[ccp4bb] PhD position at the Membrane Protein Laboratory (Diamond)
Applications are invited for a 3-year PhD position at Imperial College London which will be based in The Membrane Protein Laboratory (MPL) at Diamond Light Source, Didcot, Oxfordshire and the Centre for Structural Biology, Imperial College London. The successful applicant, for the position which is immediately available, will work on structural studies of membrane transport proteins using microfocus X-rays and X-ray Free Electron Laser beams. The project “NanoMem” is supported by the Marie Curie Initial Training Network (European Commission Framework Seven Programme). The MPL operates both as a facility for users and as a cutting edge research laboratory. It is a high-throughput protein crystallisation facility that aims to enhance the rate of success in the crystallisation of medically significant membrane proteins. Its location at Diamond Light Source facilitates high-throughput crystal screening and high quality data collection. The student will be co-supervised by Prof. So Iwata, Dr Isabel Moraes and Prof. Xiaodong Zhang at Imperial College. The student will be trained in state-of-the-art methods of membrane protein expression, purification, crystallization and structure determination using both synchrotron microfocus X-Ray beamlines and X-Ray free electron lasers. The successful candidate, as a member of the Marie Curie Initial Training Network, will participate in collaborative experiments, secondments and training events across the network (The NanoMem network compromises ten laboratories from five European countries). Studentship Details Applicants should hold a Masters degree in Biochemistry or a related subject. To apply: applications should include a cover letter describing relevant research experience to date, a CV, and the names and addresses of two referees. These should be sent to Dr Isabel Moraes (i.mor...@imperial.ac.ukmailto:i.mor...@imperial.ac.uk) by email by 31th May 2013. This studentship is only available to applicants who are not UK citizens or have not lived in the UK for more than 12 months during the last three years. For informal enquires please contact Dr Isabel Moraes (i.mor...@imperial.ac.ukmailto:i.mor...@imperial.ac.uk) Dr Isabel De Moraes, MRSC Head of the Membrane Protein Laboratory Diamond Light Source Ltd, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire, OX11 ODE, UK -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
[ccp4bb] Scaling problem with multiple crystals
Dear All, I have been trying to scale (using SCALA) the diffraction data set collected from multiple crystals. Data was indexed using mosflm but fails to scale with an error saying negative scale factors when scaling was tried in a constant mode whereas it fails to scale with an error saying No observations when scaling was tried in a batch mode. Damping shifts were tried from 0.1 to 0.5 and also unit weighting, but the scaling still fails with same errors. Can someone provide any suggessions to deal with the problem. Kind Regards, Amit
Re: [ccp4bb] Scaling problem with multiple crystals
Depending on the Space (Laue) group, there may be alternative indexing possibilities which will be sorted out in Pointless - did you run that program? I would recommend using the ccp4i task Symmetry, Scale, Merge (Aimless) which uses Aimless instead of Scala - Aimless may be a bit more robust than Scala in some cases Other things to try. If you have several crystals, one (or more) may just be bad. You can try scaling them separately, or using the scale constant option just to diagnose which crystals would be better omitted Phil On 10 May 2013, at 16:04, amit sharma amit_sps2...@yahoo.co.uk wrote: Dear All, I have been trying to scale (using SCALA) the diffraction data set collected from multiple crystals. Data was indexed using mosflm but fails to scale with an error saying negative scale factors when scaling was tried in a constant mode whereas it fails to scale with an error saying No observations when scaling was tried in a batch mode. Damping shifts were tried from 0.1 to 0.5 and also unit weighting, but the scaling still fails with same errors. Can someone provide any suggessions to deal with the problem. Kind Regards, Amit
Re: [ccp4bb] Membrane Protein Optimisation
Hi Rhys, The bOG concentration of 1.0% is quite acceptable and if your protein is stable and happy in OG, then you're in luck. At 30% PEG 600, I'm not surprised you're seeing the phase separation or detergent 'blob's for lack of a better term. I believe you have the latter, which many of us working with memb proteins experience. You are probably at above 1% OG though. When you concentrate, do you use 100kDa cutoff membrane. Based on the size of your protein, this would be a safe concentrator to use. FYI, I have several proteins that are ~50kDa, and I can use 100kDa concentrators without ill effects. If you are using 100kDa membrane, then you might try 'concentrating' via an ion-exchange column before actual concentration. Lastly, as Jim mentioned, you might want to change your construct by a few residues. This might help with the resolution. -john On Thu, May 9, 2013 at 3:49 PM, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Thanks for the suggestions so far, I should have given a little more information in my initial post. The protein I'm working on is an Gram-Neg Beta-Barrel about 100kDa, likely with 22 strands. I'm currently crystallising in bOG, although my next optimisation step is to try a range of detergents. I'm using a refolding protocol which relies heavily in LDAO (one of the only cost effective detergents for the volumes I'm using), I refold, nickel purify (N-terminal tag, signal peptide removed) , do a size exclusion step (S200), then detergent exchange into bOG using a nickel column. The BOG concentration is obviously high 0.8-1%, which might be causing the gels? The gels don't look like classic phase separation to me (from soluble proteins that is, this is my first membrane protein), they are not spherical, the tend to float or sit on the bottom and are semi-solid, around 30-100uM diameter. These screens I've set up are classic screens for membrane proteins (mem-plus, mem-start, PGA etc.) and the drops aren't dried up. The gels are strongly birefringent, but the drop is not. Additionally the conditions in my initial screen which yielded crystals seemed biased for the formation of the gels. Thanks again for the help, I feel I might have a long raod to optimisation ahead of me. Rhys From: Jim Fairman [fairman@gmail.com] Sent: 09 May 2013 20:58 To: RHYS GRINTER Cc: ccp4bb Subject: Re: [ccp4bb] Membrane Protein Optimisation With information you've provided I have multiple suggestions for you, some of which you may have already tried: 1. OMPs can be fairly particular about which detergents they will crystallize in. Try exchanging the protein into a different detergent/detergent mixture and then set up new trays in your favorite broad matrix screens. DDM, DM, LDAO, OG, and C8E4 are a few of my favorites for OMPs, but there are many others. This can be done using a size exclusion column as the final purification step for your protein where the column is equilibrated with the appropriate detergent. This step will also let you know how well behaved the protein is in that detergent via the shape/height of the peak. This won't help you optimize your current condition, but it may lead to different/new conditions with even better crystals. As Pascal suggested, try to carefully choose which MW cut-off concentrator you end up using. Minimizing the amount of detergent concentration that occurs during your concentration step(s) is optimal. 2. Attempt crystallization using DHCP/CHAPSO bicelles. You can buy them pre-made from MemX Biosciences or make them yourself using a published protocol from David Bowie's lab. These have been used to crystallize the mitochondrial beta barrel protein VDAC and I had success crystallizing intimin in them. David Bowie also has a JoVE article on the bicelle crystallization method. 3. Attempt crystallization using Lipidic Cubic Phases. This was the saving grace for my post-doc project. Neither detergent nor bicelle crystallization produced crystals that were of sufficient quality, but the crystals from LCP all diffracted to the 2.0 angstrom resolution range. If you're unfamiliar with the technique, there are several nice videos on JoVE describing it by Vadim Cherezov and Martin Caffrey. 4. Alter your construct. Small changes in the construct (ie: deletion or addition of 1-2 residues on the N- or C-terminus) often led to drastically different crystallization behavior of several OMPs in my hands. Cheers, Jim On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER r.grinte...@research.gla.ac.ukmailto:r.grinte...@research.gla.ac.uk wrote: Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of
Re: [ccp4bb] Membrane Protein Optimisation
Hello Rhys, I suggest using an assay such as that described in Anal. Biochem. 336:117 to measure the amount detergent in your concentrated samples. I found this assay accurate and easy to use with DDM. It should work with b-OG too. Good luck, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
[ccp4bb] 43rd Mid-Atlantic Macromolecular Crystallography Meeting
*43**rd** Mid-Atlantic Macromolecular Crystallography Meeting* *May 30 – June 1, 2013* *Duke University* *Durham, NC* *ANNOUNCEMENT: *We have extended the deadline for submitting abstracts to be considered for presentations until midnight Sunday, May 12. Registration and details can be found at http://www.mid-atlantic.org. The 43rd Mid-Atlantic Macromolecular Crystallography Meeting, will be held at Duke University in Durham, North Carolina. This year will feature keynote speaker Tom Terwilliger from Los Alamos National Lab, workshops on PHENIX and MolProbity, plus many talks posters on new and exciting research. The best presentation and poster by a Graduate Student Postdoctoral Fellow will be awarded (total of 4). We encourage you to submit your abstracts and sign up for the workshops as these are separate processes from registration. *DEADLINES (updated):* Abstract submission for presentation - May 12th Abstract submission for poster - May 27th *No deadline for registration With best wishes on behalf of the primary Organizing Committee, Charlie Pemble _ Charles W. Pemble IV, Ph.D. Duke University Medical Center 308 Research Drive LSRC, A06 Durham, NC 27708 charles.pem...@duke.edu We thank our current sponsors for their generous contributions: Rigaku Americas Corporation, Agilent Technologies Inc., GlaxoSmithKline, Art Robbins, Bruker, HKL Research Inc., Molecular Dimensions, Hampton Research, Labcyte, GenScript, Genentech, Emerald BioSystems, TTP LabTech, Oxford Cryosystems, PhD Posters, Duke University School of Medicine, Duke Human Vaccine Institute, and Avanti Polar Lipids Inc.
