Re: [ccp4bb] Phase separation and membrane protein crystallization
Lin, consider yourself lucky if you only have one problem...;-) regarding your question, poor reproducibility is a common problem with membrane protein crystallography and is most likely caused by different amounts of lipids copurifying with your protein. If you want to avoid it (may not be possible altogether) do your preps as reproducible as possible, especially using the same amount of detergent per cell weight, stirring at the same speed, for the same time etc, etc. Even then it may be hard to control. I tend to use this feature as a screening variable and always do several preps to hopefully get one that works well and will allow you to solve a structure. good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yibin Lin [yyb...@gmail.com] Sent: Monday, December 17, 2012 11:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Phase separation and membrane protein crystallization Dear all, I am working with one membrane protein. I met one problem. From different purification protein (following same methods, never changing anything except date and time), I setup crystal plate using same conditions, why sometime I can get crystals and sometime only phase separation? Thanks, Lin
[ccp4bb] PEG refinement
Dear all, is there any consensus about PEG refinement? I'm currently refining several structures with a lot of bound PEG molecules. However I'm completely confused now which ligand description to use. For example, a search in Hic up database reveals 12 possible descriptions for PEG, 5 of them used for PEG400, but with completely different length ranging from C8 (number of carbon atoms), ligand ID PG4 up to C24, ligand ID 12P. Other descriptions are also quite confusable, for example PEG 8K (ligand id PEU) is C55H112O28, but apparently shorter PEG1500 (ligand id 15P) is C69H140O35. Shall the crystallography community come up with some standardised list of descriptions for PEG molecules? With best regards, Albert
Re: [ccp4bb] PEG refinement
Hi, I usually pick up the one that fits best the map (there are several PEG monomers in the ccp4 monomer library to choose from). You should bear in mind that PEG's are a mixture of molecules, quite often floppy, so whatever fits best is the best choice. Good luck, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Albert Guskov [albert.gus...@googlemail.com] Sent: Tuesday, December 18, 2012 1:05 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] PEG refinement Dear all, is there any consensus about PEG refinement? I'm currently refining several structures with a lot of bound PEG molecules. However I'm completely confused now which ligand description to use. For example, a search in Hic up database reveals 12 possible descriptions for PEG, 5 of them used for PEG400, but with completely different length ranging from C8 (number of carbon atoms), ligand ID PG4 up to C24, ligand ID 12P. Other descriptions are also quite confusable, for example PEG 8K (ligand id PEU) is C55H112O28, but apparently shorter PEG1500 (ligand id 15P) is C69H140O35. Shall the crystallography community come up with some standardised list of descriptions for PEG molecules? With best regards, Albert
[ccp4bb] Position available at DESY: Protein Nanocrystallography
DESY, Hamburg location, is seeking a Scientist for Protein Nanocrystallography (f/m) DESY is one of the world’s leading centres for the investigation of the structure of matter. DESY develops, runs and uses accelerators and detectors for photon science and particle physics. The Center for Free-Electron Laser Science (CFEL) is a jointly operated research cooperation between DESY, the Max Planck Society and the University of Hamburg. DESY supports three CFEL divisions with leaders jointly appointed with the University of Hamburg. The Coherent Imaging Division of CFEL is developing novel methods in macromolecular structure determination, using intense X-ray laser pulses to out-run radiation damage and thereby removing the biggest bottleneck in structural biology: the need to grow large well-diffracting crystals. The Division is leading a consortium to deploy serial femtosecond crystallography at the European XFEL. For this we are seeking a scientist. The position * Lead the design, development and construction of the Serial Femtosecond Crystallography (SFX) instrument at the European XFEL. * Develop beamline equipment, diagnostics, and analysis methods for serial crystallography, and to critically evaluate new concepts for the instrument. * Pursue independent and collaborative research in serial crystallography. * Communicate developments within the consortium and the broad community. Requirements * A PhD in experimental physics or a related field * An ability for teamwork in a group of scientists and engineers * Expert knowledge in crystallographic theory and methods and X-ray physics * Strong background in development of instrumentation * Excellent English For further information please contact Prof. Henry Chapman, +49 40 8998 4155, henry.chap...@cfel.de The position is limited to 3 years. Salary and benefits are commensurate with those of public service organisations in Germany. Classification is based upon qualifications and assigned duties. DESY operates flexible work schemes. Handicapped persons will be given preference to other equally qualified applicants. DESY is an equal opportunity, affirmative action employer and encourages applications from women. There is an bilingual kindergarten on the DESY site. The deadline for applications is 28 February 2013. Please send your application, quoting reference code EM242/2012, by post or e-mail as follows: Deutsches Elektronen-Synchrotron DESY Human Resources Department Notkestraße 85 22607 Hamburg Germany E-Mail: recruitm...@desy.de PDF version of these details: http://www.desy.de/v2/docs/1354184737-e.pdf Phone: +49 40 8998-3392 www.desy.de
Re: [ccp4bb] PEG refinement
I doubt there is a consensus on this. Your PEG solution is a mixture of lengths of polymer and normally you can only see a small fraction of the PEG molecule. PEG400 will be between 8 and 10 ethylene glycols. Bigger PEGS probably vary more. My experience is that you can often only see density for 3-6 ethylene glycol units (and this is probably not related to whether the PEG is 400, 4K or 20K). You select the length of PEG that matches your density rather than being a function of what is in the crystallisation The argument would be I guess that a hydrophobic patch makes a section of the ethylene glycol ordered enough to be seen as density but the extensions occupy a sufficient range of conformations as to appear disordered. I have put short stretches of PEG as it seems to be the best interpretation of difference density but I do always wonder if it is real or some kind of Fourier termination effect or partially ordered waters etc. I would be cautious about basing too much biology on it. Nick -- Prof Nicholas H. Keep Executive Dean of School of Science Professor of Biomolecular Science Crystallography, Institute for Structural and Molecular Biology, Department of Biological Sciences Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G54a Office) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
