[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 ), 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)
What we usually do is to grow cells in this media: 400ml H2O milli-Q Autoclaved 120ml AAmix (5X) Filtered 60ml M9 (10X) Autocl. 6ml TES (100X) Filtr. 12ml Glucose 20% Filtr. 0.6ml MgSO4 (1M) Autocl. 0.18ml CaCl2 (1M) Autocl. 1.2ml Biotine (5mg/ml) Filtr. 0.6ml Thiamine (10mg/ml) Filtr. 0.1 ml Met (50mg/ml). x ml antibiotic (2 x 4)ml pre-cultures in LB + antibiotic Then grow until the right OD (or just a little bit less) is achieved, and add to the culture: 1.5ml SeMet (50mg/ml) Filtr. (This will stop any inherent synthesis of Methionine) 6ml Glucose 20% 60ml AAmix (optative) 0.6ml Biotine 0.3ml Thiamine Then grow for half an hour more and then add IPTG. With this we get 100% SeMet incorporation and good protein expression levels. Good luck! Raquel > 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. > * > Raquel Garcia-Castellanos, Ph.D. Dept. of Structural Biology Molecular Biology Intitute of Barcelona (IBMB-CSIC) 08034 Barcelona (Spain) Phone: +34-934006100 Ext. 269
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] Hints on oil phase in the presence precipitants?
Hello, I've got a protein that forms oil phase in almost all conditions in the screen. No xtals thus far. I was wondering if there are general hints on dealing with such cases. Basically, I am trying to understand what direction to go from here. E.g. decrease or increase [protein], [salt] and general precipitant strength? High concentration of some solvent everywhere? Detergents? The protein is small, forms dimer via short coiled coils region and and various prediction servers say that the rest is likely to be disordered/not globular. In low to moderate salt without precipitants it behaves well - single peak on gel-filtration, does not aggregate, can be concentrated to at least 30 mg/ml. Thanks, Dima
Re: [ccp4bb] Summary - Stop Refmac from refining B factors?
Eva, If you do have NCS then you should be aware of a bug in the CNS1.1 release. PMB has a patch to fix this, and I hope that CNS1.2 will have fixed it also. The problem is with the NCS restraints on B-factors in CNS. The NCS groups, usually molecules, are restrained to have identical B-factors. This does not allow for the affect of local environment on the motions of the separate molecules. The PMB patch basically applies an overall B-factor to each NCS-related group to account for differences in the local environment of each group, such as differing crystal contacts. The effect is sometimes quite significant (B-factors ~10), but on other occasions the overall B-factors can be ~0, indicating that the molecules are practically identical. Good Luck, Mark On Thu, 2007-04-19 at 10:07 +0200, Eva Kirchner wrote: > 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 > ), 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 Sincerely yours, Mark A. White, Ph.D. Assistant Professor, Dept. Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics X-ray Crystallography Laboratory, Basic Science Building, Room 6.660 C University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 Fax. (409) 747-4745 mailto://[EMAIL PROTECTED] http://xray.utmb.edu http://xray.utmb.edu/~white
Re: [ccp4bb] How to run SIGMAA twice in a row? (fwd)
Thank Eleanor and others for making suggestions. We have tested SIGMAA in CCP4i(1.4.4.1) again carefully. In the first run, we used "combine two sets of MIR phases" mode to combine 2 sets of MAD phases, and output column labels changed to PHCMBtest and WCMBtest. The second run was in "combine isomorphous phase with partial structure" mode. We had no problem assigning other column labels such as FP, PHIBP, ... because the pull-down menu of each label includes "list all labels" option. But FOM pull-down menu had no "list all labels" option, we could not select the label WCMBtest generated from the previous run. It seems to me there is a bug in the GUI that limits the option in FOM pull-down menu. It only occurs in the "combine isomorphous phase with partial structure" running mode of SIGMAA. This is the run we really wanted to combine the model phases with the MAD phases before going through further density modifications with SOLOMON or DM. Regards, Huiying On Thu, 19 Apr 2007, Eleanor Dodson wrote: Sorry about that It is ages since I have used SIGMAA The MAD recombnation gives you PHCMB WCMB HLAC HLBC etc In that run I would make sure I renamed PHCMB WCMB PHCMBmadsWCMBmads or something You might have to use the option Run and view com file, then edit in a line: LABOUT PHCMB=PHCMBmads WCMB= etc Then I think you may be able to do what you want - I suspect the poor program is getting lost between what is a default output label, and something already in the header. Can you try that? Or you use the Clipper utility to combine phases.. it is more mindless but you do have to convert your PHIC FOM to HLA HLB (HLC HLD ==0) using another clipper utility first Eleanor Huiying Li wrote: My previous posting did not get answer, it might be a bit confusing. My questions is if I want to conbine phases from three different sources, 2 MAD data sets and a partial model, can I run SIGMAA twice? Combining MAD phases first, and then add in the model phases to the pre-combined MAD phases. When I run SIGMAA the second time CCP4i GUI allows me to choose labels PHCMB, HLAC, HLBC, HLCC, HLDC, but doesn't let me use WCMB as the input FOM. Is this a bug in the GUI or SIGMAA simply does not allow to use WCMB as an input FOM? Thanks in advance. Huiying - Huiying Li, Ph. D Department of Molecular Biology and Biochemistry Natural Sciences I, Rm 2443 University of California at Irvine Irvine, CA 92697, USA Tel: 949-824-4322(or -1953); Fax: 949-824-3280 email: [EMAIL PROTECTED] -- -- - Huiying Li, Ph. D Department of Molecular Biology and Biochemistry Natural Sciences I, Rm 2443 University of California at Irvine Irvine, CA 92697, USA Tel: 949-824-4322(or -1953); Fax: 949-824-3280 email: [EMAIL PROTECTED] --
[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
