com/nprot/journal/v1/n1/full/nprot.2006.62.html In addition to these protocols, here are some of my suggestions: First, it is not always necessary to make gradient gels. 8~12% PAGE gels will usually work for regular sized proteins (20~200KD). Also it is not necessary to make discontinuous gels - meaning you can take the staking gel out. You might get a silightly broader band, but a continuous gel system (no staking gel, and the upper running buffer and loading buffer has same pH as the gel) is easier to design and you do not need to worry about artifacts caused by the compression effect. Another thing is, in some of the protocols, the authors suggested using water to make the G-250 stock solution. But you may find that this G-250 stock is actually a very poor suspension - so the final concentration in the upper buffer may never be what it is supposed to be. My solution is to make 0.5% EtOH or DMSO solutions of G-250, and add it 1:50 to the upper buffer to make 0.01% working concentration. The resulting concentration of alcohol or DMSO should not be high enough to harm most proteins. Finally, You may try using those SDS-PAGE protein ladders for the BN-PAGE as size marker. As to my experience, most bands of the fermentas PageRuler will run to approximately the same position as native proteins of the same size. Zhijie - Original Message - From: KN kn...@auckland.ac.nz To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, February 12, 2009 10:30 PM Subject: [ccp4bb] native gel Dear all, I am wondering if anyone is working on Blue Natve PAGE or other alternate methods. I need some help to troubleshoot my Native PAGE experiments. It will be great if anyone can help me in this regard. I am working on membrane proteins which have pI around less than 7. i need to run Native PAGE for my samples which are purified with nonionic detergents. When i tried to run inhouse 4-16% gradient Native gel at running buffer pH of 8.9, the samples are getting stuck at the wells itself. I just found out that invitrogen Blue Natvie PAGE may be a good option but due to the cost if anyone can suggest me some other alternate methods, I will really appreciate it. Thanks in advance, regards, Jothi,
