Re: [ccp4bb] resolution limit stuck in Refmac5
Hi Jon On 30 Oct 2010, at 00:46, Tom Huxford wrote: Hi all, I'm working with good quality relatively complete x-ray diffraction data collected to a resolution limit 2.6 Å from a crystal of a protein with a small molecule ligand bound. I ran MR from 10-4 Å and then did maximum likelihood rigid body refinement in Refmac5 against data from 50-3.5 Å. Now I would like to run restrained refinement from 50-3 Å. The reason for doing this is that, in order to minimize the divergence between R-cryst and R-free during refinement my advisor who, by the way, is forwarding this e-mail for me (and editing it so please don't bash him too mercilessly) suggested I first build in the ligand and newly positioned polypeptide loops and refine against data at a lower resolution limit before opening it up to all the available data. I personally would not first build in the ligand and refine against low res data... Assuming the ligand is what you're after, I would: (a) leave the ligand out of the time being (b) refine the model using all data (c) build the ligand in (d) refine the model using all data I'm not clear why doing restrained refinement using limited data first should help.. Apparently this has worked well for him in the past (and it has!). The problem is that I'm to the point where I'd like to extend the resolution down to 3 Å during restrained refinement but even if I set the range from 50-3 Å in the ccp4i window refinement only happens from 50-3.5 Å. If I take a step back and do the rigid body refinement from 50-3 Å and then carry out restrained refinement from 50-3 Å it works fine. Why would the limits imposed by rigid body refinement cause the subsequent restrained refinement to be stuck at the rigid body refinement's resolution limits? Are you using the original reflection file? Best Roberto Thanks for any thoughts, Jon Fleming Graduate Student Structural Biochemistry Laboratory (Huxford Lab) Department of Chemistry Biochemistry San Diego State University --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] Deposition of riding H [was: Deposition of riding H]
Dear Pavel, Stressing Ethan's point about TLS refinement becoming practical with Refmac implementation, Winn et al. (2001) Acta D, 57, 122-133 (I know you like references) states: Derivatives of the residual with respect to the TLS parameters are expanded in terms of the derivatives with respect to individual anisotropic U values, which in turn are calculated using a fast Fourier transform technique. TLS refinement is therefore fast and can be used routinely. Best wishes Roberto On 15 Sep 2010, at 19:34, Pavel Afonine wrote: Dear George, a small correction if I may: However you will not find TLS in the index, because the credit for implementing this very useful concept should be given to Martin Winn, Garib and Ethan, long after the current version of SHELXL (and its manual) were released in 1997. Acta Cryst. (1985). A41, 426-433 Restrained structure-factor least-squares refinement of protein structures using a vector processing computer I. Haneef, D. S. Moss, M. J. Stanford and N. Borkakoti Abstract: A least-squares refinement program RESTRAIN has been developed, which is capable of refining macromolecular structures using structure amplitudes, phases from isomorphousreplacement or anomalous scattering and pseudo-energy restraints. In addition to positional parameters and isotropic temperature factors, anisotropic mean-square displacements may be refined either as individual atomic U tensors or as TLS tensors applied to groups of atoms. Anharmonic effects may be handled by coupling together occupancies to enable the electron density of an atomic group to be distributed over more than one subsite. A novel way of restraining groups of atoms to be planar has been developed that does not require dummy atoms and does not restrain the plane to lie in its current orientation. One can find other, earlier programs, but they are small molecule specific. Regards, Pavel. --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] turn granular to crystal
Dear Rui, I can only support Patrick's view on MMS. We've got some very good results with this technique in one tricky case in which the starting point wasn't much better than yours. In addition to the papers Patrick suggested the following might also be worth a look: The role of bias in crystallization conditions in automated microseeding. St John FJ, Feng B, Pozharski E. Acta Crystallogr D Biol Crystallogr. 2008 Dec;64(Pt 12):1222-7. Cheers Roberto On 25 Aug 2010, at 12:37, rui wrote: Hi, All, I'm trying to crystallize a soluble protein and got something like granular, they are rounded shaped and not so regular and also don't have sharp edges. See the attached pic. The current condition is PEG4000 and pH around 5. How can I improve this condition? Thanks a lot. granular.JPG --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] TLSMD Alignments
Dear Micheal, As you say, the effect is probably arising from the different packing environment of your NCS-related molecules. Once you've taken TLS groups into account NCS restraints might even perform better. Cheers, Roberto On 24 Aug 2010, at 21:15, Michael Thompson wrote: Hi All, I have a question for those of you familiar with the TLSMD webserver. I am working on a structure with slightly imperfect 3- fold rotational NCS. My most recent .pdb file has been generated using Refmac (followed by a little tinkering in Coot), and during refinement I have been imposing medium main chain and loose side chain NCS restraints, and my R-factors don't really improve if I loosen the restraints further. This is the .pdb file I've also used an input to TLSMD. The results of TLSMD do show that the residuals appear to slowly plateau when breaking the chains into 19 or 20 groups (all three A,B,C seem to converge similarly). When I look at the alignments, the TLS groups created for each chain do not align with each other well. The alignment of groups gets slightly better as more groups are added, which is partially just an issue of the groups being smaller and looking closer I think, but there is still significant stagger between neighboring groups. Is this typical for a structure with NCS-related chains? It seems somewhat counterintuitive to my understanding of how symmetric proteins should work (if the TLS motions reflect actual motions of the molecule). Perhaps the difference in TLS grouping between chains results from differences in Biso for NCS-related atoms that result from crystal packing? Maybe someone can shed some light on the situation? Thanks a lot, Mike Thompson -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
[ccp4bb]
Dear Changyi, Yes it can. You should have a xxx.cif file which Refmac uses to know about your ligand. Please have a look at it and check that it is actually representing the ligand you want to refine. You can then play with standard deviations in the file to put more weight on idealised geometry (assuming it is correct!) in case ligand distortion is caused by limited resolution. In general, if ligand description is correct playing with SD should not be required. Clearly, make sure that distortion does not have a functional meaning. Best Roberto On 4 Aug 2010, at 20:15, Changyi Xue wrote: Dear all, In my structure, there is a ligand, which contains a sugar ring. In the process of refinement, refmac always tried to distort the sugar ring to fit into the density. Is there any way to fix or restrain the ring conformation more tightly? I know CNS has such function, just wandering if refmac could do it also. all suggestions are welcome. changyi --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] Deposition of a BUSTER refined structure
Dear Melanie On 2 Mar 2010, at 15:09, Melanie Vollmar wrote: Hi all I did my first deposition of a BUSTER refined structure and I was very surprised when I was asked for an estimated error of free R value and estimated error of bin free R value as well an ESD based on Luzzati Plot during the deposition process. I had a detailed look into the refinement log file and I tried the Buster documentation but I have absolutely no clue where I can find these values. Can someone give me a hint where I can find some information about this or where the values are in the log file? If they are not listed anywhere, in which way do they have to be estimated? And what is a Luzzati Plot? I have never heard of such a plot before... For example, page 305-307 of Jan Drenth 'Principles of Protein X-ray Crystallography' Second Edition, Springer. However, as pointed out by Cruickshank* (1999) a Luzzati (plot of R versus 2sin theta/lambda) was not really developed to give final errors. *Acta Crystallogr D Biol Crystallogr. 1999 Mar;55(Pt 3):583-601. Remarks about protein structure precision. Best wishes Roberto Thank you very much. Cheers Melanie
Re: [ccp4bb] Low Bfactor values for waters after TLS
Hi Alasdair On 11 Sep 2009, at 09:46, Alasdair K. Mackenzie wrote: Greetings CCP4-ers, I have been running some TLS refinement which does wonders for my R/ Rfree values, but seems to give some spurious Bfactors/ADPs for the waters, e.g. B= 2. These low Bfactors appear relatively consistent with the residual protein B-factors (i.e. the pdb output from REFMAC), but when i use TLS analyse to apply the TLS contribution to the total B-factors for the model, the low water B values seem crazy...i apparently have a water with a B- factor of 2 interacting with a side chain with a B-factor of 60 Has anyone encountered this type of thing before? Yes. I have reported this to Garib some time ago and I believe the active Refmac people are working on this. and does anyone have a good solution for getting around the problem? Mhh... Latest Refmac versions seem to consider waters directly bound to the protein as members of the various TLS groups. You can override this by using TLSD WATERS EXCLUDE You can give it a go and see if it helps. Best Roberto cheers, Al
Re: [ccp4bb] Alternative conformation in Mac Refmac5
Hi Heidi, I have noticed the same thing. Went back to Refmac v93 (mac version) and side-chains stopped flying. Needs a fix. Best Roberto On 11 Sep 2009, at 10:59, Heidi Tuominen wrote: Hi all, I have updated lately to ccp4-6.1.2. I have Macintosh. Now I have problem with alternative conformations. After Refmac5 run all alternative conformation atoms are fully unordered without any chemical restraints. If I put same pdb to Windows Refmac5.5.0102 there is no any problems except R-factors are some higher. Do I have some problem or is there bug in Mac version of Refmac5.5.0102? How problem could be solved? Thank you, Heidi Tuominen University of Turku, Finland
Re: [ccp4bb] cyseteine modification
You can also have a look at Active site structural features for chemically modified forms of rhodanese. Gliubich F, Gazerro M, Zanotti G, Delbono S, Bombieri G, Berni R. J Biol Chem. 1996 Aug 30;271(35):21054-61. Best Roberto On 19 Aug 2009, at 15:41, Debajyoti Dutta wrote: Dear Sir, Is there any other oxidation states of cysteine other than cysteine sulphinic acid and cysteine sulphonic acid. In my protein, the cysteine molecule is definitely overoxidized but the electron density is not corresponding to the sulphinic or the sulphonic acid. The positive density looks as if it can accomodate only one oxygen atom and not more. Thank you for reply in advance. Sincerely Debajyoti Dutta
Re: [ccp4bb] quaternary structure comparison
Hi Eike, PISA (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) or SCOPPI (http://www.scoppi.org ) might be useful. Interested in hearing about other alternatives. Best wishes Roberto On 15 Jun 2009, at 21:28, Eike - Christian Schulz wrote: Dear All, I solved a structure that has a very common tertiary structure motif but it appears as if the arrangement of the monomers is somewhat ‘non-standart’. Of course I performed a DALI search, which resulted in a great number of hits due to the similarity of the tertiary structure (Z’s: 250 8; 100 10). In order to check whether this arrangement that I see is something new I would like to perform a quaternary structure comparison. Is there any such tool available? A Google search wasn't very conclusive. Or is there a clever combination of available resources? Kind regards Eike --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] issues with TLS refinement
I might be wrong but it seems Garib has implemented some sort of line minimization for the tls part which causes the observed behavior. Garib? Roberto On 21 May 2009, at 00:39, Dima Klenchin klenc...@facstaff.wisc.edu wrote: The issue is: While running tls and restrained refinement, the program doesn't perform the given no. of tls refinements. For e.g., if you ask it to perform 10 TLS and 5 restrained refinement rounds, it'll run may be 2 or 3 rounds of TLS and then jump to restrained refinement rounds and finish the job. It is, however, finishing the given no. of restrained refinements every-time. There is no error message or any indication of abrupt ending to tls refinements in the log file either. FWIIW, I am getting the same thing. Sometimes no TLS refinement is done at all, sometimes the program does 2-3 cycles and sometimes the requested number of cycles. Seems to be PDB input-dependent but I could never find what causes the behavior. Dima
[ccp4bb] postdoctoral position at King's College London
Dear ccp4bb readers, --- King's College London Cardiovascular Division and Randall Division of Cell and Molecular Biophysics A postdoctoral position is immediately available for a highly motivated molecular biologist/biochemist with an interest in structural biology (macromolecular crystallography and NMR) to perform structure-function studies of proteins and protein complexes involved in control of reactive oxygen species (ROS) in redox signalling. The position is supported by a British Heart Foundation Centre of Excellence scheme and is part of a research collaboration between the groups of Drs Sasi Conte and Roberto Steiner (Randall Division of Cell and Molecular Biophysics) and Professor Ajay Shah (Cardiovascular Division). Excellent infrastructure support is available to carry out the project. The salary is Grade 6, SP31 which translates into: £30,594 plus £3,323 (London weighting). The position is initially funded for 18 months. For informal enquiries please contact: roberto.stei...@kcl.ac.uk http://www.kcl.ac.uk/schools/medicine/research/cardio/pi/shah-a.html http://www.kcl.ac.uk/schools/biohealth/research/randall/ --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
[ccp4bb] postdoctoral position at King's College London (2)
Dear ccp4bb readers, £1,000 worth correction on my previous postdoc ad: London weighting is £2,323 and not £3,323. Apologies (all the rest is correct..), Roberto On 9 Mar 2009, at 09:49, Roberto Steiner wrote: Dear ccp4bb readers, --- King's College London Cardiovascular Division and Randall Division of Cell and Molecular Biophysics A postdoctoral position is immediately available for a highly motivated molecular biologist/biochemist with an interest in structural biology (macromolecular crystallography and NMR) to perform structure-function studies of proteins and protein complexes involved in control of reactive oxygen species (ROS) in redox signalling. The position is supported by a British Heart Foundation Centre of Excellence scheme and is part of a research collaboration between the groups of Drs Sasi Conte and Roberto Steiner (Randall Division of Cell and Molecular Biophysics) and Professor Ajay Shah (Cardiovascular Division). Excellent infrastructure support is available to carry out the project. The salary is Grade 6, SP31 which translates into: £30,594 plus £3,323 (London weighting). The position is initially funded for 18 months. For informal enquiries please contact: roberto.stei...@kcl.ac.uk http://www.kcl.ac.uk/schools/medicine/research/cardio/pi/shah-a.html http://www.kcl.ac.uk/schools/biohealth/research/randall/ --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] Twin refinement - selecting FreeR
Hi Sabine, If your question is: Is it possible to refine twinned structures with Refmac? My answer is: Yes. Very well. Best wishes, Roberto On 20 Feb 2009, at 16:32, Sabine Schneider wrote: Hi everyone, I got good data to 2.4A on a protein-ligand complex. I want to solve the structure by MR using a model with 45% seq. ID and 55% similarity. Initially the data appeared to be P622 (pointless, selfrotation function etc). Unit cell: 145 145 65, MW protein is 28kDa I prepared the MR model with chainsaw, choping of non-conserved residues at the C gamma and reset the B-factors. Phaser found a solution, but with negativ LLG. I than processed the data in P3, P321, P312 and P6 and run Phaser searching all possible alternative spacegroups. The best solution is P3 (4mol/asu) followed by P321 (2mol/asu),both with a LLG of above 400. But when trying to refine the models in Refmac the R/Rfree stays at ~48%. Looking at the truncate output and phenix xtriage, twinning is suggested with the merohedral twin law -h, -k, l and a twin fraction of 40.3% Using twin refine option in Refmac (5.5.0070), the R/Rfree for the P321 solution drops to around 33%, but the difference between R/Rfree is only 1%. For the solution found in P3 the R/Rfree stays at around 47%. So I assumed that P321 is the better solution. Is the difference in R/Rfree only 1%, because the free and work reflections are related through the twin law? I used phenix to assign the free reflections putting in the twin operators. Doing simulated annealing in phenix, I get a rather large difference in R/Rfree of ~7%. Well, I guess I need to do some tweaking of the parameters in phenix (running the latest phenix and cci_apps). When I than use these free reflections assigned by phenix in Refmac I still get only 1.5% difference between R and Rfree? So is it doable in Refmac, or is my best bet phenix? Any advice what is the best way to proceed is much appreciated! Sabine -- Dr. Sabine Schneider Ludwig-Maximilians-University Department of Chemistry and Pharmacy Butenandtstrasse 5-13, Building F 81377 Munich Germany Phone: +49 (0)89 2180 77846 Fax: +49 (0)89 2180 77756 http://www.carellgroup.de/ --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] Off topic: Crystal degredation with age
Hi Ed, A bit late on the subject I have collected atomic resolution data (around 0.97A) on both bovine and porcine phospholipase A2 crystals which at the time of data collection were between 10-16 years old. Crystallization setup was liquid-liquid diffusion in glass capillaries. Differently from the other stories, here there's no degradation involved...just rock-solid Xtals. In the paper we stated: 'Crystals are stable in the crystallization solution...' which I guess well reflects their behavior. Best wishes, Roberto On 5 Feb 2009, at 19:11, Edward Snell wrote: /lurk_mode_off /dumb_question_on Dear All, I was recently trying to find references on how age may degrade a crystal, i.e. grow them and use them or preserve them as fresh as possible. I seem to remember seeing a couple of papers on this but my memory is fading and I have been unable to locate them. Can anyone jog my memory or tell me if I'm imagining things? I've found plenty on the protein prep etc. but nothing on the crystal. Thanks, Eddie. Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here!Crystallization, how quaint! /dumb_question_off /lurk_mode_on --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail roberto.stei...@kcl.ac.uk
Re: [ccp4bb] Choosing MR solutions in the case of perfect twinning with P41212?
Hi Francis, I would consider the possibility of a orthorhombic sg (a~b with twinning). If unlucky maybe even monoclinic. Cheers Roberto On 5 Feb 2009, at 18:10, Francis E Reyes francis.re...@colorado.edu wrote: It seems like this space group will be the death of me. I'm working on a structure in SG P41212 one molecule per asu that was solved with experimental SAD phases. The resolution is to 2.5 and the refinement is stuck at an R/Rfree of 30 and 33 with bonds rmsd of 0.011 and angles of 1.597 . The unit cell is 73.604 73.604 114.279 90.00 90.00 90.00. I'm considering the case of perfect twinning where the real s.g. is P41 masked under the higher symmetry in P41212. It seems to be the case in perfect twinning that the approach is to molecular replace the refined model into the lower space group. I reindexed my data to the lower space group P41 and molecular replaced into the reindexed data with Phaser. A single was solution was found with 2 mol per asu (39.6% solvent content) related by NCS. I've refined the now two fold ncs related structure in P41 to a much more respectable R/Rfree of 25.2 and 28.6 with rmsd bonds at 0.004 and angles at 0.865 refining with a twin law and NCS as implemented by phenix.refine. However I'm not happy: [1] a simmulated anneal omit map one of the monomers in P41 where 5 residues in a non crystal contact region of the molecule (I wanted to challenge the omit map) shows nearly no density. (the SA OMIT map was generated with phenix.autobuild using the same refine parameters as the final round of refinement) [2] the NCS selection is a little bit troubling. (maybe the phenix developers can chime in on this) reference = chain 'B' and (resseq 243:293 or resseq 310:370 ) selection = chain 'A' and (resseq 243:293 or resseq 310:370 ) seems as if resseq 243:293 is behaving differently than 310:370? [3] the densities of the side chains of a helix (not an xtal contact) are poorly defined, with geometry for the backbone not so good. There's talk about choosing the correct MR solution (see On the molecular-replacement problem in the presence of merohedral twinning: structure of the N-terminal half-molecule of human lactoferrinW. A. Breyer, R. L. Kingston, B. F. Anderson and E. N. Baker ) . I use phaser to pick my MR solution for P41. Could phaser possibly have chosen poorly? Thanks! FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] generating omit maps
There used to be a program called OMIT in CCP4. Seems to be still supported. I believe I used it years ago but I vaguely remember problems with some space groups (might be wrong though...) Regards, Roberto On 11 Dec 2008, at 16:20, rajan sreekanth wrote: Hi What about the SFcheck omit map calculation in 'Map and Mask utilities' module in CCP4? R.Sreekanth On Wed, 10 Dec 2008 Kathleen Frey wrote : Hi Everyone, Can anyone tell me a relatively easy way to generate an omit density map for a ligand? I know that CNS can do this, but I was wondering if there's a CCP4 related program to generate omit maps. Thanks, Kathleen --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
[ccp4bb] Coot and Hs
Dear all, a problem possibly at the coot/mmdb interface... If one uploads a pdb file (from phenix.refine in the example below) that contains Hs into Coot and then writes it out (with or without any modification done on it) Coot shifts the HG2n of THR on the right by one column space. Because column 17 is kept empty the result is three identical HG2 THR protons. Example: Uploaded: CRYST1 45.460 166.970 167.200 90.00 90.00 90.00 P 21 21 21 SCALE1 0.021997 -0.00 -0.000.0 SCALE2 0.00 0.005989 0.000.0 SCALE3 0.00 0.00 0.0059810.0 ATOM 1 N THR A 2 27.439 61.155 24.800 1.00 70.37 AN ANISOU1 N THR A 2 9721 7539 9478 1186 2103 297 AN ATOM 2 CA THR A 2 26.489 62.215 24.456 1.00 77.21 AC ANISOU2 CA THR A 210790 8621 9925 1145 1971 285 AC ATOM 3 CB THR A 2 26.960 63.618 24.975 1.00 73.93 AC ANISOU3 CB THR A 210519 8251 9322 1244 1638 409 AC ATOM 4 OG1 THR A 2 28.259 63.512 25.579 1.00 73.16 AO ANISOU4 OG1 THR A 210309 7971 9516 1332 1549 489 AO ATOM 5 CG2 THR A 2 25.969 64.197 25.999 1.00 64.65 AC ANISOU5 CG2 THR A 2 9547 7174 7845 1451 1403 582 AC ATOM 6 C THR A 2 26.200 62.288 22.934 1.00 71.92 AC ANISOU6 C THR A 210101 8073 9151869 2179 63 AC ATOM 7 O THR A 2 25.172 61.797 22.455 1.00 64.74 AO ANISOU7 O THR A 2 9210 7258 8131790 2339 -23 AO ATOM 8 HA THR A 2 25.639 62.015 24.900 1.00 71.84 AH ATOM 9 HB THR A 2 27.009 64.232 24.226 1.00 69.26 AH ATOM 10 HG1 THR A 2 28.232 62.991 26.212 1.00 67.50 AH ATOM 11 HG21 THR A 2 26.275 65.055 26.303 1.00 61.19 AH ATOM 12 HG22 THR A 2 25.104 64.300 25.596 1.00 61.19 AH ATOM 13 HG23 THR A 2 25.894 63.607 26.753 1.00 61.19 AH Written: CRYST1 45.460 166.970 167.200 90.00 90.00 90.00 P 21 21 21 SCALE1 0.021997 -0.00 -0.000.0 SCALE2 0.00 0.005989 0.000.0 SCALE3 0.00 0.00 0.0059810.0 ATOM 1 N THR A 2 27.439 61.155 24.800 1.00 70.37 AN ANISOU1 N THR A 2 9721 7539 9478 1186 2103 297 AN ATOM 2 CA THR A 2 26.489 62.215 24.456 1.00 77.21 AC ANISOU2 CA THR A 210790 8621 9925 1145 1971 285 AC ATOM 3 CB THR A 2 26.960 63.618 24.975 1.00 73.93 AC ANISOU3 CB THR A 210519 8251 9322 1244 1638 409 AC ATOM 4 OG1 THR A 2 28.259 63.512 25.579 1.00 73.16 AO ANISOU4 OG1 THR A 210309 7971 9516 1332 1549 489 AO ATOM 5 CG2 THR A 2 25.969 64.197 25.999 1.00 64.65 AC ANISOU5 CG2 THR A 2 9547 7174 7845 1451 1403 582 AC ATOM 6 C THR A 2 26.200 62.288 22.934 1.00 71.92 AC ANISOU6 C THR A 210101 8073 9151869 2179 63 AC ATOM 7 O THR A 2 25.172 61.797 22.455 1.00 64.74 AO ANISOU7 O THR A 2 9210 7258 8131790 2339 -23 AO ATOM 8 HA THR A 2 25.639 62.015 24.900 1.00 71.84 AH ATOM 9 HB THR A 2 27.009 64.232 24.226 1.00 69.26 AH ATOM 10 HG1 THR A 2 28.232 62.991 26.212 1.00 67.50 AH ATOM 11 HG2 THR A 2 26.275 65.055 26.303 1.00 61.19 AH ATOM 12 HG2 THR A 2 25.104 64.300 25.596 1.00 61.19 AH ATOM 13 HG2 THR A 2 25.894 63.607 26.753 1.00 61.19 AH Does anyone know (Paul?) the reason for the above? The only solution right now (that I know of) is to remove all Hs and generate them again (molprobity for example) prior to refinement. Cheers, Roberto --- Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
I have a similar problem Matthew. Just got some NVoy from Novexin in which some claim can help if hydrophobic patches are the main problem. Will see. Regards, Roberto On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote: Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/ contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] A babyish question on coot
Hi Yanming, I am glad that at least one other person has encountered this problem so I do not feel too guilty for having asked Paul to do something about it. Anyway, the MAN was kind enough to add a masking function under the Extensions Menu. It is something called 'Mask map bla bla..'. (I am not in the office so I can't read the proper name of the function). It uses mmdb code to define Chains/atoms/ that you want to exclude from map calculations and produces a map masked by them. (For example if all your metal centres are chain M something like M/* removes all of them). Very useful imo for refining metal centers at low resolution. Roberto Quoting Yanming Zhang [EMAIL PROTECTED]: Hi, After JED's email I tried immediately: case: a metal site which has very strong density and a HIS co-ordinating with the ion. After I click 'Real space refine zone' and then the residue HIS, the HIS will move to the density which belongs to the ion(clash with the ion) no matter how low the weight was set. PS, I remember that Xfit of XtalView has the beauty to avoid this kind of problem, but now I am a regular user of coot. Thanks Yanming On Tue, 29 May 2007, Debreczeni, Judit wrote: Unreasonable geometry can be avoided by setting the refinement weight to a lower value -- either using Extensions-Set Matrix (Refinement Weight) or in your .coot file: (set-matrix value) There's also a coot-bb, btw. JED -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of Yanming Zhang Sent: 29 May 2007 01:50 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] A babyish question on coot Hi, I use coot almost around the clock. One thing troubles me is that: when clicking on 'real space refine zone', coot seems only care about the 'real space'(Electron density), sometimes it will bring the model to the density no matter whether the density was already claimed by other model atoms or not, resulting in clash and unreasonable geometry. How can I avoid this? Sorry for an old crystallographer to ask so babyish question. But your help can save me lots of time. Thanks Yanming -- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] Stop Refmac from refining B factors?
On 18 Apr 2007, at 14:39, Eva Kirchner wrote: (But I'm still curious about the B-factor refinement when there is no REFI BREF ISOT in the com-file...) Eva, Refmac internal default is REFI BREF ISOT that's why even if you remove the above line from the com file (or deselect that option from the interface) it still does ISOT BREF refinement. It does tell you that though if you look at the log file there's a bit that says ... Method of minimisation : Sparse Matrix Experimental sigmas used for weighting Number of Bins and width:20 0.0080 Refinement of individual isotropic Bfactors .. what do you have there when you deselect the B fact option from the interface? I agree with Herman that at 3.2 A isotropic B values refinement can be useful. Roberto Eva 2007/4/18, Mischa Machius [EMAIL PROTECTED]: Like Harry said, it is not justified to do individual B factor refinement at that resolution. Well, you can do it, but you'll end up with funny results, such as what are observing right now. Still, from a pragmatic point of view, individual B factor refinement in cases like these can have a positive effect on the electron density. However, keep in mind that the resulting B factors may physically not be very meaningful. In the end, you'll have to switch to grouped B factor refinement, or you risk nasty comments from an attentive mentor or reviewer (and rightly so). Hope that helps. Best - MM On Apr 18, 2007, at 7:20 AM, Eva Kirchner wrote: 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 -- -- Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] Question about glove boxes for protein crystallization
Hi Mathews, An anaerobic chamber from Belle Technology (http://www.belle- technology.com/) served me well in the past. Belle's glove boxes are made of acrylic material. That offers relatively low-cost and all-round visibility. Cheers, Roberto On 23 Feb 2007, at 02:03, Mathews, Irimpan wrote: Dear Friends, Sorry for the non CCP4 question. We are planning to purchase a small glove box to setup crystallization trays under anaerobic conditions. If you have used glove boxes for crystallization, would you please give me some idea? We are thinking of getting the 815 series from Plas-labs (link below). http://www.plas-labs.com/ Thank you very much, Mathews Ps: If others are interested, I will post a summary. --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] how many stuck datasets are actually twinned?
Hi Sue, This reference has very useful info Lebedev AA, Vagin AA, Murshudov GN. Intensity statistics in twinned crystals with examples from the PDB. Acta Crystallogr D Biol Crystallogr. 2006 Jan;62(Pt 1):83-95. Epub 2005 Dec 14. PMID: 16369097 [PubMed - indexed for MEDLINE] Roberto On 21 Feb 2007, at 15:22, Sue Roberts wrote: Hello A partially philosophical, partially pragmatic question. I've noticed a trend, both on ccp4bb and locally, to jump to twinning as an explanation for data sets which do not refine well - that is data sets with R and Rfree stuck above whatever the person's pre-conceived idea of an acceptable R and Rfree are. This usually leads to a mad chase through all possible space groups, twinning refinements, etc. and, in my experience, often results in a lot of time being spent for no significant improvements. Just out of curiosity, does anyone have a feel for what fraction of stuck data sets are actually twinned? (I presume this will vary somewhat with the type of problem being worked on). And a sorta-hypothetical question, given nice-looking crystals; images with no visible split spots, extra reflections, or streaks; good predictions; nice integration profiles; good scaling with reasonable systematic absences; a normal solvent content; and a plausible structure solution, and R/Rf somewhat highish (lets say . 25/.3 for 1.8 A data), how often would you expect the Stuck R/Rf to be caused by twinning (or would you not consider this a failed refinement). (My bias is that such data sets are almost never twinned and one should look elsewhere for the problem, but perhaps others know better.) Sue Sue Roberts Biochemistry Biopphysics University of Arizona [EMAIL PROTECTED] --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
