[ccp4bb] 9th International NCCR Symposium on New Trends in Structural Biology - Registration now open
Dear colleagues, please be informed that the registration slot for the 9th International NCCR Symposium on New Trends in Structural Biology 1 + 2 September 2011, University of Zurich, Irchel Campus, Lecture Hall Y24-G-45, Zurich, Switzerland is now open. Online registration is possible directly from the symposium website: http://www.structuralbiology.uzh.ch/symposium2011 where you will also find further information about this event. Confirmed plenary lecturer to date: Jamie Cate, James J. Chou, Vadim Cherezov, Jennifer Doudna, Youxing Jiang, Alan E. Mark, Tom Muir Please do not hesitate to contact us anytime if you need further information (stic...@bioc.uzh.ch). With best regards, Patrick Sticher The NCCR Structural Biology is a research initiative of the Swiss Science Foundation. Its research encompasses the fields of recombinant protein technologies, macromolecular structure determination and computational biomolecular sciences with a special focus on membrane proteins and supramolecular assemblies/interactions. 14 research groups from Swiss Universities and Research Institutions participate in this network. www.structuralbiology.uzh.ch/ -- Dr. Patrick Sticher Scientific Officer NCCR Strucutral Biology Institute of Biochemistry University of Zurich Winterthurerstrasse 190 CH - 8057 Zurich Switzerland
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi folks, crystallized a lot of SRP RNAs - never had success with synthetic RNA, even with small 30mers T7 RNA pol in vitro transcribed was always extremely better - moreover: I always use a 3' hammerhead which yields in the end 100% pure material in mg amounts - Kiyoshi Nagais protocol is a perfect basis. Greetings Klemens Martin Hällberg wrote: Hi, I'll second Israels's comment. Since the yield per coupling in synthesis is lower for RNA than for DNA it gets really expensive over 30-35 nucleotides. However, you can stitch together several 30 nt oligos using either T4 RNA ligase or T4 DNA ligase (with a DNA splint). Regarding suppliers of synthetic RNA, Dharmacon is still very reliable in terms of actual delivered amount and quality. If you are going for very large scale then oligofactory.com is a good choice. For a 90 nt oligo without any modifications I'd definitely go for T7 in-vitro transcription. On top of Nagai's classic that Israel referred to, I can also recommend the more recent method of native purification developed by Robert Batey and Jeffrey Kieft. See: Batey, R.T. Kieft, J.S. Improved native affinity purification of RNA (2007) RNA, 13, 1384-1389. You are going to need milligrams of T7 RNA polymerase in the end so forget about transcription kits, make your own. Cheers, Martin On Mar 13, 2011, at 10:16 AM, Israel Sanchez wrote: Hi Michael, we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. Hope it helps J Mol Biol. 1995 Jun 2;249(2):398-408. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Price SR, Ito N, Oubridge C, Avis JM, Nagai K. MRC Laboratory of Molecular Biology, Cambridge, UK. Abstract In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. 2011/3/13 Michael Thompson mi...@chem.ucla.edu Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] Reporting average B values for solvent, chain, ligand etc
Dear CCP4BB Thank you to everyone who sent me answers to my problem. I obtained the average B factors using Baverage. It was also suggest that I could have used act, a script from the CCP4 homepage, MOLEMAN, phenix.model_vs_data or copying the data into Excel using the paste special option and then calculating it manually. Thanks again James
Re: [ccp4bb] Psedo-translation detected
On 03/12/2011 05:29 AM, Vandu Murugan wrote: Dear all, Recently I collected a data for my 20Kda protein in C2 space group, and ran SFcheck in ccp4 suite. It is giving an indication for pseudo-translation as 'Pseudo-translation is detected: 17.6% Pseudo-translation vector: 0.000 0.000 0.078. Is it a significant amount? Any comments on this?? Thanks in advance. cheers wandu No - thatt will be an origin ripple Eleanor
[ccp4bb] Off topic: How to identify a unknown ligand
Dear all, Sorry for this off topic question. We are working on protein/inhibitor complex structure although we can not get our inhibitors in. However, we did find a strange density at the active site, it looks really like GSH, the natural co-enzyme of thiis protein.We tried to use very simple solution to get crystal then exclude the possiblity of buffer moleculors,but that density is aways there. I am wondering how this ligand (if it is GSH) can survive all purification steps and want to indentify it. Are there any methodes to do this work? Let's say to pick up a crystal and do some analysis? Many many thanks!!! Xiaopeng
[ccp4bb] Crosslinking of Protein-Protein complex for crystallisation
Dear CCP4bb, I am trying to solve the structure of a protein-protein complex. The two proteins are co-expressed and co-purified and I get a homogeneous 1:3 complex, which is the predicted composition. I get several hits using commercial crystallisation screens (including the MD ProPlex screen), but all crystals only contain the trimeric, more stable protein of the two. I am now considering to crosslink them, but my problem is that I cannot modify them separately to get a specific crosslink, since one of the proteins precipitates by itself. I would appreciate all protocols for crosslinking of proteins aimed for crystallisation, or references to structures solved of crosslinked proteins. Thank You, Catrine -- Catrine Berthold Siöberg, PhD Stockholm Center for Biomembrane Research Department of Biochemistry and Biophysics Stockholm University S-106 91 Stockholm Sweden e-mail: catrine.siob...@dbb.su.se phone: +468-162451
Re: [ccp4bb] Crosslinking of Protein-Protein complex for crystallisation
Dear Catrine can you comment on the pH range of your crystallization hits compared to the pH of the sample you use for your crystallization trials? The pH can be a major issue depending on the nature of the interaction interface. best regards Savvas On 14 Mar 2011, at 13:21, Catrine Berthold Siöberg wrote: Dear CCP4bb, I am trying to solve the structure of a protein-protein complex. The two proteins are co-expressed and co-purified and I get a homogeneous 1:3 complex, which is the predicted composition. I get several hits using commercial crystallisation screens (including the MD ProPlex screen), but all crystals only contain the trimeric, more stable protein of the two. I am now considering to crosslink them, but my problem is that I cannot modify them separately to get a specific crosslink, since one of the proteins precipitates by itself. I would appreciate all protocols for crosslinking of proteins aimed for crystallisation, or references to structures solved of crosslinked proteins. Thank You, Catrine -- Catrine Berthold Siöberg, PhD Stockholm Center for Biomembrane Research Department of Biochemistry and Biophysics Stockholm University S-106 91 Stockholm Sweden e-mail: catrine.siob...@dbb.su.se phone: +468-162451
[ccp4bb] Scientific programmer positions at CCP4
Dear All, CCP4 is happy to announce 3 open vacancies for Scientific Programmer positions at CCP4 core team. Last year, the team has relocated to the newly built Research Complex at Harwell (RCaH), Rutherford Appleton Laboratory (RAL) near Oxford, UK, and is now seeking to expand. CCP4 has identified its strategic goals in the modernisation of the Suite in accordance with modern IT standards and making it more automated and user-friendly. This includes the development of a new-look GUI (CCP4i2), further automation of structure solution process, further cross-platform unification, and modernisation of the release and update mechanisms. We are looking for skilful and enthusiastic individuals who would contribute to a major improvement and modernisation of the Suite. In particular, we require an expert in Windows operating systems to be responsible for the modernisation, maintenance, update and distribution of the Suite on Windows platforms. A similar person is sought to be responsible for Suite operations on Unix-like platforms (various Linux flavours and Mac OS systems). An additional post will be recruited to assist in the development of the new GUI, with major focus on the design of graphical interfaces to CCP4 components and automated crystallographic pipelines. All posts will be based at RCaH-RAL. The jobholders will be part of CCP4 core team and will share all basic core activities, such as user support on Helpdesk, software maintenance and fixing bugs, contribution to CCP4 educational and outreach programmes, incorporating new code from external developers and others. All jobholders will be expected to conduct their own development project(s) related to CCP4 and to be available for travel within the UK and to overseas. The posts offer an excellent opportunity for enthusiastic persons to acquire a range of new skills and make a significant impact in bringing crystallographic software to the vibrant community of protein crystallographers. Closing day for applications is 15 April 2011, with interviews to be held on 4-5 May 2011 or shortly thereafter. Full description of the positions, required qualifications and application details are found at http://www.ccp4.ac.uk/vacancies. Everybody interested is welcome to apply! I would greatly appreciate if you can pass this message to your colleagues and acquaintances who are looking for computation/IT/PX-related job and may be interested in joining us. Many thanks and best of luck to all prospective candidates, Eugene. __ Eugene Krissinel, PhD Group Leader for CCP4 Research Complex at Harwell Rutherford Appleton Laboratory Harwell Science Inoovation Campus Didcot, Oxfordshire OX11 0FA United Kingdom Phone: +44 (1235) 56 7725 Email: eugene.krissi...@stfc.ac.uk
Re: [ccp4bb] Synthetic RNA for Crystallization
Hi Michael, If the problem is the absence of a purine on the 5' end, try using the Marc Dreyfus' mutant T7 RNA polymerase. It can accommodate any nucleotide at the 5' end. Guillerez, J., Lopez, P. J., Proux, F., Launay, H., and Dreyfus, M. (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance, Proceedings of the National Academy of Sciences of the United States of America 102, 5958-5963. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrievedb=PubMeddopt=Citationlist_uids=15831591 With best regards, Eric -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Martin Hällberg Sent: Sunday, March 13, 2011 9:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Synthetic RNA for Crystallization Hi, I'll second Israels's comment. Since the yield per coupling in synthesis is lower for RNA than for DNA it gets really expensive over 30-35 nucleotides. However, you can stitch together several 30 nt oligos using either T4 RNA ligase or T4 DNA ligase (with a DNA splint). Regarding suppliers of synthetic RNA, Dharmacon is still very reliable in terms of actual delivered amount and quality. If you are going for very large scale then oligofactory.com is a good choice. For a 90 nt oligo without any modifications I'd definitely go for T7 in-vitro transcription. On top of Nagai's classic that Israel referred to, I can also recommend the more recent method of native purification developed by Robert Batey and Jeffrey Kieft. See: Batey, R.T. Kieft, J.S. Improved native affinity purification of RNA (2007) RNA, 13, 1384-1389. You are going to need milligrams of T7 RNA polymerase in the end so forget about transcription kits, make your own. Cheers, Martin On Mar 13, 2011, at 10:16 AM, Israel Sanchez wrote: Hi Michael, we normally produce synthetic RNAs following this classic paper if the size is more than let say 40-50 nucleotide, otherwise we buy the RNAs from Dharmacon and the quality is totally OK. Hope it helps J Mol Biol. 1995 Jun 2;249(2):398-408. Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Price SR, Ito N, Oubridge C, Avis JM, Nagai K. MRC Laboratory of Molecular Biology, Cambridge, UK. Abstract In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs. 2011/3/13 Michael Thompson mi...@chem.ucla.edu Hello All, I am looking for some advice from some experienced RNA crystallographers. I would like to order some relatively short (90 bases) synthetic RNAs for crystallization trials. I was wondering if anyone could comment on the use of synthetic RNAs for crystallization. Specifically, what is the longest synthetic RNA that can be used for crystallization trials? I've seen some structures in the PDB that are up to 88 bases and are reported to have been obtained with synthetic constructs (3OWI - glycine riboswitch), but I don't really know if that's routine or if it's an exceptional case. Also, for those who have experience with the use of synthetic RNAs, I was wondering where people generally order their synthetic constructs from? Our resident expert in RNA crystallography recommended a company called Dharmacon (part of ThermoFisher), but I was hoping that I might get some other opinions as to which companies make the best quality oligonucleotides, provide samples with the highest purity, and have the most reasonable prices. Thanks in advance for the help! Mike -- Michael C. Thompson Graduate
[ccp4bb] registration open for beginners hands-on course on x-ray structural biology
The X6A workbench: Advanced Structural Biology Tools?? winter session (http://protein.nsls.bnl.gov)?? Location: NSLS Date: March 29 through April 1 We would like to invite you to participate in this four day hands-on course at the National Synchrotron Light Source. This course is intended for beginners who would like to obtain an overview on macromolecular structure determination. This course will discuss the basic concepts of macromolecular crystallography beam lines at synchrotron facilities, crystal growth and handling, data collection and processing, phasing and the first steps in refinement. Most time will be spent at the beam line actually handling crystals and screening for cryoprotectants, collecting and processing data at lower energies and at the Se edge. Significant amount of time will be spent phasing and obtaining the first electron density map.?? Registration; http://protein.nsls.bnl.gov; you must have an active guest appointment at Brookhaven National Laboratory at the time of the course.
Re: [ccp4bb] Jan 15, 2011 deadline- User proposal submission for Collaborative Crystallography at BCSB
Dear Users, The deadline for May/June 2011 Collaborative Crystallography proposals will be *Mar 15, 2011.* Through the Collaborative Crystallography Program (CC) at the Advanced Light Source (ALS), scientists can send protein crystals to Berkeley Center for Structural Biology (BCSB) staff researchers for data collection and analysis. The CC Program can provide a number of benefits to researchers: * Obtain high quality data and analysis through collaborating with expert beamline researchers; * Rapid turn around on projects; and * Reduced travel costs. To apply, please submit a proposal through the ALS General User proposal review process for beamtime allocation. Proposals are reviewed and ranked by the Proposal Study Panel, and beamtime is allocated accordingly. BCSB staff schedule the CC projects on Beamlines 5.0.1 and 5.0.2 to fit into the available resources. Only non-proprietary projects will be accepted. As a condition of participation, BCSB staff researchers who participate in data collection and/or analysis must be appropriately acknowledged - typically being included as authors on publications and in PDB depositions. Please consult the website for additional information at: http://bcsb.als.lbl.gov/wiki/index.php/Collaborative_Crystallography - How To Apply: To learn more, go to: http://www.als.lbl.gov/als/quickguide/becomealsuser.html To submit a proposal, go to: http://alsusweb.lbl.gov/. Scroll down to *Structural Biology beamlines (includes protein SAXS)* and click on New Proposal. Enter your proposal information, with attention to the following details: * For question 3/First choice, select 5.0.1 (Monochromatic); for question 3/Second choice, select 5.0.2 (MAD). * Check box (yes) in response to question (7) Do you want collaborative crystallography (beamline 5.0.1 or 5.0.2 only) * In question 4, please describe a specific research project with a clear end point. In order to request CC time for May/June 2011 allocation period, proposals must be submitted by *Mar 15, 2011.* The deadline for CC proposals for the time period July/Aug 2011 will be May 15, 2011. Regards, Banumathi Sankaran
[ccp4bb] Mar 15, 2011 deadline- User proposal submission for Collaborative Crystallography at BCSB
Dear Users, The deadline for May/June 2011 Collaborative Crystallography proposals will be *Mar 15, 2011.* Through the Collaborative Crystallography Program (CC) at the Advanced Light Source (ALS), scientists can send protein crystals to Berkeley Center for Structural Biology (BCSB) staff researchers for data collection and analysis. The CC Program can provide a number of benefits to researchers: * Obtain high quality data and analysis through collaborating with expert beamline researchers; * Rapid turn around on projects; and * Reduced travel costs. To apply, please submit a proposal through the ALS General User proposal review process for beamtime allocation. Proposals are reviewed and ranked by the Proposal Study Panel, and beamtime is allocated accordingly. BCSB staff schedule the CC projects on Beamlines 5.0.1 and 5.0.2 to fit into the available resources. Only non-proprietary projects will be accepted. As a condition of participation, BCSB staff researchers who participate in data collection and/or analysis must be appropriately acknowledged - typically being included as authors on publications and in PDB depositions. Please consult the website for additional information at: http://bcsb.als.lbl.gov/wiki/index.php/Collaborative_Crystallography - How To Apply: To learn more, go to: http://www.als.lbl.gov/als/quickguide/becomealsuser.html To submit a proposal, go to: http://alsusweb.lbl.gov/. Scroll down to *Structural Biology beamlines (includes protein SAXS)* and click on New Proposal. Enter your proposal information, with attention to the following details: * For question 3/First choice, select 5.0.1 (Monochromatic); for question 3/Second choice, select 5.0.2 (MAD). * Check box (yes) in response to question (7) Do you want collaborative crystallography (beamline 5.0.1 or 5.0.2 only) * In question 4, please describe a specific research project with a clear end point. In order to request CC time for May/June 2011 allocation period, proposals must be submitted by *Mar 15, 2011.* The deadline for CC proposals for the time period July/Aug 2011 will be May 15, 2011. Regards, Banumathi Sankaran
