[ccp4bb] Protein aggregation and crystallization
Hi All, I am working on a protein which has a membrane spanning region and as cytosolic domain.I have made various deletion constructs of the protein, so that I can have a crystallizable fragment. There is no homologues mentioned in the pdb for this protein. All of these constructs are purified successfully but when concentrated and loaded on a gel filtration column Superdex-200, they elute in the void volume. But the proteins donot precipitate out !! Is it worth while to go ahead for crystallization trials?? Any other suggestion is most welcome. Thanks Anita
[ccp4bb] waters shell analysis
Once waters have been located and refined is there a program that analyses their positions in terms of solvation shells? Can the results be compared easily with those from related known protein structures? Rex Palmer http://www.bbk.ac.uk/biology/our-staff/emeritus-staff http://rexpalmer2010.homestead.com
[ccp4bb] Postdoc Position at EMBL Grenoble
The Berger laboratory at the European Molecular Biology Laboratory (EMBL) Grenoble, France seeks to recruit an outstanding postdoctoral scientist in structural biology with a research focus directed towards the structure of macromolecular assemblies. The major theme within the group is the structural molecular biology of multiprotein complexes in human gene expression. For this purpose, we are also very active in the development and implementation of advanced recombinant expression technologies (Fitzgerald et al. Nat. Methods 2006; Bieniossek et al. Nat. Methods 2009; Kriz et al. Nat. Communications 2010). Notably our MultiBac technology is our unique asset for producing large multisubunit transcription factors in the quality and quantity required for structural analyses (Berger et al. Nat. Biotechnol. 2004, Imasaki et al. Nature 2011). A number of highly purified complexes are available in the laboratory. Information about our work can be obtained from http://www.embl.fr/research/unit/berger/index.html. This position requires a Ph.D. in biochemistry or a related field, with a strong background in molecular biology and protein biochemistry. The applicant has experience in structure determination projects from start to finish, experience in expressing and purifying protein complexes and /or Electron Microcopy or X-ray crystallography are advantageous. The candidate is a highly motivated individual who enjoys working as part of a collaborative and multidisciplinary, dynamic and interactive team. The laboratory is excellently situated in the structural biology environment at the Polygone Scientifique in Grenoble. Access to our Eukaryotic Expression Facility (EEF), synchrotron X-ray radiation (ESRF), modern biophysical instrumentation (surface plasmon resonance, dynamic light scattering, isothermal calorimetry, analytical ultracentrifugation), and to electron microscopy is provided. Complementary collaborations with leading laboratories in the field are in place. The position is available immediately and funded for 2 years. It can be extended dependent on performance. To apply please send your CV, a statement of research interests, and names (including email address) of at least two referees by email to Dr. Imre Berger (iber...@embl.fr). Applications will be accepted until the position is filled.
[ccp4bb] identifying hinges in flexible proteins
We have structures of two states of a large multi-domain protein in which domain movements of up to 30 degrees are observed. Is there a programme available which attempts to determine rigid-body units and hinge regions in an 'unbiased' way? thanks for your suggestions, Stephen Cusack -- ** Dr. Stephen Cusack, Head of Grenoble Outstation of EMBL Group leader in structural biology of protein-RNA complexes and viral proteins Joint appointment in EMBL Genome Biology Programme Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI) ** Email: cus...@embl.fr Website: http://www.embl.fr Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 Fax:(33) 4 76 20 7199 Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, 6 Rue Jules Horowitz, 38042 Grenoble, France **
Re: [ccp4bb] identifying hinges in flexible proteins
Dear Stephen, I find DynDom very good (http://fizz.cmp.uea.ac.uk/dyndom/) otherwise the morph server (http://molmovdb.mbb.yale.edu/molmovdb/morph/) also performs similar analysis, cheers, Matt. On 26/08/2011 11:25, Stephen Cusack wrote: We have structures of two states of a large multi-domain protein in which domain movements of up to 30 degrees are observed. Is there a programme available which attempts to determine rigid-body units and hinge regions in an 'unbiased' way? thanks for your suggestions, Stephen Cusack -- Matthew Bowler Structural Biology Group European Synchrotron Radiation Facility B.P. 220, 6 rue Jules Horowitz F-38043 GRENOBLE CEDEX FRANCE === Tel: +33 (0) 4.76.88.29.28 Fax: +33 (0) 4.76.88.29.04 http://go.esrf.eu/MX http://go.esrf.eu/Bowler ===
Re: [ccp4bb] identifying hinges in flexible proteins
DynDom version 1.5 is part of the CCP4 suite. There is an interface for it under Program List. Although Steve Hayward appears to have a new version DynDom3D which we don't have. No idea if it would be better for your case. HTH Martyn On Fri, 2011-08-26 at 11:28 +0200, Matthew BOWLER wrote: Dear Stephen, I find DynDom very good (http://fizz.cmp.uea.ac.uk/dyndom/) otherwise the morph server (http://molmovdb.mbb.yale.edu/molmovdb/morph/) also performs similar analysis, cheers, Matt. On 26/08/2011 11:25, Stephen Cusack wrote: We have structures of two states of a large multi-domain protein in which domain movements of up to 30 degrees are observed. Is there a programme available which attempts to determine rigid-body units and hinge regions in an 'unbiased' way? thanks for your suggestions, Stephen Cusack -- *** * * * Dr. Martyn Winn * * * * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * * Tel: +44 1925 603455E-mail: martyn.w...@stfc.ac.uk* * Fax: +44 1925 603634Skype name: martyn.winn * * URL: http://www.ccp4.ac.uk/martyn/ * ***
Re: [ccp4bb] number of cycles in refmac
Frank, Point #1 - fair point; the reason Rfree is popular, though, is because it is a *relative* metric, i.e. by now we have a sense of what good is. So I predict an uphill fight for LLfree. Why? I don't see any difference. As you say Rfree is a relative metric so your sense of what 'good' is relies on comparisons with other Rfrees (i.e. it can only be 'better' or 'worse' not 'good' or 'bad'), but then the same is true of LLfree (note that they both assume that exactly the same data were used and that only the model has changed). So when choosing between alternative model parameterisations in order to minimise over-fitting we compare their Rfrees and choose the lower one - same with LLfree, or we compare the observed Rfree with the expected Rfree based on Rwork and the obs/param ratio to check for problems with the model - same with LLfree. In fact you can do it better because the observations in LLfree are weighted in exactly the same way as those in the target function. Point #2 would hold if we routinely let our refinements run to convergence; seems common though to run 10 cycles or 50 cycles instead and draw conclusions from the behaviour of the metrics. Are the conclusions really much different from the comparison-at-convergence you advocate? Which is in practice often less convenient. You might do 10 cycles for a quick optimisation of the coordinates, but then I wouldn't place much faith in the R factors! How can you draw any conclusions from their behaviour: there's no way of predicting how they will change in further cycles, the only way to find out is to do it. I'm not saying that you need to refine exhaustively on every run, that would be silly since you don't need to know the correct value of the R factors for every run; but certainly on the final run before PDB submission I would regard stopping the refinement early based on Rfree as implied in Tim's original posting as something akin to 'cheating'. Cheers -- Ian
[ccp4bb] Crystals with Organic solvents
Dear All I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like *broccoli *shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same is there is any idea to increase the size and shape of my protein crystals. Any suggestions will be helpful for me Thanks in Advance
Re: [ccp4bb] Crystals with Organic solvents
Depending on how big your broccoli is I would simply crush them and mount as big of a piece as possible and see how well it diffracts. I assume you have tried already: - glycerol - changing protein:reservoir ratios - temperature - adding oil to either / and the reservoir, your drop -crushed up some broccoli and recreened your original sparse matrix screen while seeding with chunks of broccoli seeds - when you say the His-tag needs to be cleaved otherwise it will precipitate, that mitt also just be either leaching Ni from your column or simply the imidazole - You don't say how you purify your protein, I hope you are not just setting up after the Ni capture step and at least have some other method following e.g. anion or SEC Just a few incomplete suggestions, Jürgen On Aug 26, 2011, at 6:56 AM, eswar reddy wrote: Dear All I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like broccoli shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same is there is any idea to increase the size and shape of my protein crystals. Any suggestions will be helpful for me Thanks in Advance .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Crystals with Organic solvents
I would definitely try gelfiltration (how do you get rid of the cleaved tag anyway, sample buffer exchange?) but especially ion exchange. A homogeneous sample on SDS-PAGE and/or gelfiltration is often not (at all) homogeneous in ion exchange. Beyond that i would make some point mutations on surface residues and focus on those (ie try the surface entropy method). Good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of eswar reddy [eswar.uo...@gmail.com] Sent: Friday, August 26, 2011 6:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystals with Organic solvents Dear All I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like broccoli shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same is there is any idea to increase the size and shape of my protein crystals. Any suggestions will be helpful for me Thanks in Advance
[ccp4bb] Cadmium sites and co-ordinations in structure
Hi, I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and determined its structure. It turns out that I see several metal sites in the structure, mostly cadmiums. Is there any information published (preferably a review) which summarises data on cadmium sites in proteins such as for example the possible coordination numbers of cadmium, distances, type of side chains found to coordinate with cadmium, etc.? I could extract all this from the PDB, but a nice review would be simpler to start with. Thank you in advance for your help Sandeep
Re: [ccp4bb] Cadmium sites and co-ordinations in structure
Hi Sandeep, if someone sends one, kindly share the references. In general, Ca2+ could have more Asp, Asn kind of coordination and distorted pentagonal bipyramidal geometry with waters (about 2.5A), Cd can also have S- since it is softer, I guess Co might have N/O/S (i.e all three with paired electrons). An inorganic chemistry textbook like Greenwood Earnshaw or Cotton Wilkinson could be handy.. or a bioinorganic chemistry book. HTH, Partha On Fri, Aug 26, 2011 at 7:24 PM, Sandeep s.talapa...@beatson.gla.ac.ukwrote: Hi, I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and determined its structure. It turns out that I see several metal sites in the structure, mostly cadmiums. Is there any information published (preferably a review) which summarises data on cadmium sites in proteins such as for example the possible coordination numbers of cadmium, distances, type of side chains found to coordinate with cadmium, etc.? I could extract all this from the PDB, but a nice review would be simpler to start with. Thank you in advance for your help Sandeep
Re: [ccp4bb] Cadmium sites and co-ordinations in structure
Hello Sandeep, I have been in this situation many times before but with different metal ions.. I have found papers published by Marjorie M Harding very useful in such situations. In fact, there are lots of information on-line on which is available here (including all the references for his papers): *METAL COORDINATION SITES IN PROTEINS* * * http://tanna.bch.ed.ac.uk/ http://tanna.bch.ed.ac.uk/qg3.htm http://tanna.bch.ed.ac.uk/newtargs_06.html You will at least find information for Ca and Co here for sure. All the best Simanshu On Fri, Aug 26, 2011 at 10:21 AM, Partha Chakrabarti ppc...@gmail.comwrote: Hi Sandeep, if someone sends one, kindly share the references. In general, Ca2+ could have more Asp, Asn kind of coordination and distorted pentagonal bipyramidal geometry with waters (about 2.5A), Cd can also have S- since it is softer, I guess Co might have N/O/S (i.e all three with paired electrons). An inorganic chemistry textbook like Greenwood Earnshaw or Cotton Wilkinson could be handy.. or a bioinorganic chemistry book. HTH, Partha On Fri, Aug 26, 2011 at 7:24 PM, Sandeep s.talapa...@beatson.gla.ac.ukwrote: Hi, I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and determined its structure. It turns out that I see several metal sites in the structure, mostly cadmiums. Is there any information published (preferably a review) which summarises data on cadmium sites in proteins such as for example the possible coordination numbers of cadmium, distances, type of side chains found to coordinate with cadmium, etc.? I could extract all this from the PDB, but a nice review would be simpler to start with. Thank you in advance for your help Sandeep -- Dhirendra K Simanshu Memorial Sloan-Kettering Cancer Center Structural Biology Program New York, NY, USA 10065
Re: [ccp4bb] Cadmium sites and co-ordinations in structure
While I believe there is plenty written about metal coordination, the best approach, IMHO, is to search PDB for metal of your choice at resolution as high as you can get and compare to your case. Vaheh From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dhirendra K Simanshu Sent: Friday, August 26, 2011 10:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Cadmium sites and co-ordinations in structure Hello Sandeep, I have been in this situation many times before but with different metal ions.. I have found papers published by Marjorie M Harding very useful in such situations. In fact, there are lots of information on-line on which is available here (including all the references for his papers): METAL COORDINATION SITES IN PROTEINS http://tanna.bch.ed.ac.uk/ http://tanna.bch.ed.ac.uk/qg3.htm http://tanna.bch.ed.ac.uk/newtargs_06.html You will at least find information for Ca and Co here for sure. All the best Simanshu On Fri, Aug 26, 2011 at 10:21 AM, Partha Chakrabarti ppc...@gmail.commailto:ppc...@gmail.com wrote: Hi Sandeep, if someone sends one, kindly share the references. In general, Ca2+ could have more Asp, Asn kind of coordination and distorted pentagonal bipyramidal geometry with waters (about 2.5A), Cd can also have S- since it is softer, I guess Co might have N/O/S (i.e all three with paired electrons). An inorganic chemistry textbook like Greenwood Earnshaw or Cotton Wilkinson could be handy.. or a bioinorganic chemistry book. HTH, Partha On Fri, Aug 26, 2011 at 7:24 PM, Sandeep s.talapa...@beatson.gla.ac.ukmailto:s.talapa...@beatson.gla.ac.uk wrote: Hi, I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and determined its structure. It turns out that I see several metal sites in the structure, mostly cadmiums. Is there any information published (preferably a review) which summarises data on cadmium sites in proteins such as for example the possible coordination numbers of cadmium, distances, type of side chains found to coordinate with cadmium, etc.? I could extract all this from the PDB, but a nice review would be simpler to start with. Thank you in advance for your help Sandeep -- Dhirendra K Simanshu Memorial Sloan-Kettering Cancer Center Structural Biology Program New York, NY, USA 10065 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.
[ccp4bb] Closed density map
Dear all, Do you know how to generate some closed density map (mesh) that can wrap ligand without tangling with electron density map of other residues? Many Thanks! Hui
Re: [ccp4bb] Cadmium sites and co-ordinations in structure
Hi, I recall seeing some (old) ConA structures with Cadmium substituting for one of the native metals (Ca+2 or Mn+2). They must be in the PDB database. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sandeep [s.talapa...@beatson.gla.ac.uk] Sent: Friday, August 26, 2011 4:54 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cadmium sites and co-ordinations in structure Hi, I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and determined its structure. It turns out that I see several metal sites in the structure, mostly cadmiums. Is there any information published (preferably a review) which summarises data on cadmium sites in proteins such as for example the possible coordination numbers of cadmium, distances, type of side chains found to coordinate with cadmium, etc.? I could extract all this from the PDB, but a nice review would be simpler to start with. Thank you in advance for your help Sandeep
Re: [ccp4bb] Closed density map
I would calculate an omit map. Delete your ligand and use the resulting pdb file to calculate a map. Density for your ligand will show up as positive difference density. Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RONG hui Rong Sent: Friday, August 26, 2011 4:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Closed density map Dear all, Do you know how to generate some closed density map (mesh) that can wrap ligand without tangling with electron density map of other residues? Many Thanks! Hui
Re: [ccp4bb] number of cycles in refmac
Dear Dr Ian from your argument i could not understand how many cycles to refine before submitting the coordinates to the PDB. what is the upper limit 100 or thousand or million according to my understanding, its more logical to stop the refinement when over refinement is taking place (when R and Rfree are going in opposite directions and LLG is stabilized ) AR
Re: [ccp4bb] Closed density map
I think the number one criterion for choosing a map for a figure should be What am I trying to show in this figure? If you simply want an electron density mesh that covers your ligand calculate an Fcalc map. This type of figure is usually worthless in a research paper but may have value in a tutorial setting. More common is a figure whose intent is to justify your identification of the ligand and its conformation. In that case you want a map that has as little model bias toward you interpretation as possible. As Herman noted, this is often calculated as an omit map with some additional effort to removed secondary model bias (e.g. refining the model with the ligand omitted). My preference is to show the very map that convinced me, since that map is certainly not biased by models that were built later. You should be the harshest critic of your own models so that map should have been very convincing to have convinced even you. If the density in that map bleeds a little into the protein density, so be it. That is not important. Dale Tronrud On 08/26/11 07:44, RONG hui Rong wrote: Dear all, Do you know how to generate some closed density map (mesh) that can wrap ligand without tangling with electron density map of other residues? Many Thanks! Hui
[ccp4bb] reindexing monoclinic data
Hi all, We have several primitive monoclinic datasets for the same protein with various ligands, with essentially the same unit cell parameters. We would like to have these with the molecules/density oriented the same way for easy comparison, but as chance would have it, some have effectively the opposite k index, which of course puts these molecules/density (relatively) upsidedown. I was wondering how people typically deal with this. I found what I believe to be the answer of reindexing monoclinic h, k, l to -h, -k, h+l to keep the system right handed and flip k. For this it seems that SFTOOLS would be appropriate? Is this reindexing commonly done at the stages of integration (altering rotx roty rotz in HKL2000) or scaling? Thanks, Greg
Re: [ccp4bb] reindexing monoclinic data
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Greg, with XDS I normally set REFERENCE_DATA_SET to the first one indexed / integrated in order to maintain consistent indexing between data sets. Not sure whether similar options are available in other integration programs, but if I remember correctly, pointless also offers reindexing. Cheers, Tim On 08/26/2011 05:55 PM, Gregory Bowman wrote: Hi all, We have several primitive monoclinic datasets for the same protein with various ligands, with essentially the same unit cell parameters. We would like to have these with the molecules/density oriented the same way for easy comparison, but as chance would have it, some have effectively the opposite k index, which of course puts these molecules/density (relatively) upsidedown. I was wondering how people typically deal with this. I found what I believe to be the answer of reindexing monoclinic h, k, l to -h, -k, h+l to keep the system right handed and flip k. For this it seems that SFTOOLS would be appropriate? Is this reindexing commonly done at the stages of integration (altering rotx roty rotz in HKL2000) or scaling? Thanks, Greg - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOV8MbUxlJ7aRr7hoRApbGAKD7kSUGiz9jYT9B51YVjraSMxzulQCdFyt+ MiTKc3vKUFvj140JsybDQN8= =aiWT -END PGP SIGNATURE-
Re: [ccp4bb] number of cycles in refmac
Dear Protein Chemistry (?), When R and R-free drift off you are probably refining with suboptimal weights. If anything, it proves you still have work to do. At convergence R and R-free do not really change anymore so neither does the difference. If you have already done a lot of rebuilding and refinement 20 cycles is usually enough (but more cycles shouldn't hurt). Cheers, Robbie Date: Fri, 26 Aug 2011 20:29:59 +0530 From: proteinchemistr...@gmail.com Subject: Re: [ccp4bb] number of cycles in refmac To: CCP4BB@JISCMAIL.AC.UK Dear Dr Ian from your argument i could not understand how many cycles to refine before submitting the coordinates to the PDB. what is the upper limit 100 or thousand or million according to my understanding, its more logical to stop the refinement when over refinement is taking place (when R and Rfree are going in opposite directions and LLG is stabilized ) AR
Re: [ccp4bb] Cadmium sites and co-ordinations in structure
Cadmium(II) prefers softer Lewis bases (e.g. thiolates and imidazole ligands more than carboxylates) and is commonly found in a four-coordinate environment, similar to zinc(II). Cheers, -- Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 8/26/2011 10:46 AM, Boaz Shaanan wrote: Hi, I recall seeing some (old) ConA structures with Cadmium substituting for one of the native metals (Ca+2 or Mn+2). They must be in the PDB database. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sandeep [s.talapa...@beatson.gla.ac.uk] Sent: Friday, August 26, 2011 4:54 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cadmium sites and co-ordinations in structure Hi, I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and determined its structure. It turns out that I see several metal sites in the structure, mostly cadmiums. Is there any information published (preferably a review) which summarises data on cadmium sites in proteins such as for example the possible coordination numbers of cadmium, distances, type of side chains found to coordinate with cadmium, etc.? I could extract all this from the PDB, but a nice review would be simpler to start with. Thank you in advance for your help Sandeep
[ccp4bb] Postdoctoral position at the University of Chicago
Postdoctoral position at the University of Chicago The Keenan lab in the Department of Biochemistry Molecular Biology at the University of Chicago seeks to recruit an outstanding postdoctoral scientist with a strong interest in membrane protein biogenesis. The main goal of the lab is to understand, in molecular mechanistic detail, how membrane proteins are targeted to, and inserted into biological membranes. We do this using a combination of structural, biochemical and genetic approaches. This position offers an excellent opportunity for an experienced protein biochemist to complement his or her expertise using modern tools for structural analysis. The focus of the project is on a series of soluble and integral membrane proteins (and their complexes), which coordinate targeting and insertion of eukaryotic membrane proteins. The postdoc will benefit from the highly collaborative University of Chicago community, and our regular access to the nearby Advanced Photon Source (APS) at Argonne National Laboratory. Candidates should have (or expect) a Ph.D. in biochemistry or a related field, and have strong experience in molecular biology, protein expression, purification, crystallization and functional analysis. A significant publication record is an advantage. To apply, please email a PDF of your CV to bkee...@uchicago.edu. For more information, please visit the lab website: http://keenanlab.bsd.uchicago.edu
Re: [ccp4bb] reindexing monoclinic data
Hi Yes, in the CCP4 world, pointless is the program for you. On 26 Aug 2011, at 17:00, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Greg, with XDS I normally set REFERENCE_DATA_SET to the first one indexed / integrated in order to maintain consistent indexing between data sets. Not sure whether similar options are available in other integration programs, but if I remember correctly, pointless also offers reindexing. Cheers, Tim On 08/26/2011 05:55 PM, Gregory Bowman wrote: Hi all, We have several primitive monoclinic datasets for the same protein with various ligands, with essentially the same unit cell parameters. We would like to have these with the molecules/density oriented the same way for easy comparison, but as chance would have it, some have effectively the opposite k index, which of course puts these molecules/density (relatively) upsidedown. I was wondering how people typically deal with this. I found what I believe to be the answer of reindexing monoclinic h, k, l to -h, - k, h+l to keep the system right handed and flip k. For this it seems that SFTOOLS would be appropriate? Is this reindexing commonly done at the stages of integration (altering rotx roty rotz in HKL2000) or scaling? Thanks, Greg - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOV8MbUxlJ7aRr7hoRApbGAKD7kSUGiz9jYT9B51YVjraSMxzulQCdFyt+ MiTKc3vKUFvj140JsybDQN8= =aiWT -END PGP SIGNATURE- Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] number of cycles in refmac
Hi AR Please define what you mean by 'over-refinement' as it's not a term I use: does it mean 'convergence', or 'over-fitting', or 'over-optimisation' (whatever that means) or something else? If by LLG is stabilized you mean it has converged then I agree that's a possible stopping criterion, but then so must all the refinement indicators including R and Rfree ( RMSDs etc) since by definition at convergence there can be no further significant changes in the parameters to cause R and Rfree to change further. You say R Rfree are going in opposite directions when LLG has stabilized. It's not possible for R and Rfree to continue to change if the refinement has converged, since that clearly implies that it hasn't converged. Cheers -- Ian PS 3 copies of your email is 2 too many (or if this is the list server acting up again, my apologies). On Fri, Aug 26, 2011 at 3:55 PM, protein chemistry proteinchemistr...@gmail.com wrote: Dear Dr Ian from your argument i could not understand how many cycles to refine before submitting the coordinates to the PDB. what is the upper limit 100 or thousand or million according to my understanding, its more logical to stop the refinement when over refinement is taking place (when R and Rfree are going in opposite directions and LLG is stabilized ) On Fri, Aug 26, 2011 at 4:01 PM, Ian Tickle ianj...@gmail.com wrote: Frank, Point #1 - fair point; the reason Rfree is popular, though, is because it is a *relative* metric, i.e. by now we have a sense of what good is. So I predict an uphill fight for LLfree. Why? I don't see any difference. As you say Rfree is a relative metric so your sense of what 'good' is relies on comparisons with other Rfrees (i.e. it can only be 'better' or 'worse' not 'good' or 'bad'), but then the same is true of LLfree (note that they both assume that exactly the same data were used and that only the model has changed). So when choosing between alternative model parameterisations in order to minimise over-fitting we compare their Rfrees and choose the lower one - same with LLfree, or we compare the observed Rfree with the expected Rfree based on Rwork and the obs/param ratio to check for problems with the model - same with LLfree. In fact you can do it better because the observations in LLfree are weighted in exactly the same way as those in the target function. Point #2 would hold if we routinely let our refinements run to convergence; seems common though to run 10 cycles or 50 cycles instead and draw conclusions from the behaviour of the metrics. Are the conclusions really much different from the comparison-at-convergence you advocate? Which is in practice often less convenient. You might do 10 cycles for a quick optimisation of the coordinates, but then I wouldn't place much faith in the R factors! How can you draw any conclusions from their behaviour: there's no way of predicting how they will change in further cycles, the only way to find out is to do it. I'm not saying that you need to refine exhaustively on every run, that would be silly since you don't need to know the correct value of the R factors for every run; but certainly on the final run before PDB submission I would regard stopping the refinement early based on Rfree as implied in Tim's original posting as something akin to 'cheating'. Cheers -- Ian
Re: [ccp4bb] Protein aggregation and crystallization
Hi, I do have 10% glycerol in my buffers, and still the constructs come in the void volume. and I have sarkosyl in the lysis buffer. but none in the elution or dialysis buffer. So do I still need detergents please suggest. reg. Anita On Sat, Aug 27, 2011 at 12:13 AM, Pius Padayatti ppadaya...@gmail.comwrote: Answer is simply no. aggregates are of no value why would you try it? you could try adding glycerol to 10 % during the preparation( all the way from homogenization) itself and try detergents like c8e4 and series of that. i know one membrane associated protein need detergent from the begining itself. Please do not chop off the membrane part keep it and chop some of the unstructured cytosolic part if you want. all the best. let us know if any of these worked Padayatti On Fri, Aug 26, 2011 at 3:03 AM, anita p crystals...@gmail.com wrote: Hi All, I am working on a protein which has a membrane spanning region and as cytosolic domain.I have made various deletion constructs of the protein, so that I can have a crystallizable fragment. There is no homologues mentioned in the pdb for this protein. All of these constructs are purified successfully but when concentrated and loaded on a gel filtration column Superdex-200, they elute in the void volume. But the proteins donot precipitate out !! Is it worth while to go ahead for crystallization trials?? Any other suggestion is most welcome. Thanks Anita -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] Protein aggregation and crystallization
Hi Yury, I have done dynamic light scattering and it shows its polydispersed. Please let me know if it is still ok for setting trays. reg. anita On Fri, Aug 26, 2011 at 10:21 PM, Yuriy Patskovsky yuriy.patskov...@einstein.yu.edu wrote: Anita, an assembly may be quite large - I would check it somehow, maybe by light scattering or centrifugation Good luck Yury -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of anita p [ crystals...@gmail.com] *Sent:* Friday, August 26, 2011 3:03 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Protein aggregation and crystallization Hi All, I am working on a protein which has a membrane spanning region and as cytosolic domain.I have made various deletion constructs of the protein, so that I can have a crystallizable fragment. There is no homologues mentioned in the pdb for this protein. All of these constructs are purified successfully but when concentrated and loaded on a gel filtration column Superdex-200, they elute in the void volume. But the proteins donot precipitate out !! Is it worth while to go ahead for crystallization trials?? Any other suggestion is most welcome. Thanks Anita
[ccp4bb] [off-topic] Rigaku MicroMax-007 system available
Hi everyone, Due to recent downsizing, a complete Rigaku MicroMax-007 system needs a new home! R-Axis IV++ detector, computer, software and all other components included. Please contact me off-list for details. Happy Friday! Erin 650-804-7008 ress...@gmail.com
Re: [ccp4bb] Cadmium sites and co-ordinations in structure
While it's not a review on protein binding sites, the following review might be helpful in understanding its chemistry: Holloway Melnik (1995) Main Group Met. Chem. 451-585. Also, David Giedroc has written a couple of useful reviews that include discussions of cadmium binding proteins. Cadmium is kind of a generalist. It will bind to sites tailored for calcium, manganese and zinc quite readily, accepting a wide range of protein ligands. Biologically, most cadmium-specific proteins use softer ligands (cysteines) since other metals are relatively less attracted to those sites. You may see some coordination positions taken up by chloride if you have a high enough concentration in your buffer. Good luck, Arthur On Aug 26, 2011, at 6:54 AM, Sandeep wrote: Hi, I crystallised a protein in the presence of Calcium, Cobalt, and Cadmium and determined its structure. It turns out that I see several metal sites in the structure, mostly cadmiums. Is there any information published (preferably a review) which summarises data on cadmium sites in proteins such as for example the possible coordination numbers of cadmium, distances, type of side chains found to coordinate with cadmium, etc.? I could extract all this from the PDB, but a nice review would be simpler to start with. Thank you in advance for your help Sandeep
Re: [ccp4bb] reindexing monoclinic data
Gregory Bowman wrote: Hi all, We have several primitive monoclinic datasets for the same protein with various ligands, with essentially the same unit cell parameters. We would like to have these with the molecules/density oriented the same way for easy comparison, but as chance would have it, some have effectively the opposite k index, which of course puts these molecules/density (relatively) upsidedown. I was wondering how people typically deal with this. I found what I believe to be the answer of reindexing monoclinic h, k, l to -h, -k, h+l to keep the system right handed and flip k. For this it seems that SFTOOLS would be appropriate? Is this reindexing commonly done at the stages of integration (altering rotx roty rotz in HKL2000) or scaling? Thanks, Greg If you want to redo in HKL, look at the denzo/scalepack manual, scalepack scenario 5, reindexing. I think your case is the first example, switch a and c in p21 HKL MATRIX 0 0 1 [reindexing matrix: h - l] 0 -1 0 [this is -1 to keep determinant = 1] 1 0 0 [l - h] If you just want to reindex the data, you can use the previous scalepack output (.sca) as input. If you rescale the denzo output files (.x) with this HKL matrix, then in the log file, just below the unique occurance of the word mosaicity in the file, is a line (or several lines if fit batch) which gives the post-refined missetting angles and cell param, which can be edited into the corresponding lines of integ.inp if you want to re-integrate in the re-indexed setting. (I don't know if there is any good reason to do so, however).
[ccp4bb] HTP ligand screening
Dear Crystallographers, I have ~30 data sets from ligand soaks of my protein of known structure (all approximately the same cell. Can anyone suggest a high throughput method which would do molecular replacement, refine, then output new blobs, perhaps as a water pdb file? I am sure they do this or better in pharma every day... Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Methods for dehydrating crystals
Hi all, What are the most successful methods you know of for dehydrating a crystal prior to freezing it? I am trying to push the resolution of my crystals. Thanks, Andrea
Re: [ccp4bb] reindexing monoclinic data
Hi Greg, you could also CAD them into one huge mtzfile with different labels e.g. FP_Lig1, FP_Lig2 etc. Then they would be the way you need them for direct comparison in Coot, Pymol or whatever program you wish to use. Jürgen On Aug 26, 2011, at 11:55 AM, Gregory Bowman wrote: Hi all, We have several primitive monoclinic datasets for the same protein with various ligands, with essentially the same unit cell parameters. We would like to have these with the molecules/density oriented the same way for easy comparison, but as chance would have it, some have effectively the opposite k index, which of course puts these molecules/density (relatively) upsidedown. I was wondering how people typically deal with this. I found what I believe to be the answer of reindexing monoclinic h, k, l to -h, -k, h+l to keep the system right handed and flip k. For this it seems that SFTOOLS would be appropriate? Is this reindexing commonly done at the stages of integration (altering rotx roty rotz in HKL2000) or scaling? Thanks, Greg .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] HTP ligand screening
See message to Greg :-) CAD them first, run MR with one and then just refine using different labels, inspect your difference density maps and enjoy your success rate of 6% :-) You can certainly write a tiny script which would use different labels and do a quick phenix.refine or Rafmac. I would skip the water picking as you can't skip the part where you actually look at the density maps. So I'd rather do a few cycles of refinement and visually inspect using Coot find unmodelled blobs and go from there. For 30 data sets that shouldn't take much more than a day and at the end you know which ones look promising and focus on those first. Jürgen On Aug 26, 2011, at 4:43 PM, Jacob Keller wrote: Dear Crystallographers, I have ~30 data sets from ligand soaks of my protein of known structure (all approximately the same cell. Can anyone suggest a high throughput method which would do molecular replacement, refine, then output new blobs, perhaps as a water pdb file? I am sure they do this or better in pharma every day... Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Methods for dehydrating crystals
Andrea L Edwards wrote: Hi all, What are the most successful methods you know of for dehydrating a crystal prior to freezing it? I am trying to push the resolution of my crystals. Thanks, Andrea First of all, be aware that not all crystals are improved by dehydration. Some need to be drier, some need to be wetter. If you have long or large crystals that extend outside the loop, you can get a clue by comparing diffraction at the outside tip (driest) with the center of the loop (wettest). We've seen dramatic differences in both directions, and the ones that diffract best outside (beef orthorhombic cyt bc1) were greatly improved by dehydration, while for the ones that diffract best in the center (chicken complex II) we got the best diffraction when they were so wet we started to get ice rings. Dehydration destroyed them. Our best dehydration results were with simply holding the crystal in the air 30 sec to 2 min after mounting before plunging into LN2. Also something about pushing the crystal to the edge of the drop where the peg is getting sticky before fishing. be aware the result may depend on relative humidity and temperature. My grand plan for optimizing dehydration (if the FMS is not available) was to mount crystals on cryocap pins and screw the caps into vials containing 0.5 ml of different concentrations of glycerol. (Pin being short enough that the crystals are suspended in the air over the solution) Wait for a few hours at RT or 4* then unscrew the cap and rapidly freeze. Never really got good results though. And if the humectant is really wet, the crystal soaks up so much water it falls out of the loop into the humectant! Ed
