[ccp4bb] Summary - Stop Refmac from refining B factors?
Hi all, thanks for all your help so far, and as we ended up in a more general discussion about temperature factor refinement at not-so-great resolution, here is a quick summary of what I'll try out: 1.) Refine overall B's instead of isotropic B's. 2.) Use isotropic B's with the following (combinable) options: a) Isotropic in the beginning, grouped B's in the end. b) Use tight geometric restraints (I'm doing this anyways). c) Use tight B restraints rather than grouped CNS B's (not geometrically restrained, and most likely not restrained by NCS). 3.) Use not so tight B restraints, but tight geometric restraints with individual or grouped B's, plus TLSMD server and multi-group TLS refinement. 4.) Use CNS and try Mark White's tools, and simulated annealing. 5.) Use phenix with weighted nearest-neighbor restraint. ...and some remarks: * Of course I never wanted not to refine any B factors! I just wanted to see their contribution/influence on the refinement and explain their strange behaviour. * Luckily, I have NCS :o) Thanks for your good wishes, Mark. Eva 2007/4/18, Eva Kirchner [EMAIL PROTECTED]: Hi, I have a little problem with B-factor refinement. I'm using the CCP4i interface, Refmac 5.2.0019, a resolution of 30-3.2 A (I tried 8-3.2 A as well, it doesn't make a big difference for this problem), and a current Rfree of 30.4%. Refmac refines the B-factors so that they are nearly the same for main chain and side chain, and I don't like that (or could it make sense in any way?). Moreover, my structure is a protein complex, and Refmac is mainly doing this for one component of the complex. If I take the B-factors from the original uncomplexed protein (around 18, 1.75 A) and add 44 to them with moleman to get them in the range they are in the complex, Refmac flattens them remarkably in only 5 cycles of restricted refinement. Does anyone have an explanation for this? I am pretty sure that the complex components are in the right place, I see beautiful density and everything I should see at this resolution. Here is what I tried further: * I de-selected Refine isotropic temperature factors in the Refmac interface. There was no REFI BREF ISOT any more in the com file. But there was also no difference in the B-factors compared to when there _was_ REFI BREF ISOT in the com file... So does Refmac just _ignore_ my wish not to refine B-factors? (The REFI keywords were as follows: type REST - resi MLKF - meth CGMAT - is there any B-factor-thing hidden in this?) * I played around with the geometric parameters. If I select the B-factor values there (the keywords are TEMP|BFAC wbskalsigb1sigb2sigb3sigb4), it does not make _any_ difference, what values I fill in there, the resulting B-factors are always the same (but different from when I don't use the TEMP keyword, and even flatter). Default for WBSCAL is 1.0, I tried 10, 1.0, 0.1, 0.01, and the equivalent numbers for the sigbs. Thanks for any thoughts on this, Eva
Re: [ccp4bb] Protein expression in Minimal media (M9)
I had a similar situation, i.e. the cells wouldn't grow past an OD of 0.1 on minimal media, but in my case I did get protein expression. The way I got around it was as follows. I grew the cells up in minimal media plus the amino acids that suppress methionine biosynthesis, plus L-methionine. For whatever reason, the cells behaved much better in not quite minimal media. Then, when they were near 0.6 OD, I spun them down and resuspended them in media with Selenomethionine instead of L-Met. It worked for me (i.e. ~100% SeMet incorporation, structure solved, etc.), but I only needed to solve a growth problem, not an expression problem. It might work for you, and you could try a dry run without burning up precious SeMet. _ Eric A. Toth, Ph.D. Assistant Professor Department of Biochemistry and Molecular Biology Marlene and Stewart Greenebaum Cancer Center University of Maryland School of Medicine 108 North Greene St. Baltimore, MD 21201 Email: [EMAIL PROTECTED] Phone: x-410-706-5345 Fax: x-410-706-8297 http://www.umaryland.edu/bmb/faculty/toth.html http://crystal.umaryland.edu -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Wednesday, April 18, 2007 10:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein expression in Minimal media (M9) Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
[ccp4bb] Post doctoral positions in Structural Immunology
--- STRUCTURAL IMMUNOLOGY -- Positions are now available to study the structure, function and therapeutic application of T cell costimulatory molecules in the Nathenson and Almo laboratories at the Albert Einstein College of Medicine. We employ a multi-disciplinary approach that includes traditional high resolution crystallography, fluorescence microscopy, FRET, cell-based and whole animal studies. The ideal candidate will have considerable experience in protein production in both bacterial and eukaryotic platforms and a strong interest in defining the fundamental mechanisms that modulate the cellular immune response. These positions are supported by an NIH-funded Training Grant in Immuno-Oncology and require either US citizenship or permanent residence status. Interested individuals should send a CV and arrange for three letters of reference to be sent to [EMAIL PROTECTED] Representative examples of our program are provided by the following references: Schwartz, et al., (2001) Structural Basis for Costimulation by the Human CTLA-4/B7-2 Complex. Nature 410: 604-608. Zhang, X., et al. (2004) Structural and Functional Analysis of the Costimulatory Receptor Programmed Death-1. Immunity 20: 337-347. Bhatia, S., et al. (2005) Different Cell Surface Oligomeric States of B7-1 and B7-2: Implications for Signaling. Proc. Natl. Acad. Sci. USA 102: 15569-15574. Cao, E., et al. (2006) NTB-A Crystal Structure: Implications for Homophilic Interactions and Signaling by the SLAM Family of Receptors. Immunity 25:559-570. Chattopadhyay, K., et al. (2006) Structural basis of inducible costimulator ligand costimulatory function: determination of the cell surface oligomeric state and functional mapping of the receptor binding site of the protein. J Immunol. 177:3920-3929. Cao, E., et al. (2007), T cell immunoglobulin mucin-3 crystal structure reveals a galectin-9-independent ligand-bindng surface, Immunity 26:311-21.
Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish, I also had a similar problem getting cells (C41 (DE3) in my case) to grow in minimal media. To get around this problem, I took cells from an agar plate and grew them in a small volume (5 mL) of the minimal media. Once that culture got thick, I then inoculated 200 mL of minimal media with 0.5 mL of the 5 mL culture. I would let this grow, and I used this as my overnight culture to inoculate my large flasks. Long story short, the cells seemed happier adjusting to the minimal media in a smaller volume first. Cheers, Jamie Wallen Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
Re: [ccp4bb] Protein expression in Minimal media (M9)
Are you adding vitamins to your M9 minimal? RosettaBlue is thiamin requiring and can not grow in the absence of thiamin. The thiamin requirement is so low that you can often get slow growth to a low OD based on residual thiamin in the cells, but you will not get robust growth. Also, minimal recipes vary pretty drastically from one another, your system may prefer one of the other recipes out there. Personally, I have never really liked the standard M9 minimal recipe. Contrary to another poster, I found that my cells tended to grow better in minimal medium if I pre-adapted them to minimal by growing my starters in the same minimal that I intended to use (with Met instead of SeMet). However, I prefer to stick with high dilutions (1:1000-1:100) so that may be the difference. For years I used the MM/CA recipe from Pryor and Leiting, Protein Expression and Purification, 1997 v10 pg 309. This recipe works well and can be adapted for SeMet growth by subbing out the casamino acids for a mixture of amino acids. For the past few years I've been using the autoinduction recipes from Studier, Protein Expression and Purification, v41 pg 207. I have found that these work fantastically well (as long as you don't need to express at really reduced temperature...I only use them down to 20° C). There are recipes for SeMet incorporation in there as well. Best of luck, Cynthia On Apr 18, 2007, at 10:34 PM, [EMAIL PROTECTED] wrote: Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. * Cynthia Kinsland, Ph.D. Cornell University Protein Facility Director 607-255-8844
[ccp4bb] I/sigma in XDS output
Dear all, I'm a bit confused from the output of the CORRECT step in XDS. In one of the first tables I can read the mean I/sigma for each resolution shell, but these values are much different from the I/sigma reported in the table at the end of the output files, titled completeness and quality of data set for the full data range with signal/noise -3.0. For example, from the first table I have I/sigma = 2 at 3.6 A, while from the second table I have I/sigma = 2 at 2.8 A! What is exactly the difference between the two values? And which one is reliable to decide the resolution cutoff? Thank you in advance, Michele Lunelli MPI for Infection Biology Berlin - Germany
Re: [ccp4bb] I/sigma in XDS output
Michele Lunelli wrote: Dear all, I'm a bit confused from the output of the CORRECT step in XDS. In one of the first tables I can read the mean I/sigma for each resolution shell, but these values are much different from the I/sigma reported in the table at the end of the output files, titled completeness and quality of data set for the full data range with signal/noise -3.0. For example, from the first table I have I/sigma = 2 at 3.6 A, while from the second table I have I/sigma = 2 at 2.8 A! What is exactly the difference between the two values? And which one is reliable to decide the resolution cutoff? Thank you in advance, Michele Lunelli MPI for Infection Biology Berlin - Germany Michele, the first table, which is long and fine-grained in resolution, has (citing CORRECT.LP): I/Sigma = mean intensity/Sigma of a reflection in shell and is thus talking about individual reflections _before_ merging symmetry equivalents. Later tables have (again citing CORRECT.LP): I/SIGMA = mean of intensity/Sigma(I) of unique reflections (after merging symmetry-related observations) and are thus giving statistics _after_ merging. The latter tables are suitable for deciding about the resol cutoff. HTH, Kay -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: [EMAIL PROTECTED]Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz smime.p7s Description: S/MIME Cryptographic Signature
[ccp4bb] MSc in Structural Biology and Biophysics at Manchester, UK
[Posted on behalf of Lydia Tabernero. Full text of this posting available here: http://www.ccp4.ac.uk/martyn/MSc-Leaflet.doc ] 1 year MSc in Structural Biology and Biophysics at Manchester, UK, starting in September 2007 Aims Objectives: Structural and biophysical analyses are essential in understanding modern biology. The Faculty of Life Sciences has an excellent representation of high quality research groups whose main emphasis is the application of structural biology and biophysical techniques to biologically important problems such as regulation of cellular processes, cell signaling, mechanistic enzymology, biological membranes and cell-matrix interactions. Research is supported by state-of-the-art facilities and we aim to take advantage of our combined portfolio to offer an integrated multidisciplinary training that will engage students from different career backgrounds and will allow them to broaden their knowledge and research expertise and to interact with other future professionals. Further Information: http://www.ls.manchester.ac.uk/postgraduate Enquiries: Tel: 0161 275 5608; Fax: 0161 275 5657 Email: [EMAIL PROTECTED] Apply on-line: http://www.manchester.ac.uk/postgraduate/howtoapply/
Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish, Assuming that you need the minimal medium for Se, have you tried the regular heavy atom derivatives? You already have diffracting crystals... M9 medium can be slightly difficult. However, no one says that you *have* to use minimal medium such as M9 (again, assuming that you're doing Se-Met). There are numbers of defined media compositions out there, many with very good growth characteristics. Finally, you can spike the M9 with a small quantity of yeast extract. This will give your culture a decent initial boost and the amount of normal methionine in the extract should be relatively small so it will all get eaten up while the cells are still in early log phase. Regards, Artem Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish, In practice, we have found that it is very helpful to grow up an overnight starter culture in minimal media to acclimatize the cells for growth under minimal conditions. (Cells transferred from LB to M9 do not perform well for overexpression). We inoculate 1 L of modified M9 (see below) with 10 mL of an overnight culture (centrigued, washed, and ressuspended in M9) in the same medium. In addition--and even though it should not be required for many strains of E. coli--we have found that he inclusion of thiamin, 5-10 ug/L, greatly enhances the rate of cell growth, but it may still takeup to 8 hr for cells to grow to OD600 = 0.6 for induction. Overexpression overnight (12-18 hr) may be required to get optimal cell density and overexpression yield. Using this protocol we get excellent overexpression yields of protein. Cheers, ___ Roger S. Rowlett 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: [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Thursday, April 19, 2007 10:31 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein expression in Minimal media (M9) Manish, Assuming that you need the minimal medium for Se, have you tried the regular heavy atom derivatives? You already have diffracting crystals... M9 medium can be slightly difficult. However, no one says that you *have* to use minimal medium such as M9 (again, assuming that you're doing Se-Met). There are numbers of defined media compositions out there, many with very good growth characteristics. Finally, you can spike the M9 with a small quantity of yeast extract. This will give your culture a decent initial boost and the amount of normal methionine in the extract should be relatively small so it will all get eaten up while the cells are still in early log phase. Regards, Artem Hello everybody, Sorry for an offtopic question. I am trying to express a protein in M9 minimal media for Selenomet incorporation. When grown in LB this protein expressed very well and got good crystals. Diffraction was upto 2 A. I am having a hard time expressing the same protein in Minimal media. It took nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in minimal media and eventually got no protein expression. It looks like the cells are not growing or taking very long to grow. The cell line I am using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use RosettaBlue (DE3). It worked very well in LB, but having a hard time while expresing the same in minimal media using Rosetta Blue. Has anybody tried expression in minimal media using Rosetta Blue cell line? I am planning to try overnight induction. Any suggestions would be greatly appreciated. Thanks, Manish * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
[ccp4bb] recommendation for heavy atoms used in lithium sulfate and ammonium sulfate
Dear all, Could any one recommend some heavy atoms used for crystals grown in 0.1M tri-sodium citrate pH 5.6, 1M Lithium sulfate and 0.5M ammonium sulfate? I read from Hampton user guide of heavy atom kit that high salt concentrations are not the ideal medium for heavy atom reactions with macromolecules, so is there any type of heavy atom we should avoid using? We do not have any experience in preparing heavy atom derivative, so any suggestions, experience or references will be greatly appreciated. Thanks in advance! Tiancen Hu Shanghai Institute of Materia Medica
