[ccp4bb] job offer - slightly off topic
Argonne National Laboratory has the following job offer: Requisition Number: 405283 Position Title: Postdoctoral Appointee Career Level: 700 Division: ASD-Accelerator Systems Division Location: Lemont, IL Offsite Work Location: ANL Shift: 8:30 - 5:00 Hours of Work: 40 The Accelerator Systems Division (ASD) of the Advanced Photon Source (APS) is offering a Postdoctoral position. You will work with a multidisciplinary team of scientists and engineers to carry out magnetic measurements and tuning of APS insertion devices (ID) and develop your own program in the enhancement of magnetic measurement capabilities at the APS. You will also have the opportunity to work in the areas of novel ID design as well as participate in the development and implementation of customized measurement techniques for future generation light sources. We expect you to have: A strong background in experimental system design, build-up, and commissioning. Good skills in running measurements, data collection and analysis. Familiarity with programming languages including Python, Matlab, C/C++, and LabVIEW. Please, contact Joseph Xu (x...@anl.gov) with informal inquires. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Beamline Scientist position at ANL
Dear all, I'd like to draw your attention to the beamline scientist position currently open at the Structural Biology Center, ANL. Job ID: 403448 Job Title: Beamline Scientist/Physicist Job Details Link: http://www.anl.gov/careers/apply-job/external-applicants?locale=en-us=https%3A%2F%2Fcareers.peopleclick.com%2Fcareerscp%2Fclient_argonnelab%2Fexternal%2Fen-us%2Fgateway.do%3FfunctionName%3DviewFromLink%26jobPostId%3D5827%26localeCode%3Den-us For informal inquiries, please contact Andrzej Joachimiak, andrz...@anl.gov Best, Karolina
Re: [ccp4bb] mystery feature near a PLP substrate
Hi, The Lys-PLP adduct looks like a perfectly happy internal aldimine. I do not see any indication of a covalent bond between PLP and the density in question indicating any other intermediate. Which, to my knowledge, would be rather hard to get accidentally. I do not know distances between these fairly well resolved peaks in the 2Fo-Fc map but I am guessing they would nicely accommodate solvent molecules. The peak closer to PLP is weaker, maybe just another partly occupied solvent molecule? Could the continuity of your difference map be coming from too low contour level? Best, Karolina W dniu 2017-05-24 09:16, Eleanor Dodson napisał(a): > Any ideas?
Re: [ccp4bb] cryoprotectant
Hi Reza, Check the following reference: Cryoprotection properties of salts of organic acids: a case study for a tetragonal crystal of HEW lysozyme. Bujacz G, Wrzesniewska B, Bujacz A. Acta Crystallogr D Biol Crystallogr. 2010 Jul;66(Pt 7):789-96. Cheers, Karolina W dniu 2014-12-01 10:59, Reza Khayat napisał(a): Hi, Has anyone used citrate as the sole cryoprotectant? If so, what concentration was needed? Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org [1] Links: -- [1] http://www.khayatlab.org
Re: [ccp4bb] High Salt Cryo
4M NaCl should work too. It worked for the conditions with 1.8 - 2.0 M NaCl. Karolina W dniu 2014-02-19 06:38, Mooers, Blaine H.M. (HSC) napisał(a): For crystals grown out of a 2 uL drop of 1.2-1.8 M LiSO4 or 1.6-2.4 M AmmSO4, we do in situ cryoprotection with sodium malonate. We add 2-4 uL of 1.9 M Na malonate to the crystallization drop, wait 10 seconds and add 2-4 uL of 2.4 M sodium malonate, repeat with 2.8 M and then 3.4 M. We do not bother withdrawing aliquots to maintain a fixed volume. You may need to tweak the volumes to optimize the resulting diffraction. You can also break the additions at given concentration into smaller aliquots to reduce the osmotic shock. This approach is much gentler than transferring the crystal directly to 3 M sodium malonate. Do not leave the drop exposed to the air for more than 3 minutes or so because salt crystals will start to grow. When there are multiple crystals in a drop, often the unused crystals in the very high salt solution will still diffract well up to a year later if the crystallization chamber is resealed well; their diffraction might even improve with the prolonged exposure to high salt. Blaine Mooers Assistant Professor Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center S.L. Young Biomedical Research Center Rm. 466 Shipping address: 975 NE 10th Street, BRC 466 Oklahoma City, OK 73104-5419 Letter address: P.O. Box 26901, BRC 466 Oklahoma City, OK 73190 office: (405) 271-8300 lab: (405) 271-8313 fax: (405) 271-3910 e-mail: blaine-moo...@ouhsc.edu Faculty webpage: http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d- [1] X-ray lab webpage: http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory [2] Small Angle Scattering webpage: http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0 [3] From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katherine Sippel [katherine.sip...@gmail.com] Sent: Tuesday, February 18, 2014 12:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] High Salt Cryo Hi all, I'm looking for a cryo condition for high NaCl (3+ M) crystallization condition. I would do it the proper way, but our beam/cryostream is down. I've tried a bunch of things at the moment. Ethylene glycol and PEG 400 nuke the crystals immediately even at low concentrations. Prolonged exposure to glycerol and sucrose starts to break them down so I'm thinking that the diffraction will probably suffer. I can't find any reports of NaCl's viability as a cryosalt. I've got Paratone/Paraffin oil/Mitegen's LV cryo oil on tap but I was hoping to not put all my eggs in one basket. I tried the ISRDB database through archive.comhttps://urldefense.proofpoint.com/v1/url?u=http://archive.comk=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0As=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e [4] without any luck (no search function). I've gone to the PDB searching for similar crystallization conditions and looked up the papers for their cryos, but they are all glycerol. Google gives me the same. I thought I'd see if anyone on the bb has an anecdotal this worked for us story. I would love to hear it. Thank you for your time, Katherine -- Nil illegitimo carborundum - Didactylos Links: -- [1] http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d- [2] http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory [3] http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0 [4] https://urldefense.proofpoint.com/v1/url?u=http://archive.comamp;k=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Aamp;r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Aamp;m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0Aamp;s=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e
[ccp4bb] which dimer?
Hi all, I'm working with a protein that appears to be a dimer in solution, on SEC in runs as 24 kDa, while the actual mass of a dimer is 30. And I am trying to figure out which dimer is the biological one (it is a regulatory protein but details are uknown). The crystal structure gives me a few options (mol A and B in ASU plus P6122 symmetry), but none of them is really convincing: for deltaGdiss PISA goes from -0.4 to 1.5 kcal/mol. Theoretically, I have two compact dimers (1 and 2) and one elongated (3). In 1, I have two hydrophobic helices interacting and four hydrogen bonds, 1550 A2 buried area (out of 14200 total). This interface applies only to molecule A and its crystallographic mate, equivalent molecules B are too far from each other. Moreover, even in the dimer made of mol A there are channel at the interface. Interface 2 is purely hydrophobic, but at least it's consistent, i.e there are comparable interactions for A-A and B-B pairs, 1850 A2 buried area Interface 3 involves non-crystallographic copies, buried area is 1040 A2. The interacting elements are proline-rich, and there are four main-chain - main chain hydrogen bonds plus two main-chain - side chain ones. Formally, these fragments are not classified as beta-strands, but the association does look like an intermolecular beta-sheet. This dimer is not consistent with the SEC data though. I'm assuming that with an elongated shape it would run as a bigger particle than it actually is, not as a smaller one. So I think I can discard first option but I am still debating on 2 and 3. I'll appreciate your comments on this. Karolina
[ccp4bb] small proteins that do not crystallize
Hello everybody, I'm looking for small proteins (up to 150 aa) that are known to express reasonably in E. coli, are soluble and folded but failed to crystallize. Can you provide some examples, especially of proteins that might be interesting for a broad community? Thanks, Karolina
Re: [ccp4bb] The effect of His-tag location on crystallization
You may want to have a look at 3ur7 and 3ur8. I have also another example of a glycohydrolytic enzyme with an N-terminal His-tag sitting in the active site of a symmetry-related molecule (not deposited yet). Karolina On 27/6/2012, Brad Bennett bradbennet...@gmail.com wrote: I think it was an N-terminal RGS-type His tag in 3O8Y (human lipoxygenase) that mediated crystal contacts with a symmetry related molecule. As I recall, this tag composed a B-strand that formed a nice interface with a native B-strand of the symmetry related molecule. Pretty cool... -Brad On Wed, Jun 27, 2012 at 11:00 AM, Phoebe Rice pr...@uchicago.edu wrote: With Flp recombinase - DNA complexes, a C-terminal His tag triggered a different (but sadly not better) crystal form, and the His side chains packed against the bases at the end of a neighboring DNA duplex. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 27 Jun 2012 10:14:58 -0400 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of R. M. Garavito rmgarav...@gmail.com) Subject: Re: [ccp4bb] The effect of His-tag location on crystallization To: CCP4BB@JISCMAIL.AC.UK Most of the comments you will get will be anecdotal in that people will report the successful results and do not take the time or effort to characterize the less successful results. This often occurs because the tagged portion of the protein is most often disordered, even in the best crystals. Thus, other than saying tagging on this end works, but tagging on that end doesn't, there is little more you can say. Each case will be different, and it is almost impossible to arrive at any generalized conclusion. We prefer C-terminal tagged proteins for a number of reasons, but if an N-terminally tagged protein crystallizes well, so be it. Of the dozens of N- and C-tagged protein structures we have solved in my lab and with collaborators, I have only seen one case of an ordered His-tag: the His residues had coordinated Cd ions, which proved essential for getting good crystals. However, beyond that there was not much more to say. For your protein and the resulting crystals, an N-terminally tagged protein crystallized well. Whether you can draw any more conclusions from these results depends on characterizing crystals of both N- and C-tagged proteins. Just assuming that the C-tagged protein is trying to crystallize in the same or related crystal form as the N-tagged protein is an unwarranted assumption without experimental evidence to back it up. That is why most groups just run with the winner. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com On Jun 26, 2012, at 9:06 PM, weliu wrote: Dear all, We crystallized a protein and found that crystal quality greatly depended on the location of His-tag. When a His-tag was added at the C-terminus, only crystalline precipitate or spherical quasi crystals were grown. However, when the His-tag was moved to the N-terminus, single crystals were grown under a number of conditions, and the best one diffracted to 1.7 angstrom after optimization. I was wondering if there were published reports describing similar cases. Thank you in advance Wei Liu
[ccp4bb] ligand density
Hi all, I have a ligand-corresponding density which I cannot identify: http://s1194.photobucket.com/albums/aa376/dziuba22/?action=viewcurrent=coot1.png Here is the story. I collected a couple of datasets, three of them belong to methylated protein and one to non-methylated. The protein is putative chitinase. Methylation reaction was carried out in a buffer composed of HEPES, NaCl, BME, imidazole, glycerol. For the reaction, formaldehyde and dimethylamine borane complex were added and reaction was quenched with glycine. After that, buffer was exchanged to HEPES, NaCl and DTT. The non-methylated protein was stored in the same buffer. Both proteins are His-tagged. The density that I'm showing here is present only in the crystals of methylated protein, both were grown form PEG3350 and either NaI or NaSCN. I have been trying to model something and histidine nicely fits on the left side, but right fragment seems to be more complicated. In terms of geometry, D-Thr-L-His would fit: http://s1194.photobucket.com/albums/aa376/dziuba22/?action=viewcurrent=coot4.png But the problem is that instead of CB from Thr I need a proton donor to interact with a neighboring Asp (unless it is C-H...O interaction). Moreover, if such a compound were grabbed from bacterial cells I would expect it to be present also in non-methylated crystals, which is not the case. This made me think of some sort of intermediate of methylation reaction as dimethylamine borane complex would fit in Thr position with nitrogen atom replacing CB, but with borane I could not find an explanation for an equivalent of Thr amino group. Moreover, I do not know what was the source of free histidine in the methylated protein prep. A separated big peak on right corresponds to an anion and it is also conserved in methylated crystals. In case of crystals obtained from NaI it is an iodide anion. I will appreciate any suggestions, Karolina
