[ccp4bb] A SAXS REFINEMENT PUZZLE
We have collected SAXS curves for two multiprotein complexes (C1 and C2) where each complex is a specific combination of two kinds of Protomers (P1 and P2). P1 and P2 are well-folded structures and their atomic coordinates can be treated as rigid bodies. Both C1 and C2 can be thought of as a string of protomers from N- to C-terminus, where adjacent protomers are connected by flexible linkers of 15 amino acids. The sequence of protomers in C1 and C2 are P1P1P2P1P1 and P1P2P1 respectively. C2 is a subset of C1 in solution. Additionally we have collected a third SAXS curve of multiprotein complex3 (C3) that is composed of two sets of C2 (C2 forms stable dimers under certain conditions). There are no linker restrains between the two sets of C2 in C3 as the interaction between the sets is purely electrostatic in solution. Taken together, C2 is a subset of both C1 and C3. The structural arrangement of the protomers in C2 is preserved in C1 and C3. Since there is a presumed C2 commonality between the three multiprotein complexes, we consider it best to refine C1, C2 and C3 simultaneously as it provides cross validation across the incremental steps of the refinement process. Our program of choice so far is CORAL. We were successful in refining C1 and C2 simultaneously. The PDB of C1 (P1P1P2P1P1) was represented as a single chain and the middle constituents (P1P2P1) between the flanking P1 protomers were defined as C2. In this instance, the input files were a single PDB and two SAXS curves of C1 and C2. We are at a loss for a method to incorporate the SAXS curve of C3. One possibility is to somehow define C3 as two sets of C2 and note the absence of linker constrains between the sets, in which case the input for refinement will consist of the original PDB and 3 SAXS curves. Another possibility is to represent C3 in a separate PDB where each set of C2 is specified in a discrete chain, in which case the input for refinement will consist of two PDBs and 3 SAXS curves. In any case we lack the CORAL-proficiency in testing these possibilities. All help is welcome and most appreciated. Amongst other things, we have no knowledge of the symmetries in the above complexes. Kratky analysis along with a variety of biophysical and biochemical data assures us of the structural stability and the stoichiometry of the multiprotein complexes. -- Balendu Avvaru Postdoctoral Fellow Cytoskeletal dynamics and Motility Laboratory of Structural Enzymology and Biochemistry (LEBS) CNRS Bâtiment 34, Avenue de la Terrasse FRANCE 91198
Re: [ccp4bb] Improving microcrystals
I've had some success in the past following Terese Bergfors' advice: http://xray.bmc.uu.se/terese/tutorial3.html However, the last time I dealt with spherulites I just tuned the pH and it worked like a charm. Good luck, Jon 2012/8/25 RHYS GRINTER r.grinte...@research.gla.ac.uk Hi Samuel, I've has good success going from sphereulites to crystals using an additive screen (the 96 condition Hampton one is good) with the conditions giving the spherulites. Just watch for salt crystals as you'll be adding some compounds that might cause your Ca ions to form insoluble CaSO4. http://hamptonresearch.com/product_detail.aspx?cid=1sid=36pid=27 Cheers, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Samuel Johnson [samueljohnson...@yahoo.in] Sent: 25 August 2012 02:11 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Improving microcrystals Hi everyone, I have been working on a protein for the past year. After a number of trials at crystallizing the protein i have identified conditions for getting spherulites/micro-crystaline material under micro batch method. I have confirmed that the crystalline material is protein, by using Izit-dye test. The condition is 50mM CaCl2, Mes pH 6.5 and 40% PEG 400. I will be happy to get suggestions on improving conditions to obtain single crystals. I have already tried varying a number of parameters like salt, precipitant concentration and buffer pH but that didn't help. Thanks. -- Jon Agirre, PhD Unit of Biophysics (CSIC-UPV/EHU) http://www.ehu.es/jon.agirre http://sourceforge.net/projects/projectrecon/ +34656756888
[ccp4bb] Dear ccp4
Dear CCp4 Thank you for all your and information suggestions regarding question Best Regards Rana
[ccp4bb] Small Molecule Effector Discovery for Hemoglobin
--Small Molecule Effector Discovery for Hemoglobin-- We seek an experienced scientist to join our multidisciplinary program directed at the discovery of new small molecule effectors of hemoglobin function. The successful candidate must have documented experience in hemoglobin crystallization and high resolution X-ray structure determination. The ability to communicate with computational chemists and medicinal chemists is also critical. We seek a Ph.D. level scientist, but exceptional candidates at all levels will be considered. Please send a CV and the names of 2-3 references to steve.almo(at)einstein.yu.edu.
Re: [ccp4bb] Off-topic: Duet vectors with C-terminal GST tag
How about just co-transfecting with two different plasmids which have different selection markers? I've done it a lot, and it seems to work fine... JPK On Fri, Aug 24, 2012 at 10:39 PM, Lye, Ming ming_...@hms.harvard.eduwrote: Dear CCP4bb, We would like to co-express proteins under Se-Met conditions for de-novo phasing of a complex. One of the proteins expresses much better with a GST-tag compared with a his-tag. As its binding site is at its N-terminus, we are hoping to co-express it as a C-terminal GST tag recombinant. So far however, we haven't found any commercially available duet vectors with such a tag. We would truly appreciate if anyone knows of the availability of such vectors, or has any suggestions on similar vectors. Thanks so much! Ming -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Off-topic: Duet vectors with C-terminal GST tag
Hi Jacob, Thanks for your message! I was thinking about that too, but I wasn't sure if it was something that just sounded good in theory.. I guess the two different vectors should have different antibiotic resistance to select for the 'co-transformation.' Thanks so much! Ming From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, August 27, 2012 9:31 AM To: Lye, Ming Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Off-topic: Duet vectors with C-terminal GST tag How about just co-transfecting with two different plasmids which have different selection markers? I've done it a lot, and it seems to work fine... JPK On Fri, Aug 24, 2012 at 10:39 PM, Lye, Ming ming_...@hms.harvard.edumailto:ming_...@hms.harvard.edu wrote: Dear CCP4bb, We would like to co-express proteins under Se-Met conditions for de-novo phasing of a complex. One of the proteins expresses much better with a GST-tag compared with a his-tag. As its binding site is at its N-terminus, we are hoping to co-express it as a C-terminal GST tag recombinant. So far however, we haven't found any commercially available duet vectors with such a tag. We would truly appreciate if anyone knows of the availability of such vectors, or has any suggestions on similar vectors. Thanks so much! Ming -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] sealing tapes
Hello Flip, I saw your post on the ccp4bb about UV transparent seals. Is it possible for you to send me the compilation that formulatrix has put together Thanks Hari Constellation Pharmaceuticals On Mon, Mar 7, 2011 at 4:41 AM, Flip Hoedemaeker f...@formulatrix.comwrote: Hi Jean-Luc, We have complied a list of UV compatible cover media and plates. The list is by all means not complete yet, but the best seals we found are all sheets (that does not mean all sheets are UV compatible!). If you want I can send you a PDF file outside of the BB. Flip On 3/7/2011 10:31, ferrer wrote: Dear all, Sorry for this slightly off-topic email, but I am looking for a transparent sealing tape for 96-well crystallization plates, with the following properties: - high sealing performances - compatible with UV screening - optionally, available as rolls Thanks for your help JL
Re: [ccp4bb] Improving microcrystals
Samuel Clearly you should try the rMMS random microseeding approach where you add a seed stock made from the spherulites to a random screen. See refs: Acta Crystallographica section D63 (2007), 550–554. On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652 Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442. http://www.douglas.co.uk/SER-CAT09_1.html On 25 August 2012 02:11, Samuel Johnson samueljohnson...@yahoo.in wrote: Hi everyone, I have been working on a protein for the past year. After a number of trials at crystallizing the protein i have identified conditions for getting spherulites/micro-crystaline material under micro batch method. I have confirmed that the crystalline material is protein, by using Izit-dye test. The condition is 50mM CaCl2, Mes pH 6.5 and 40% PEG 400. I will be happy to get suggestions on improving conditions to obtain single crystals. I have already tried varying a number of parameters like salt, precipitant concentration and buffer pH but that didn't help. Thanks. -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] Postdoctoral position
Dear all, I have an exciting postdoctoral position opened in my lab, available immediately. Please find attached the description and if possible, please forward to interested candidates. Best wishes, Svetla Svetla Stoilova-McPhie, PhD Assistant Professor, Department of Neuroscience and Cell Biology Scientist, Sealy Centre for Structural Biology and Molecular Biophysics University of Texas Medical Branch at Galveston 301 University Boulevard, Galveston, Texas 77555-0620 Lab: (+1) 409-747-2159 Cell: (+1) 979-319-1349 Fax: (1+) 409-747-2200 Email: svmcp...@utmb.edu www.svetla-mcphie-cryoem.com Postdoctoral Position 2012 - SCSB-UTMB.doc Description: Postdoctoral Position 2012 - SCSB-UTMB.doc
[ccp4bb] Meeting Announcement for the 4th Roadmap Meeting for Membrane Protein Structures and Complexes
*Meeting Announcement for the 4th Roadmap Meeting for Membrane Protein Structures and Complexes* The 4th NIH Roadmap meeting for Membrane Protein Structures and Complexes will be held at the San Francisco Westin Hotel in downtown San Francisco on Wednesday November 28th 2012, 8:00 AM through Friday, November 30th, noon. This Meeting will be hosted by the Membrane Protein Expression Centerhttp://mpec.ucsf.edu/, one of the centers funded by the NIH Common Fund Structural Biology Programhttp://commonfund.nih.gov/structuralbiology/index.aspx, organized by Dr. Robert Stroud, UCSF. This series of meetings have been very successful in generating open exchange and sharing of technologies, materials, and ideas focused on targeting membrane protein structure determination. This 4th meeting in the series provides a unique opportunity for all investigators engaged in developing and applying new technologies for structural biology of membrane proteins and complexes to focus on technologies that facilitate membrane protein structure determination, and recent structures of membrane proteins. This meeting will include all investigators supported by the NIH common fund program for structural biology, and invites all PSI member membrane protein groups, and all investigators of membrane protein structure to participate fully. Presentations and discussions chosen from submitted abstracts will focus on progress as well as overcoming barriers that could transform functional expression of membrane proteins and complexes, and structure determination at atomic resolution. Corporate participation is encouraged for those companies who are actively working on membrane protein targets, and those producing equipment and reagents for membrane protein research. Corporate involvement in abstract submission, presentations or display for the meeting is encouraged. Industry participation is encouraged for companies who are actively working on membrane protein targets, or producing equipment and reagents for membrane protein research. Meeting sponsorships for industry partners are available. Hands-on workshops covering technologies such as robotic Lipidic Cubic Phase (LCP) crystallization methods, the latest LCP crystallization experiment visualization instrumentation, gene design for optimized protein expression, high throughput thermostability assay of protein stability for protein purification and crystallization, tetra-detector characterization of membrane proteins, and more are planned for November 27, 2012, the day before the main meeting. Please contact Suzan Betheil at rmi2...@msg.ucsf.edu for more information.
