Re: [ccp4bb] partial density
Dear Kumar, This is perfectly normal. Long and flexible side chains e.g. lysines, facing the solvent move around and have weak electron density. In fact, I have not seen a structure where this is not the case. If you get strong Fo-Fc density back, I would fit the side chains and not worry about the electron density being below 1 sigma. best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Putcha, Balananda Dhurjati Kumar Sent: Wednesday, October 07, 2009 6:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] partial density Dear all, I have refined a structure where several sidechains have only partial 2Fo-Fc density ( 1 X sigma). I see strong Fo-Fc density in both omit maps and when I delete the sidechains, suggesting the presence of the sidechain. However, the density is never complete and sometimes only covers the C-beta atoms. The structure is at 2.9A and the Rfree is approximately 25% using BUSTER-TNT (29% with Refmac). I am not sure how to interpret this and was wondering if it suggested a disordered or dynamic structure. Are there any reports of such structures in the literature? Thank you Kumar
Re: [ccp4bb] xds and cell refinement in cP
Hi Jan, Always worth a try is to process the data in P1 then assigning the symmetry in CORRECT - that way the cell constants can refine to what they want to. It also means you can check that the symmetry actually is cubic. For the majority of data sets in my experience it makes little difference whether you assign the lattice constraints during integration, unless they're wrong. Cheers, Graeme 2009/10/8 Jan Abendroth jan.abendr...@gmail.com: Hi all, I am running into a strange behaviour fo xds for a primitive cubic data set. Neither in the INTEGRATE nor in the CORRECT step the cell parameters are refined and stay exactly at the value specified in XDS.INP. R-factors and I/sigmas of the XSCALE run look suspiciously high/low. When I reduce the symmetry to tetragonal and run an otherwise identical XDS.INP script, the cell parameters are refined again. Any ideas? Thanks a bunch Jan -- Jan Abendroth deCODE biostructures Seattle / Bainbridge Island WA, USA work: JAbendroth_at_decode.is home: Jan.Abendroth_at_gmail.com
Re: [ccp4bb] Nobel prize for chemistry 2009
A Leslie wrote: BB members may be interested to know that the 2009 Nobel prize for chemistry has been awarded to Venki Ramakrishnan, Tom Steitz and Ada Yonath for their structural work on the bacterial ribosome. FYI: http://yalepatents.org/2009/05/26/patented-therapy-based-on-ribosome-structure/ Determination and uses of the atomic structures of ribosomes and ribosomal subunits and their ligand complexes : http://patft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO1Sect2=HITOFFd=PALLp=1u=%2Fnetahtml%2FPTO%2Fsrchnum.htmr=1f=Gl=50s1=7504486.PN.OS=PN/7504486RS=PN/7504486 Hmm. Paul.
[ccp4bb] Beamtime at X06SA, SLS, PSI - deadline: 15th October
= SYNCHROTRON BEAM TIME FOR MACROMOLECULAR CRYSTALLOGRAPHY AT THE UNDULATOR BEAMLINE X06SA AT SLS FROM JANUARY TO APRIL 2010 = - Automatic sample changer (IRELEC CATS) is available now with SPINE pucks, vials, and caps. - PILATUS 6M pixel detector available for user operation at High Resolution Diffractometer. Continuous, fine phi-sliced data acquisition (12.5 frames per second) with 20 bit dynamic range, see http://pilatus.web.psi.ch/ or www.dectris.com http://www.dectris.com/ for further information - mar225 CCD installed at Micro-Diffractometer MD2. This MAD-compatible diffractometer allows for data collection with a focussed beam size of 15 x 5 micrometers, and down to 10 x 5 micrometer using an apertures. - SLS is able to provide support for travel and accomodation for two users per trip: http://sls.web.psi.ch/view.php/users/experiments/eusupport/index.html - For recent highlights: http://sls.web.psi.ch/view.php/beamlines/px/research/index.html PROPOSAL SUBMISSION DEADLINE: THURSDAY, 15TH OCTOBER You need send proposals both X06SA(PX1) and X06DA(PX3) individually if you would like to have beamtimes on both beamlines. For further information and / or proposal submission: http://sls.web.psi.ch/view.php/users/experiments/proposals/opencalls/PX/index.html Looking forward to seeing you at SLS, TOMIZAKI, Takashi on behalf of MX crew at the SLS
Re: [ccp4bb] Nobel prize for chemistry 2009
The patent #7504486 you cited actually only covers 'A method of growing a crystal of a 50S ribosomal subunit from Haloarcula marismortui', i.e. the crystallisation method for the 50S subunit from this single organism (from my school Latin I deduce 'haloarcula' = 'small box of salt' and 'marismortui' = 'from the Dead Sea'!), not the crystal nor the resulting macromolecular structure. However other patents (e.g. notably #6,638,908) quoted in the article you cite do cover pretty well any untwinned crystals (i.e. in this case the crystals themselves not the crystallization method) of the large ribosomal subunit from *any* organism (again not AFAICS the structures though). Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Paul Emsley Sent: 08 October 2009 09:25 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Nobel prize for chemistry 2009 A Leslie wrote: BB members may be interested to know that the 2009 Nobel prize for chemistry has been awarded to Venki Ramakrishnan, Tom Steitz and Ada Yonath for their structural work on the bacterial ribosome. FYI: http://yalepatents.org/2009/05/26/patented-therapy-based-on-ribosome- structure/ Determination and uses of the atomic structures of ribosomes and ribosomal subunits and their ligand complexes : http://patft1.uspto.gov/netacgi/nph- Parser?Sect1=PTO1Sect2=HITOFFd=PALLp=1u=%2Fnetahtml%2FPTO%2Fsrchnum. ht mr=1f=Gl=50s1=7504486.PN.OS=PN/7504486RS=PN/7504486 Hmm. Paul. Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
[ccp4bb] cif file for NAD
Dear All, I would like to request to send me a working and correct cif file for NAD. I obtained the same from Prodrg and ebi. But seems to have an issues around O1N-PN-O2N O3-PN-O5. Thanks With best regards Rajesh Rajesh Kumar Singh, PhD Biochemical Engg. Group Chemical Engg. Division Dr. Homi Bhabha Road Pashan Road, Pune Pin - 411 08 India E-mail: rajesh.si...@ncl.res.in Phone: 00919850898175
[ccp4bb] temperature factors, mosaicity and wilson plot
Hi, I recently solved the structure of a mutant protein that contains a single amino acid substitution. Here are some data about the mutant: resolution: 2.65 A completeness: 99.9 % (100.0 %) Rwork/free: 22.3/27.8 % Rsym 15.6 (47.7) % avg. mosaicity: 0.48° now whats confusing me: Wilson b-factor: 30.34 A^2 Overall mean b-factor: 55.92 A^2 My questions are the following: Where could the large difference of the b-factors come from? As noted, the crystal shows a mosaicity of avg. 0.48°. Is there a way I could evaluate whether the high b-factors are completely explained by the high mosaicity? What other factors could explain the high b-factors? Does the b-factor correlate that clearly with the resolution? I used XDS for data processing and REFMAC for refinement. Does either of these programs consider the mosaicity so that i could exclude that the b-factors origin from the mosaicity? Thanks, Dominik
Re: [ccp4bb] mammalian cell culture on IMAC
Dear All, When mammalian cell culture is being loaded to GE HisTrap resin Ni ions are being stripped off the resin, at least in my hands. Did any of you have similar experience and if so what kind of work-around was found? Volume is fairly large (3L) and concentration/dialysis have proven to cause loss of desired protein. Please share your positive experience. Thank you for your time. Vaheh To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.
Re: [ccp4bb] mammalian cell culture on IMAC
In case of cell lysates this may be a good idea since you can adjust the salt concentration in your sample. In case of secreted proteins it is probably not so good since the media contains ~150 mM NaCl. In my case this salt prevents protein from bindinq to Q column. Thank you anyway. From: CCP4 bulletin board on behalf of Dima Klenchin Sent: Thu 10/8/2009 3:54 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] mammalian cell culture on IMAC When mammalian cell culture is being loaded to GE HisTrap resin Ni ions are being stripped off the resin, at least in my hands. Did any of you have similar experience and if so what kind of work-around was found? Volume is fairly large (3L) and concentration/dialysis have proven to cause loss of desired protein. Please share your positive experience. I get this with insect cell lysates. The solution: load onto ion exchanger first - not for purification but to get rid of whatever strips Ni2+. Crude wash with 50 mM salt (or as high as your protein allows) followed by step elution with 0.5M salt (very few proteins do not elute at 0.5M) -- load directly onto Ni-NTA. Solves the stripping problem 100%. (If you use step elution, make sure to NOT use weak exchangers or you will have pH shift of 2-3 units). If the protein binds to cation-exchangers at pH 7, even such a crude step results in significant purification in itself. If, for some reason, ion exchangers are not an option, load onto hydroxyapatite and elute with phosphate. The downside to HA is that it has a lot less capacity than ion-exchangers and that it needs frequent repacking. Good luck, Dima To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.
Re: [ccp4bb] mammalian cell culture on IMAC
I had exactly the same problem. You should switch to Talon matrix (Clontech). The cobalt ion is bound more tightly so stripping is dramatically reduced. Furthermore, the Talon matrix is more specific (then Ni Sepharose) for your His-tagged protein, so you have less contaminants. I load two to three liters of culture medium directly on a 20 mL Talon column. This works very good in my hands. Good luck, Kenneth Oganesyan, Vaheh schreef: Dear All, When mammalian cell culture is being loaded to GE HisTrap resin Ni ions are being stripped off the resin, at least in my hands. Did any of you have similar experience and if so what kind of work-around was found? Volume is fairly large (3L) and concentration/dialysis have proven to cause loss of desired protein. Please share your positive experience. Thank you for your time. Vaheh To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.
Re: [ccp4bb] mammalian cell culture on IMAC
We tried that trick, which works amazingly well in insect cells, in mammalian media, and it fails. It will depend on the exact media, obviously. Engin On 10/8/09 1:50 PM, Matthew Franklin wrote: The trick we used at Genentech (which I'm still using) was for secreted insect cell proteins, but it should work for you as well. Add 1 mM NiCl2 and 10 mM CaCl2 to your conditioned media, and adjust the pH to 7.2 - 7.5. For insect cell conditioned media, this produces a fairly heavy precipitate which appears to contain the nickel-chelating factor. Most of the time (but not always!) your protein of interest will not be precipitated by this step, and you can spin out the precipitate then load the supernatant on to your nickel column with no further processing. Hope that helps, Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems, a wholly owned subsidiary of Eli Lilly Company 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you. -- Engin Özkan Post-doctoral Scholar Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111
[ccp4bb] Coot and X11
I am trying to install the pre-compiled version of Coot on OSX 10.5.4. I downloaded the program from Bill Scott's website. When I try to run Coot, it complains about the wrong version of libXdamage.1.dylib. It needs 3.0 and I have 2.0. I currently have Xcode 3.0 and Xwindows 2.0 installed. How do I get the correct version of the library or how else can I get coot to work with my X-windows version? Thanks Ursula -- Ursula Schulze-Gahmen, PhD. QB3, Tjian Lab MCB, 16 Barker Hall #3204 University of California Berkeley Berkeley, CA 94720-3204 Phone: (510) 642 8258 uschulze-gah...@lbl.gov
[ccp4bb] mammalian cell culture on IMAC
This article discusses this effect in E. coli lysates. Please let us know if you find something that works well with insect/mammalian lysates or secreted proteins! Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-477.html ho Confometrx
Re: [ccp4bb] cif file for NAD
Dear RajeshCould you please try the attached dictionary. Please let me know if it still has a problem.What are the issues around O1N-PN-O2n and O3N-PN-O5?regardsGarib nad_exp.cif Description: Binary data On 8 Oct 2009, at 15:11, RK Singh wrote:Dear All,I would like to request to send me a working and correctcif file for NAD. I obtained the same from Prodrg and ebi.But seems to have an issues around O1N-PN-O2NO3-PN-O5.ThanksWith best regardsRajeshRajesh Kumar Singh, PhDBiochemical Engg. GroupChemical Engg. DivisionDr. Homi Bhabha RoadPashan Road, PunePin - 411 08IndiaE-mail:rajesh.si...@ncl.res.inPhone: 00919850898175
[ccp4bb] Question about the covalent Link between PLP Lysine
Dear All, I’m currently refining the structure (2.0 A) with a cofactor PLP. The PLP density is clearly indicates lysine residue is covalently bound with PLP cofactor. But I don’t know how to link the cofactor and lysine residue for further refinement. Any suggestions on how to create the link between the PLP and lysine residue and do the refinement with refmac would be greatly appreciated! Thanks Sincerely, Sampath N.
