[ccp4bb] metal binds?
Dear ccp4 users I would like to know, is there a way to check that heavy metal has bound to crystals before I take it to synchrotron which is far away? thanks Careina
Re: [ccp4bb] metal binds?
yes u can run Native page and on that if u will see shift then crystals containing heavy atom regards On Fri, Mar 25, 2011 at 1:09 PM, Careina Edgooms careinaedgo...@yahoo.comwrote: Dear ccp4 users I would like to know, is there a way to check that heavy metal has bound to crystals before I take it to synchrotron which is far away? thanks Careina -- Vandana Kukshal Postdoc Fellow structure biology group international Center For Genetic Engineering and Biotechnolgy Aruna Asaf Ali Marg 110 067 New Delhi INDIA
Re: [ccp4bb] metal binds?
a quick google search turned up: http://people.mbi.ucla.edu/sawaya/tutorials/Phasing/gel.pdf Acta Cryst. (2000). D56, 161-168[ doi:10.1107/S0907444999015188 ] Mass-spectrometry assisted heavy-atom derivative screening of human Fcinline: gamma_rmgif.gifRIII crystals P. D. Sun and C. H. Hammer Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 25 Mar 2011, at 08:39, Careina Edgooms wrote: Dear ccp4 users I would like to know, is there a way to check that heavy metal has bound to crystals before I take it to synchrotron which is far away? thanks Careina
Re: [ccp4bb] metal binds?
On Mar 25, 2011, at 08:39 , Careina Edgooms wrote: Dear ccp4 users I would like to know, is there a way to check that heavy metal has bound to crystals before I take it to synchrotron which is far away? thanks Careina Here's a useful reference: Boggon TJ, Shapiro L Screening for phasing atoms in protein crystallography Structure. 2000 Jul 15;8(7):R143-9 http://www.ncbi.nlm.nih.gov/pubmed/10903954 HTH, Luca Luca Jovine, Ph.D. Assistant Professor EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 57 Huddinge, Sweden Voice: +46.(0)8.524-81136 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org
[ccp4bb] Postdoctoral position in Grenoble, France - Living deep
*Living deep: Structural basis of piezophilic adaptation* ELMA group. Institut de Biologie Structurale J.-P. Ebel -- Grenoble -- France http://www.ibs.fr/groupes/groupe-extremophiles-et-grands/ We are recruiting a post-doctoral scientist interested in life adaptation to extreme conditions. The position is for 2 years and is funded by the french national agency for research (ANR) A high-pressure environment characterizes our planet. For more than 60 % of the biosphere pressure is greater than 100 bars and it can reach 1000 bars in the deepest sea trenches, like the Marianna's trench. However, in these extreme conditions, a vast biodiversity of adapted micro-organisms (piezophiles) can be encountered. So far, the molecular mechanisms that facilitate proteins function under high pressure remain obscurs. Beside interests to understand fundamental protein folding processes and life evolution, this information is essential to develop biotechnological application from deep sea enzymes. This work will be performed in the framework of a collaborative network including ENS Lyon and IFREMER-UBO in Brest in which a new archaeon from the Pyroccoccus phylum was recently discovered and was called Pyrococcus yayanosii CH1. This microbe is the first obligate piezophile as it is only able to grow under pressure (optimal growth: 98°C and 520 bars). The project is based on the study of two protein families, for which the biochemistry is well handled by the team: large peptidases complexes and lactate/malate dehydrogenase (LDH/MalDH). These proteins play key roles in metabolic adaptation to deep sea ecosystem and allow combining structural approaches (protein crystallography, small-angle scattering...) and biochemical approaches (oligomer stability, enzymatic activity measurements...). The candidate will study the molecular effects of high pressure/temperature by comparing the structural and functional properties of different proteases and LDH/MalDH from Thermococcus and Pyrococcus phylums (including Pyrococcus yayanossi CH1) isolated at various water depths. The ELMA team possesses a fully-fledged wet laboratory for all biochemical steps and the project will benefit from privileged access to large facilities (synchrotron and neutron sources) as well as from latest developments in high-pressure biophysics: high-pressure protein crystallography, for which the ELMA team is a world leader, small-angle x-ray scattering under high-pressure, UV-Visible spectrophotometer and fluorimeter both equipped with a high-pressure vessel. The candidate should have a background in biochemistry with a strong interest in structural approaches or a physico-chemistry background with a strong interest for biophysics. Candidatures, including CV, intention letter and the names and contact information of references, as well as informal inquiries should be sent to Eric Girard (eric.girard at ibs.fr) and Bruno Franzetti (bruno.franzetti at ibs.fr). Relevant publications from the team: Coquelle et al (2007) Activity, stability and structural studies of lactate dehydrogenases adapted to extreme thermal environments. J Mol Biol 374: 547-562. Dura et al (2009) The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea. Mol Microbiol 72: 26-40. Girard et al. (2011). Structure-function perturbation and dissociation of tetrameric urate oxidase by high-hydrostatic pressure. Biophys J 98: 2365-2373. -- *** Eric Girard Extremophiles and Large Macromolecular Assemblies (ELMA) group Institut de Biologie Structurale J.-P. Ebel UMR 5075 CEA-CNRS-UJF-PSB 41, Rue Jules Horowitz 38027 Grenoble Cedex 1, France Web site: http://www.ibs.fr/ ***
Re: [ccp4bb] Tangential Flow Filtration device
We use the Millipore Spiral-wound TFF1 and TFF2 cartridges for concentrating 10-20L of insect cell media. They work great, don't cost all that much money, and you don't need the expensive holder they sell. You can use a ring stand and clamp the tubing directly to the cartridge. Our cartridges are several years old and have seen hundreds of liters of media, if you clean them well and pre-clear the media(we use a 10,000xg spin) they will last longer and keep a good flow rate. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Thu, Mar 24, 2011 at 6:08 PM, Junyu Xiao jx...@ucsd.edu wrote: Dear all, Sorry for non-crystallography related questions. I am looking for a Tangential Flow Filtration device to concentrate large volumes of media (several liters) for protein purification. I am wondering whether I could get some suggestions on my choices. It appears that both Pall and Millipore carry this kind of products. I would really like to get some information on the pros/cons of different models. Any kind feedback will be highly appreciated. Best regards, Junyu --- Junyu Xiao, Ph.D. University of California, San Diego Leichtag Room 283 9500 Gilman Drive, 0721 La Jolla, CA 92093-0721 Lab phone: 858-822-0684
[ccp4bb] data processing deviations chisq
Hi all, I have collected one iodine soaked data in our home source, and processing the data using HKL2000. Exposure time per frame is 5min/1 degree. While processing I have noticed that the Chisq values, cell parameters and rotation change Vs frame are deviating like anything. Please find the picasa link to see the carves. I will be benefited if anyone kindly suggest me the possible cause of these deviations. https://picasaweb.google.com/118341875228875389610/Mar252011?authkey=Gv1sRgCJXe6-ncmt6KmwE# Thank you all for your suggestions, Sincerely, Debajyoti
Re: [ccp4bb] data processing deviations chisq
The most likely explanation is that you have a cracked crystal or your crystal has split from radiation damage around the frame 70. If you had a cracked crystal from the start, the spot overlap between crystals would change when the sample was rotated. In such cases, it may happen that the program starts refining a different subgroup of crystals as the one that defines crystal parameters. If these crystals are isomorphous to each other, the scaling can correct for such a variable exposure/integration. If the crystals are not isomorphous, which sometimes happens, you have a problem. In such a case it would probably be better to restrict the scaling to the initial 70 frames. Sometimes it helps to reprocess the data with different spot integration size. There are two opposing strategies that could be beneficial: 1) reduce the spot integration size to narrow down the integration to a single crystal 2) increase the spot integration size to integrate diffraction from a group of crystals with similar orientation. If these are uniformly integrated, the phasing signal should be still preserved. If your crystal has split from radiation damage, the strategy 2 may help, but frequently the cracking induced by radiation damage is a very bad sign (crystal lattice has changed so the crystal is no longer isomorphous with its initial state). Scaling provides the ultimate diagnostic of the problem's seriousness. Statistics from integration that you provided are secondary, but may help in pinpointing the source of the problem. One can disregard them if the scaling is good. The presence of iodine creates additional factors. It increases X-ray absorption and so the radiation damage, but iodine is also a quencher of radicals and tends to reduce structural changes induced by radiation. Hope that helps, Zbyszek Otwinowski Hi all, I have collected one iodine soaked data in our home source, and processing the data using HKL2000. Exposure time per frame is 5min/1 degree. While processing I have noticed that the Chisq values, cell parameters and rotation change Vs frame are deviating like anything. Please find the picasa link to see the carves. I will be benefited if anyone kindly suggest me the possible cause of these deviations. https://picasaweb.google.com/118341875228875389610/Mar252011?authkey=Gv1sRgCJXe6-ncmt6KmwE# Thank you all for your suggestions, Sincerely, Debajyoti Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
[ccp4bb] PDBe introduces Quips - QUite Interesting PDB Structures
Hi all, As part of its recent winter update, the Protein Data Bank in Europe (PDBe; http://pdbe.org) introduced Quips - QUite Interesting PDB Structures. Quips are short stories about one or more interesting or topical structures, coupled with an interactive viewer and often a tutorial that allows you to do more detailed exploration of a structure using PDBe resources. Try it out at: http://pdbe.org/quips Perhaps you have come across macromolecular structures in paper figures and on journal covers but would like to delve deeper into the structures that interest you? We hope that Quips will be a starting point for further exploration of structures in the PDB archive and will (help you) answer questions about structural data. Quips are short articles and tutorials on structures picked from the PDB archive. More often than not, as the name suggests, these articles will focus on quite interesting PDB structures rather than daunting behemoths. The tutorials assume that you have a background in biology, chemistry or medicine and have an interest in proteins, nucleic acids and ligand interactions. New Quips will be added approximately once a month. They are currently produced by PDBe annotation staff, but we would be more than happy to host Quips produced by structural biologists who would like to sell their own structures (or even someone else's). Quips articles include interactive structure displays with a number of predefined (often animated) views to highlight points made in the text. Follow the tour of each structure by clicking on links to these views and they will appear, in real time, in the molecular viewer window. The viewer is also yours to command in case you want to spin the structure around or go on a detour to a different part of the structure. Afterwards simply rejoin the Quips tour by selecting the next view link. For more information about the user-interface, see http://pdbe.org/quips Some Quips will also introduce you to PDBe resources and services that can be useful in your own explorations of the wonderful world of structures. When a particular PDBe service is especially useful for understanding a Quips structure, we will provide a walk-through to show you how it works. This will get you started in using these services for your own structural searches and analyses. Our inaugural Quips (of 14 February ...) was entitled A deadly toxin with a romantic name: Panton-Valentine Leukocidin complex and was accompanied by a tutorial on how to use PDBeFold (a.k.a. SSM) to compare the structures of two PVL components and superimpose them on alpha-hemolysin. This Quips can be found at http://pdbe.org/quips?story=PantonValentine Our March Quips commemorates the fact that it is 20 years ago that the structure of Nerve Growth Factor (NGF) was determined. The accompanying tutorial shows you how to use PDBePISA to analyse the quaternary structure of NGF. To access this Quips, go to: http://pdbe.org/quips?story=NGFstory If you would like to use the Quips format to tell a story about any interesting structures of your own then please get in touch! We welcome your comments, bug reports and feature requests on Quips. Please use the feedback button at the top of any PDBe web page. --Gerard --- Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK ger...@ebi.ac.uk . pdbe.org Secretary: Pauline Haslam pdbe_ad...@ebi.ac.uk
Re: [ccp4bb] protein aggregation
Try taking the detergents out of your solublization buffer (that is if you know it's not a membrane protein) and only extract the buffer soluble portion. If you have a tag try switching the tag to a different terminus. Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com email: kraft_proteome_resea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so. --- On Wed, 3/23/11, Mark J van Raaij mjvanra...@cnb.csic.es wrote: From: Mark J van Raaij mjvanra...@cnb.csic.es Subject: Re: [ccp4bb] protein aggregation To: CCP4BB@JISCMAIL.AC.UK Date: Wednesday, March 23, 2011, 2:03 PM - try limited proteolysis to see if you can chop off a disordered region - consider the fact that, although it purifies nicely, your protein may not be well-folded do you have a biochemical activity test? Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 23 Mar 2011, at 18:59, Van Den Berg, Bert wrote: Try different detergents. Try 10% or more glycerol. Try adding ligands (if present/known). Try varying ionic strength and/or pH. Try giving more specifics so people on the board may be able to help you better. Bert On 3/23/11 1:51 PM, gauri misra kamga...@gmail.com wrote: The protein purifies nicely there is no problem in that. Just at the last step when it is concentrated it starts precipitating beyond a concentration of 1mg/ml. Already the purification buffers have the detergent. On Wed, Mar 23, 2011 at 1:44 PM, Kornelius Zeth kornelius.z...@tuebingen.mpg.de wrote: 2 M urea and detergents On Wed, 23 Mar 2011 13:41:55 -0400 gauri misra kamga...@gmail.com wrote: Hi, What are the different methods to prevent protein aggregation while concentrating so as to increase the concentration of the protein? I have some idea of adding EDTA and charged amino acids like L-Arg and L-Glu. I would appreciate if the readers share their experiences. Thanks! Gauri -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
Re: [ccp4bb] metal binds?
Collect all your diffraction data in your home lab, solve the phase problem, fit the map, refine, deposit coordinates in the PDB. _ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Careina Edgooms Sent: Friday, March 25, 2011 2:40 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] metal binds? Dear ccp4 users I would like to know, is there a way to check that heavy metal has bound to crystals before I take it to synchrotron which is far away? thanks Careina
