Re: [ccp4bb] Kd's in Crystals
Determination of Kd in crystal using only crystallographic data look at The First Direct Determination of a Ligand Binding Constant in Protein Crystals Wu SY, Dornan J., Kontopidis G., Taylor P., Walkinshaw M.D. Angew Chem Int Ed Engl, 2001, 40, 582-586. George --- George Kontopidis Associate Professor of Biochemistry Head of Biochemistry Veterinary School, University of Thessaly Trikalon 224, Karditsa 43100, Greece Tel: +30 24410 66017 Mob: 69 342 643 75 Fax: +30 24410 66041 e-mail: gkontopi...@vet.uth.gr web site: http://www.vet.uth.gr/english/departments_biochemistry.html --- -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Steven Herron Sent: Monday, June 27, 2011 9:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Kd's in Crystals I had success using crystallography to measure the Ca2+ affinity (in the mM range) for a Ca2+ dependent enzyme. See: Characterization and implications of Ca2+ binding to pectate lyase C. Herron SR, Scavetta RD, Garrett M, Legner M, Jurnak F. J Biol Chem. 2003 Apr 4;278(14):12271-7. We measured the occupancy of the Ca2+ ion using three different pH's and 3-4 different Ca2+ concentrations. The presence of the Ca2+ ion altered the conformation of two residues in the binding pocket. In several of the Ca2+ soak experiment the occupancy was between 35% and 70%, where both orientations of the side chains could be modeled separately and their occupancy values refined (see attached picture). We confirmed our crystallographic Kd approach using tryptophan fluorescence. Since it was difficult to measure mM binding affinities using dialysis or titration calorimetry, we turned to crystallography (since we had lots of crystals and beam time). Steve Jacob Keller wrote: Dear Crystallographers, what is the dogma with regard to affinities in crystals? For example, if I soak three crystals in 1pM, 1nM, and 1uM compound X, and they all show equivalent density, does that mean that the affinity is really better than 1pM, or is the crystal of such a high local concentration (~600mg/mL) that it will be fully occupied at nearly any concentration, provided external ligand concentration does not change due to binding in the crystal? I guess there is also the problem that the crystallization solutions are very non-physiological, but neglecting that, is there any straightforward way to think of this, or is there a good reference? Jacob Keller
[ccp4bb] changing residue numbering in Coot
Hello, I am trying to use Coot to change the numbering of the residues in a PDB file. The structure contains six semi-identical lettered chains, currently described as D through I. For some reason, the feature in coot that should allow me to change them to A-F, as well as change the residue numbering, will only allow me to alter the first chain. Any ideas? The only other solution I can think of is to directly alter the text of the PDB file, but the tedious nature of the task and the possibility of introducing mistakes makes it rather unattractive. Thanks in advance, Katie Molland Purdue University, Department of Biology
Re: [ccp4bb] changing residue numbering in Coot
On 28/06/11 12:13, Katrina L Molland wrote: I am trying to use Coot to change the numbering of the residues in a PDB file. The structure contains six semi-identical lettered chains, currently described as D through I. For some reason, the feature in coot that should allow me to change them to A-F, as well as change the residue numbering, will only allow me to alter the first chain. Any ideas? The only other solution I can think of is to directly alter the text of the PDB file, but the tedious nature of the task and the possibility of introducing mistakes makes it rather unattractive. So, IIUC, you can change D - A, but E - B fails. E - B should fail if there is a B chain already - even if it has only one atom in it. If there is no B chain, then E - B should work. If it does not work you may well have discovered a bug. What does the console say? You might like to try the following (Calculate - Scripting - Scheme): (change-chain-id 0 B E 1 1 999) Where the 0 is the molecule number of the model you are trying to modify - you might need to change that and 999 is the last residue number in the chain - you might need to modify that too. Paul.
Re: [ccp4bb] Sc calculation
Hi Fahimeh, what has happened here is that the interface area in the new file is very small, particularly after the periphery is trimmed (see the original paper for how this is defined). For the old file, the area is about 1100 Angs**2, for the new it is about 110 Angs**2. As the log file states, Sc is then not a very good measure of complementarity. sincerely Mike Lawrence Hello :) I am trying to find the shape complementarity of two structures. both of them are two layer of betasheet, that I have excluded hydrogens. in one of them (old.pdb, attached to the email) I have two complete layer in which chain A are all the strands in one layer and chain B are all the strands in the other layer. and in the other structure (new.pdb) I removed two strands from the middle of betasheet. I run Sc and the results are attached in sc_old.log and sc_new.log As you can see, sc factor is higher in the second structure , which is missing two strands in the middle?! How it is possible? I am confused ;) Am I missing something? Thank you in advance Fahimeh __ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. __
[ccp4bb] AW: [ccp4bb] changing residue numbering in Coot
Hi, worth to try moleman - %moleman Choose READ_pdb_file - your_pdbfile.pdb Choose AUTO_chain_segid Choose WRITE_pdb_file - out.pdb Or the tedious NEDIT way Open your pdb.file in nedit, mark ONLY the chain ID column - put your cursor before the first X, press [Shift][Ctrl], go the the last X in your chain. Then perform a replace/Find by selecting only the Selection option ... done. Repeat for each chain. Cheers Stefan __ Dr Stefan Gerhardt Albert-Ludwigs-Universität Freiburg Inst. f. Org.Chemie u. Biochemie Raum 911 Albertstrasse 21 79104 Freiburg office: +49 761 2035970
Re: [ccp4bb] changing residue numbering in Coot
Dear Katie, the menu entry Calculate - Change Chain ID works fine, although in the current version (0.6.1), you cannot use the preselected Whole Chain radio button but have to explicitly tell coot the residue range 'from' - 'to'. Tim On Tue, Jun 28, 2011 at 07:13:56AM -0400, Katrina L Molland wrote: Hello, I am trying to use Coot to change the numbering of the residues in a PDB file. The structure contains six semi-identical lettered chains, currently described as D through I. For some reason, the feature in coot that should allow me to change them to A-F, as well as change the residue numbering, will only allow me to alter the first chain. Any ideas? The only other solution I can think of is to directly alter the text of the PDB file, but the tedious nature of the task and the possibility of introducing mistakes makes it rather unattractive. Thanks in advance, Katie Molland Purdue University, Department of Biology -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A pgpjDpB2BtbhE.pgp Description: PGP signature
[ccp4bb] Protein scientist position at Syngenta
Protein Scientist £28,000 - £35,000 + benefits Jealott’s Hill, Bracknell, Berkshire As an independent worker, you will be a part of our Protein Production Team, providing protein expression/purification support in a timely manner across a range of Crop Protection Research projects. This will involve carrying out small to medium-scale protein purification and delivering purified proteins for assays and crystallography and communicating results to the project team to influence/guide project decisions. You will also have the opportunity to conduct expression screening in E.coli (essential), insects and yeast (desirable), as well as purifying and characterising difficult to express proteins in heterologous systems with genes from a variety of sources. With a BSc/PhD in protein biochemistry and or relevant lab experience you will have the skills in isolating proteins from complex mixtures, both native and recombinant sources. Experience in working with membrane protein and kinases would be highly advantageous. Bioinformatics skills to analyse DNA and protein data and the ability to deal with academic and industrial collaborators, including CROs, are also desirable. You should also have the ability to learn quickly and apply new skills with excellent organisational skills. To apply online, please visit www.syngenta.com and navigate to our current vacancies via the career page. Closing date: 1st July 2011. Syngenta is one of the world's leading companies with more than 26,000 employees in over 90 countries dedicated to our purpose: Bringing plant potential to life. Through world-class science, global reach and commitment to our customers we help to increase crop productivity, protect the environment and improve health and quality of life. For more information about us please go to www.syngenta.com
[ccp4bb] PhD student position
Dear colleagues, I would appreciate it if you could bring this off-topic opportunity to the attention of any suitable candidates. Petri --- A PhD student position is available in the group of Dr. Petri Kursula, to use neutron scattering methods to study proteins specifically expressed in the vertebrate myelin sheath. These proteins have functions e.g. in the interactions between the myelin sheath and the axon, and in the compaction of the multilayered myelin membrane. The project will involve large-scale recombinant expression of myelin proteins and studying their structure and function using mainly neutron scattering. The student will focus on the structure and dynamics of myelin proteins and their complexes, as well as their interactions with membranes. Complementary experiments will be carried out using X-rays and other biochemical/biophysical methods. The ideal candidate will: - have an MSc degree in biochemistry, physics, or a related field - have experience in recombinant protein expression and purification and/or in X-ray and neutron scattering methods - be genuinely interested in using neutrons to study biological macromolecules - be fluent in English The selected candidate will be affiliated with the Department of Biochemistry, University of Oulu, Finland, but a large part of the work is expected to be carried out at the Centre for Structural Systems Biology (CSSB-HZI), on-site the DESY synchrotron campus, Hamburg, Germany. The work will also involve performing measurements at international neutron infrastructures, and will be carried out in collaboration with staff at such facilities, including the ILL (Grenoble). The position will be funded by the European Spallation Source (ESS). More information about our group can be found at www.biochem.oulu.fi/kursula, and informal queries by email are also welcome. To apply, please send your cv, including list of publications, plus the names and email addresses of 2-3 referees by email to petri.kurs...@oulu.fi. The application deadline is July 31st, 2011. --- Petri Kursula, PhD Group Leader and Docent of Neurobiochemistry (University of Oulu, Finland) Visiting Scientist (CSSB-HZI, DESY, Hamburg, Germany) www.biochem.oulu.fi/kursula www.desy.de/~petri petri.kurs...@oulu.fi petri.kurs...@desy.de ---
[ccp4bb] Postdoctoral position available
POSTDOCTORAL POSITION AVAILABLE Applications are invited for a postdoctoral position to investigate the structural and cellular biology of viral immune evasion tactics and assembly mechanisms of Bunyaviruses, the largest family of negative-strand RNA viruses. The family Bunyaviridae includes a number of emerging viruses of human and agricultural importance and much remains to be discovered about their life cycle and the mechanisms they use to subvert the innate immune system. The following is an example of our recent work in this area: James TW, et al. (2011). Proc Natl Acad Sci U S A. 108(6):-7. A number of exciting projects are underway and ready to carry forward. More information about our lab can be found at: http://home.cc.umanitoba.ca/~bmark/Welcome.html We seek enthusiastic applicants who hold a PhD in biochemistry or related field. Candidates must have a strong background in molecular biology and protein X-ray crystallography. Expertise in mammalian cell culture and fluorescence microscopy would be considered strong assets. Our group is located in the Department of Microbiology, University of Manitoba, Winnipeg, Canada. The laboratory includes state-of-the-art X-ray instrumentation (Rigaku) and crystallization robotics (Art Robbins). We have routine access to the Canadian Light Source Synchrotron via mail-in service, remote control of robotics, or in person (within driving distance). The University of Manitoba (http://umanitoba.ca/) is the largest University in the province (over 30,000 students, faculty, and staff) and hosts a dynamic biomedical research community. Winnipeg is a vibrant, multicultural city with numerous cultural events, festivals and a brand new NHL hockey team (http://www.destinationwinnipeg.ca/). With over 110,000 fresh water lakes in our province alone, there is also much to explore for those who love the outdoors. Salary will be in accordance with the Canadian Institutes of Health Research (CIHR) (http://www.cihr.ca/ ) standards. Fellowship funding is also available through agencies such as the CIHR and the Manitoba Health Research Council (MHRC) (http://www.mhrc.mb.ca/). All qualified candidates are encouraged to apply. Please direct formal and informal inquiries and CV's (including names of three referees) to my email address: brian_m...@umanitoba.ca = Brian Mark, MSc, PhD Associate Professor Manitoba Research Chair in Structural Biology Department of Microbiology Department of Biochemistry and Medical Genetics Mailing/Courier address: Department of Microbiology 418 Buller Building University of Manitoba Winnipeg, Manitoba CANADA R3T 2N2 Phone (204) 480-1430 Fax (204) 474-7603 Web: http://home.cc.umanitoba.ca/~bmark/Welcome.html
[ccp4bb] selenomethionine contact
Hi -- I have a case in which I have a refined structure in which the 'so-far' measured distance between the Se atom of a selenomethionine and its crystallographic neighbouring Se atom (same residue) is 2.9 A, and I see continuous density between the two atoms. Has anyone subscribing to this newsgroup come across a case in which methionine S atoms and/or selenomethionine Se atoms form disulfide/diselenide bonds in the crystal, and what does that mean in terms of its possible presence biologically? Thanks for your help Alex -- Dr. Alex Singer C.H. Best Institute 112 College St. Room 70 University of Toronto Toronto, Canada, M5G 1L6 416-978-4033
Re: [ccp4bb] selenomethionine contact
Sounds to me like an artifact of the two-fold (I think there must be one between the two atoms if it is the same residue?). I think things get dicey near the symmetry axes... JPK On Tue, Jun 28, 2011 at 6:25 PM, Alexander U. Singer alexander.sin...@utoronto.ca wrote: Hi -- I have a case in which I have a refined structure in which the 'so-far' measured distance between the Se atom of a selenomethionine and its crystallographic neighbouring Se atom (same residue) is 2.9 A, and I see continuous density between the two atoms. Has anyone subscribing to this newsgroup come across a case in which methionine S atoms and/or selenomethionine Se atoms form disulfide/diselenide bonds in the crystal, and what does that mean in terms of its possible presence biologically? Thanks for your help Alex -- Dr. Alex Singer C.H. Best Institute 112 College St. Room 70 University of Toronto Toronto, Canada, M5G 1L6 416-978-4033 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
