[ccp4bb] EMBO Course on combination of electron microscopy and x-ray crystallography
The combination of electron microscopy and x-ray crystallography for the structure determination of large biological complexes 18 - 24 October | 2009 |Grenoble| France The course aims to train junior investigators in both fields (electron microscopy and X-ray crystallography) to carry out their combined use in their home institutes. The participants will be introduced to underlying theoretical aspects of the used methods but emphasis will be given to the practical work and problem solving. Deadline for registration: 15 September 2009 For registration and full program, please have a look at the Website of the course: http://cwp.embo.org/pc09-18/ Looking forward seeing you in Grenoble! the Organisers -- -- Laurence Serre Partnership for Structural Biology Carl-Ivar Branden Building Room 018 Polygone scientifique 6, rue Jules Horowitz BP181 F-38042 Grenoble France Tel: 33 (0) 476 20 94 08 Fax: 33 !0) 476 20 94 00 --
Re: [ccp4bb] PDB Validation Server
1) By submitting my structure to the PDB Validation Server for precheck and validation, will my structure become publicly available in any way? It will not. But if you prefer, you can download the validation tools and run them on your home computer. However, most recent workstation/downloadable version of ADIT (that I've been able to find) dates from ~2003, and perhaps unsurprisingly gives slightly different results than the hosted version.
Re: [ccp4bb] PDB Validation Server
.. and ot be honest a structure that passes with flying colors from the MolProbity or WhatCheck scrutinee, is unlikely to get stack in PDB submission. A. On Jul 7, 2009, at 16:59, Pete Meyer wrote: 1) By submitting my structure to the PDB Validation Server for precheck and validation, will my structure become publicly available in any way? It will not. But if you prefer, you can download the validation tools and run them on your home computer. However, most recent workstation/downloadable version of ADIT (that I've been able to find) dates from ~2003, and perhaps unsurprisingly gives slightly different results than the hosted version. P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
[ccp4bb] Lipid content/removal
Dear Crystallographers, does anybody know of a simple, fast way to determine whether a protein sample contains lipid, and roughly to determine the quantity? As corollary, does anybody know of a quick way to extract/remove the putative lipid? In my case, I am working with an extremely robust/stable soluble protein which may contain bacterial lipids. Thanks for your consideration, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Lipid content/removal
Hi Jacob, there are ways, but simple and fast, I don't know. You can do extraction of some purified protein with organic solvents and doing TLC. Quantitation may be harder. In the case of phospholipids you can do a phosphorous determination (with molybdenum, the protocol should be easy to retrieve online) . For regular, non-P containing lipids you may have to go back to TLC with standards. For removing the lipid, any column that binds your protein would work (metal-affinity, ion exchange), just wash with a large volume to gradually push the lipid off. Gel filtratiuon is also possible, but as you can imagine less efficient. Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -Original Message- From: CCP4 bulletin board on behalf of Jacob Keller Sent: Tue 7/7/2009 1:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Lipid content/removal Dear Crystallographers, does anybody know of a simple, fast way to determine whether a protein sample contains lipid, and roughly to determine the quantity? As corollary, does anybody know of a quick way to extract/remove the putative lipid? In my case, I am working with an extremely robust/stable soluble protein which may contain bacterial lipids. Thanks for your consideration, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Lipid content/removal
Activated charcoal (see serum albumin structure references) can remove fatty acids. Good luck- Steve -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Tuesday, July 07, 2009 1:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Lipid content/removal Dear Crystallographers, does anybody know of a simple, fast way to determine whether a protein sample contains lipid, and roughly to determine the quantity? As corollary, does anybody know of a quick way to extract/remove the putative lipid? In my case, I am working with an extremely robust/stable soluble protein which may contain bacterial lipids. Thanks for your consideration, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Lipid content/removal
Also MALDI-TOF can be used to help identify the lipid (with standards). I vaguely remember one would extract the lipid, from the protein, using organic solvents then apply that to the MS. When I did it (about 10 years ago) I used non plastic vials and a Hamilton syringe so as not to contaminate the sample with plastics. Hope that's useful! Gina -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Soisson, Stephen Sent: Tuesday, July 07, 2009 2:01 PM To: Subject: Re: [ccp4bb] Lipid content/removal Activated charcoal (see serum albumin structure references) can remove fatty acids. Good luck- Steve -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Tuesday, July 07, 2009 1:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Lipid content/removal Dear Crystallographers, does anybody know of a simple, fast way to determine whether a protein sample contains lipid, and roughly to determine the quantity? As corollary, does anybody know of a quick way to extract/remove the putative lipid? In my case, I am working with an extremely robust/stable soluble protein which may contain bacterial lipids. Thanks for your consideration, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
[ccp4bb] postdoc position available
A post-doctoral associate position is available in the Institute of Molecular Virology, University of Minnesota to study the cell entry mechanism of small envelope viruses (Zhang, W., et al 2002. J. Virol. 76:11645-11658; Kielian, M., and F.A. Rey. 2006, Nature Rev. Microbiol. 4:67-76.) using cryo-electron microscopy (cryo-EM) (Baker, T. S., et al. Microbiol. Molec. Biol. Reviews 63:862-922) and other biochemical and biophysical methods. Specifically, identifying the steps and determination of the type-II viral fusion protein oligomerization, re-organization during low-pH triggered membrane fusion. Candidates should have a PhD and training in virology, structural biology, biophysics or related fields. Please send cv, research experience and three references to Dr. Wei Zhang (zhang...@umn.edu)
[ccp4bb] MX Frontiers at the One Micron Scale Workshop - July 23-24, 2009 - Late registration date July 10
Meeting Notice: We invite you to participate in the MX Frontiers at the One Micron Scale workshop, which will be held at Brookhaven National Laboratory on July 23 and 24, 2009. The workshop will include lectures and discussions about potential opportunities in macromolecular crystallography at the one micron scale. During the workshop, distinguished speakers will present lectures with regard to structural biology scientific opportunities, the physics of radiation damage, x-ray optical requirements, challenges in instrumentation, and new crystallographic methods. To view the complete agenda and list of speakers, please visit the workshop website at : http://www.nsls.bnl.gov/newsroom/events/workshops/2009/mx/ Register now for this fast paced, modestly priced, day-and-a-half workshop at : http://www.nsls.bnl.gov/newsroom/events/workshops/2009/mx/registration/registration.asp **Please note that the early registration deadline has been extended. The new registration deadline is Friday, July 10, after which a late fee will be applied** Thank you, Workshop organizers, Dieter Schneider (schnei...@bnl.gov) Lonny Berman (ber...@bnl.gov) Marc Allaire (alla...@bnl.gov)
[ccp4bb] Maximum # of input parameter files for use in CNS refinement?
Hi all,
I'm trying to refine a structure but it needs six parameter files input in
order to deal with al lthe atoms built in.
minimize.inp keeps crashing with the error : %NBUPDA-ERR: missing nonbonded
Lennard-Jones parameters %%%
I realized after switching up the order of the parameter files input below
minimize does not read the 6th one- is there any way to input a 6th parameter
file? This is what I have in now:
{=== molecular structure =}
{* molecular topology file *}
{===} structure_infile=../../dG/generate/7jul09gk.mtf;
{* parameter files *}
{===} parameter_infile_1=../param/cpq_xplor.param;
{===} parameter_infile_2=../../toppar/dna-rna_ino.param;
{===} parameter_infile_3=CNS_TOPPAR:water.param;
{===} parameter_infile_4=CNS_TOPPAR:ion.param;
{===} parameter_infile_5=../../jj/dioxane.param;
{===} parameter_infile_6=CNS_TOPPAR:protein_rep.param;
{* coordinate file *}
{===} coordinate_infile=../../dG/generate/7jul09gk.pdb;
Thanks!
Krystle
---Krystle WilliamsDept.
Biochemistry and BiophysicsSchool Of Medicine and DentistryUniversity of
Rochester601 Elmwood Ave. Box 712Rochester, NY 14642
Phone:585-276-3681
_
Show them the way! Add maps and directions to your party invites.
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Re: [ccp4bb] unknown density
Dear Bert, Your density reminded me a little bit of the paper by Pearson et al. Biochemistry 39:8575-8584(2000) which describes the water structure in the catalytic center of urease. I just went back to it and it looks like Figure 3 in that paper may hint some similarities to your case, in that you may indeed have a mixture of water models. You did not say if your data comes from cryo-cooled crystals that were manipulated in similar or different ways, or whether one of the two is a room temperature data set. One of the largely overlooked issues related to crystal cryo-cooling concerns the structure attained by the solvent. Structures of urease at 100K and 298K (pdb entries 1ejw and 1ejx) show marked differences in the water structure in the active site and elsewhere. This phenomenon has been observed in other structures as well, and not just with respect to water structure but also for loops and side-chains. Even two cryo-cooled crystals that are manipulated in similar ways may exhibit differences. Bart Hazes addressed some of these issues in an interesting paper in Acta Cryst. (2005). D61, 80-87. Nonetheless, to maximize the chances for the best interpretation possible I would first improve phase accuracy by completing the solvent model as much as possible (except from the contested density), and then use post-refinement residual Fo-Fc density to evaluate possible water models (and other possible interpretations) for the density in question. Best wishes Savvas - Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 http://www.lprobe.ugent.be/xray.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Van Den Berg, Bert Sent: Tuesday, July 07, 2009 10:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] unknown density Dear all, I have attached a jpeg of some (2fo-fc) density in the active site of a protein I'm refining (1.9 A data, Rfree 24%, still need to pick waters etc; oxygen atom placed in the approximate center for reference). It looks like there are 3 non-hydrogen atoms sandwiched between an Asp, His and Arg. The molecule seems hydrogen bonded to all these three residues. Has anybody any idea what this can be? In a map of another crystal of the same crystal form at lower res (2.3 A), there is a clear, single water molecule between the Asp and His. Are they waters at partial occupancies (there is not enough room for 3 separate waters)? The only other thing I can come up with is formate, but that wasn't used in the crystallization. Any hints appreciated! Thanks, Bert Bert van den Berg UMass Medical School Worcester, MA 01605 E-mail message checked by Spyware Doctor (6.0.1.441) Database version: 6.12770 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] low UV reading on AKTA prime
Apologies for coming late with this comment, but this A280 filter clouding problem appears to be a common feature of the AKTA machines. The UV unit on these AKTAs is the same as on the old FPLC machines and the filters are interchangeable. The filters from the 10-20 year old FPLC machines fit perfectly into the AKTA UV unit and have a much longer life (15+ years) than the new GE-made filters. Best, Petr From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Patrick Loll Sent: Wednesday, July 01, 2009 11:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] low UV reading on AKTA prime I second Scott's post. About the only problem we've had with our Akta instruments is this type of degradation of the filter. I'm not sure what the mechanism is, but the filters do seem to crap out after a while, at least in the cold room (oxidation? I have no idea of what the filter is made of...). Pat On 1 Jul 2009, at 5:07 PM, Scott Walsh wrote: Hi Matt, Check to make sure the A280 filter is clean. Ours was filthy. You might need to replace this (~$350). We were experiencing the same thing you mentioned and just had the GE technician out today. Also, make sure the A280 dial is set correctly on the lamp. Please check the manual for guidance. Best, Scott - Original Message - From: Matt Colins matt.colins2...@yahoo.commailto:matt.colins2...@yahoo.com Date: Wednesday, July 1, 2009 4:40 pm Subject: [ccp4bb] low UV reading on AKTA prime To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Hi all, We recently got low UV reading on our AKTA prime. We contacted GE healthcare, but was told that the UV reading on AKTA prime should be 20% of the spectrometer reading, because the path length of the flow cell of AKTA prime is only 2 mm (20% of the pathlength of a spectrometer cuvette). I am not sure whether that is the case, but we used to get much higher UV readings on the same AKTA prime (almost the same as the spectrometer reading). The UV reading just keeps dropping over time in the past several months. Here I have two questions. First, should the UV reading on AKTA prime be 20% of the spectrometer reading? Second, what could go wrong with our AKTA prime? (I know it is not the lamp, because we put in a new lamp and it didn't solve the problem) Thanks a lot! Matt --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edumailto:pat.l...@drexelmed.edu
Re: [ccp4bb] Lipid content/removal
At 12:01 PM -0500 7/7/09, Jacob Keller wrote: does anybody know of a simple, fast way to determine whether a protein sample contains lipid, and roughly to determine the quantity? As corollary, does anybody know of a quick way to extract/remove the putative lipid? In my case, I am working with an extremely robust/stable soluble protein which may contain bacterial lipids. Here is a reference describing an extremely robust/stable soluble protein (perhaps the same?) that when expressed as a recombinant protein in E. coli and purified will contain bacterial lipids. We used a chromatography resin that we called Lipidex VI to extract the lipids from this and related proteins without resorting to solvents that could denature the protein. McDonnell P.A. et al., Journal of Medicinal Chemistry 2006 49 (16), 5013-5017. Proteins treated with Lipidex in this way were fully competent for binding hydrophobic ligands in binding assays and in NMR and X-ray structural studies. I believe that Lipidex VI can now be obtained from Sigma (#H6258), but they really jacked up the price in recent years. You need to treat all of your columns, tubing, and tubes with ethanol to remove trace lipids that could get sucked back into/onto your freshly de-lipidated protein. You may also need to do several de-lipidation cycles to get rid of most of the lipid, depending upon the strength with which your protein binds them. Regards, - John --
