Re: [ccp4bb] Ligand fitting into density
Dipankar, What you describe occurs frequently and some times improves with refinement and other times does not. I suggest not adding ligands until the rest of the structure is well behaved - usually around 25-28% Rfree. This will tend to favor the highest quality electron density and will not have bias from the ligand in the phases. Small proteins with electron rich ligands may need the ligand fitted to cross even 30%, but it is a guideline. Also keep in mind that organic molecules can show true electron density shifts based on the chemistry present. A sulfonamide on a phenyl ring will for instance pull most of the electrons (on average) from the ring, leaving it looking more like a comma than a disc...even at quite high resolution...it is real electronics observed visually. Your second question regarding ~100A^2 for the B-factor of the ligand requires further clarification: 1. Is the resolution of your data sufficient to warrant individual B refinement? If so, what is the average B of residues near the ligand? In general, I ligand at roughly 100% occupancy should have B-factors within 10-20% of the value of the protein. Some programs do a poor job of refining B-factors of ligands. X-PLOR, for instance used to have a hard time with them. 2. What is the relative quality of the electron density of the ligand vs protein? Does it suggest less than full occupancy of the ligand in the pocket? If so, the approximate occupancy of the compound can be estimated by the comparison of the ligand B and nearby protein B factors. A ligand B twice the value of the protein residues would roughly suggest an occupancy of 50%. You can try that and see if the Bfactors stabilize. Cheers, Jim On Wed, Apr 11, 2012 at 2:11 AM, Dipankar Manna dipanka...@aurigene.comwrote: Dear Crystallographers, The protein I am working with is having SG P3121, Structure is solved at 2.5A. the protein was soaked with compound, compound density is also looking prominent except one six membered ring. There is no density at all for the particular ring, but other parts of the compound is fitting well enough into the density. The B factor of the ligand is showing 100. How can I justify this issue. Asking for suggestions. Regards, Dipankar Manna -- This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com -- * * * James Kiefer, Ph.D. * Structural Biology Genentech, Inc. 1 DNA Way, Mailstop 27 South San Francisco, CA 94080-4990
Re: [ccp4bb] zinc finger
There are review articles on various motifs. I think that I remember that you can also find motifs via sequence or structure classes on places like SWISSPROT/EXPASY. A quick search of PUBMED did not produce a single-source paper listing the various motifs...and there are several. Biochemistry texts will list a few motifs but will be far from exhaustive. Adding Zinc acetate to your fermentation and being careful not to use EDTA/EGTA in the prep may give you a more definitive answer. Similarly, DTT and TCEP can be good chelators of metals, so I would avoid those. Your initial comment indicated that you had no diffraction yet. Does that mean you have crystals??? If so, what percent of your produced protein is the putative zinc finger region? If it is high, and you already have crystals, then you may be searching for a problem that does not exist. -- James Kiefer, Ph.D. Structural Biology Genentech, Inc. 1 DNA Way, Mailstop 27 South San Francisco, CA 94080-4990
Re: [ccp4bb] Requested: Three-Day Data Fabrication Workshop
Dear Jacob, With all due respect, you have left out a key component to successful data fabrication in the modern age: software. It is quite obtuse not to have allocated at least one day of the workshop for practical applications of Photoshop to diffraction image generation and at least a passing coverage of whether or not Adobe Lightroom and crystallographic presets therein will be sufficiently capable of muddling the RCSB staff analysis of data feasibility checking. I would very much like to see Gerard Bricogne present a keynote lecture entitled something like, The R-Fake Parameter: A Maximum Likelihood Modulus to Define a Minimum Acceptable Data Drift Coefficient for Use in the Fabrication of Credibly Artificial Diffraction Data. I also believe that we are perhaps full of hubris as a crystallographic community, because an entire field of faked structural data has existed long before crystallographers even considered manufacturing their data. Specifically, the molecular modeling community has already surpassed us in their thinking on the subject. While we idly discuss how to properly generate false data, they have had the foresight to abandon ALL data...and even the starting coordinates in crystal structures - be they real or fictitious - and publish volumes of papers entirely unencumbered by reality or plausibility. My hat is off to them. Best regards, Jim On Mon, Apr 2, 2012 at 8:15 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear CCP4BB, due to increasing demand, it seems we should put together a workshop on data fabrication, covering the various important topics (chaired by JHo): --Images: the future of fabrication? How long can we rely on database Luddism? --Ways out: how to leave a trail of accidental data mix-ups --Publish large or small? Cost-benefit analyses of impact factor vs. risk of being discovered --Pushing the envelope: how significant is two [sic] significant --Crossing discipline boundaries: are data fabrication procedures universal? --Build a better hofkristallrat-trap: utilization of rhetorical bombast and indignation in reply letters --Break-out support-session with survivors: comforting words on careers after the fall --Session on the inextricably-related topic of grammatical pedantry, to be followed by a soccer (football?) match Greeks Vs. Latins Ample funding will be available from big pharma and other industry sectors Please submit further topics to the CCP4BB list JPK ps I can't believe no one mentioned the loathsome Latino-Greek multimer in the recent curmudgeonry postings. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- James Kiefer, Ph.D. Structural Biology Genentech, Inc. 1 DNA Way, Mailstop 27 South San Francisco, CA 94080-4990
[ccp4bb] OPENING: Genentech, Research Associate/Senior Research Associate in Purification and Crystallization
Dear All, We have an open position at Genentech in the Structural Biology group for an Associate/Senior Associate in our protein purification and crystallization laboratory (Job #: 380684). Prior experience in purification and/or crystallization is required. Preference will be given to candidates with a track record of success in challenging systems such as membrane proteins, multi-domain proteins, or protein:protein (or protein:nucleic acid) complexes. Please encourage qualified candidates to apply to the Genentech website (details below). All the best, Jim * James Kiefer, Ph.D. * Structural Biology Genentech, Inc. 1 DNA Way, Mailstop 27 South San Francisco, CA 94080-4990 -- Here is the official posting: Job requisition number: 380684 RA/SRA – Crystallography, Macromolecular Who we are: At the Roche Group, about 80,000 people across 150 countries are pushing back the frontiers of healthcare. Working together, we've become one of the world's leading research-focused healthcare groups. A member of the Roche Group, Genentech has been at the forefront of the biotechnology industry for more than 30 years, using human genetic information to develop novel medicines for serious and life-threatening diseases. The headquarters for Roche pharmaceutical operations in the United States, Genentech has multiple therapies on the market for cancer and other serious illnesses. Please take this opportunity to learn about Genentech, where we believe that our employees are our most important asset and are dedicated to remaining a great place to work. The Position: E2/E3Position available for a Research Associate to join the Structural Biology Department involved in exploring structural and functional properties of proteins of therapeutic interest. The successful candidate will be responsible for the purification and characterization of proteins for structural analysis by X-ray crystallography. Day-to-day activities include designing and optimizing protein expression and purification strategies for challenging structural biology targets. Additional experience in the crystallization, cloning, expression and/or use of automation methods for protein purification and characterization would be considered strong assets. Who you are: A B.S. or M.S. in biochemistry, molecular biology or related discipline and 5 (five) or more years) of experience in areas relating to protein biochemistry and crystallization are required. Experience with crystallization automation and HPLC or FPLC purification systems is preferred. The successful candidate must be motivated, capable of working independently, and enjoy working in a collaborative setting. Strong analytical, communication and organizational skills are highly desirable. To apply visit www.gene.com /careers. Genentech is an Equal Opportunity Employer with a commitment to diversity. All individuals are encouraged to apply.
