Re: [ccp4bb] Help needed in solving a MAD dataset
Hi Qing Lu, try Autorickshaw: http://www.embl-hamburg.de/Auto-Rickshaw/ It can perform the complete structure solution procedure if the quality of your data is sufficient and I consider it very user-friendly, especially for beginners . Regards, Uli Hi All, I am new to protein crystallography. I would like to know the steps involved in solving a MAD dataset by using the program in CCP4 where you determine the phases and then obtain the trace. The dataset is collected at 3 different wavelengths (peak, inflection and remote) using Se-Met as the scatterer. The crystals diffracted to resolution of 2 Angstrsoms and has a good anomalous signal. Thanks, Qing Lu
[ccp4bb] Postdoctoral position in protein structure at UCLA
A postdoctoral position is available immediately to work on structures of bacterial microcompartments and to pursue other projects involving protein design and assembly. Bacterial microcompartments are poorly characterized virus-sized protein shells that serve as organelles inside many bacterial cells by encapsulating enzymes in key metabolic pathways. Recent findings indicate that they are highly sophisticated, for example controlling the transport of metabolites by specific molecular transport through protein pores. Our structural studies on these systems are far enough advanced that protein design and computational analysis provide numerous avenues for exploration. Individuals interested in combining computational investigations with crystallographic studies are welcome. UCLA provides a rich environment for interactive and collaborative work in structural and computational biology. Please contact me directly at yea...@mbi.ucla.edu Best regards, Todd Yeates
Re: [ccp4bb] ncsfind
Hi Karin, I find PROFESSS by Kevin Cowtan very easy to use: professs xyzin hatom.pdb xyzout professs.pdb e END e I'm sure there is a CCP4i interface for it as well ... What I especially like about it is the tabulated relation between sites in the output PDB file. Cheers Clemens On Thu, May 20, 2010 at 04:47:06PM -0600, Karin van Straaten wrote: Dear all, Does anyone know if NCSFIND is still available or how I can get it. If not is there another program that I can use to determine noncrystallographic symmetry between Se-sites. Thanks in advance, Karin -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] TLSANL total B factor question
That was a bug in TLSANL, introduced in 6.1.0 and fixed in 6.1.2. So you need to update your CCP4, or at least extract a newer tlsanl binary. Apologies for that. Martyn On Fri, 2010-05-21 at 04:46 +0100, Shiva Kumar wrote: Dear Crystallographers I am trying to print out my total B factors using TLSANL (version: 6.1) in CCP4- 6.1.1. My TLSANL’s input file.pdb is coming from refmac (version: 5.5.0072) using the TLS restraint refinement option and isotropic B factors. The TLSANL’s output file.pdb contains the following ATOM and ANISOU records as an example. REMARK 3 TLS DETAILS REMARK 3 NUMBER OF TLS GROUPS :2 REMARK 3 ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS REMARK 3 ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS ATOM 88 C ASN A 14 0.748 -5.841 -6.258 1.00 35.84 C ANISOU 88 C ASN A 14 5335 4549 3734 0 0 0 C ATOM 89 O ASN A 14 0.807 -6.941 -6.845 1.00 35.04 O ANISOU 89 O ASN A 14 5229 4375 3709 0 0 0 O I am not able to understand why my ANISOU record contains ‘0 0 0’ for the anisotropic component. Something is not correct and I'm not sure why I am not able to print out my total B factors. I would appreciate it if someone could tell me what is going wrong and how can I print my total B factors. Thanks Regards Shiva Kumar -- *** * * * Dr. Martyn Winn * * * * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * * Tel: +44 1925 603455E-mail: martyn.w...@stfc.ac.uk* * Fax: +44 1925 603634Skype name: martyn.winn * * URL: http://www.ccp4.ac.uk/martyn/ * ***
Re: [ccp4bb] Help needed in solving a MAD dataset
Dear All, Not only for beginners, but also for experts who want to solve structures in hurry at the beamline and who want to complete model starting from X-ray data at resolution better than 3.0 A resolution. Santosh Quoting Ulrich Zander ulrich.zan...@strubio.uni-kiel.de: Hi Qing Lu, try Autorickshaw: http://www.embl-hamburg.de/Auto-Rickshaw/ It can perform the complete structure solution procedure if the quality of your data is sufficient and I consider it very user-friendly, especially for beginners . Regards, Uli Hi All, I am new to protein crystallography. I would like to know the steps involved in solving a MAD dataset by using the program in CCP4 where you determine the phases and then obtain the trace. The dataset is collected at 3 different wavelengths (peak, inflection and remote) using Se-Met as the scatterer. The crystals diffracted to resolution of 2 Angstrsoms and has a good anomalous signal. Thanks, Qing Lu Santosh Panjikar, Ph.D. Staff Scientist EMBL-Hamburg Outstation C/o DESY Notkestrasse 85 22603 Hamburg (Germany) panjikar at embl-hamburg.de http://www.embl-hamburg.de/~panjikar
Re: [ccp4bb] Help needed in solving a MAD dataset
Dear Jürgen, The road to perfection is a long one ... . Thank you for the feedback! With best wishes, Gerard. -- On Thu, May 20, 2010 at 10:31:37PM -0400, Jürgen Bosch wrote: I don't like the site finding option in autosharp, takes too long in most of my cases. So my approach is locate sites via SHELX, then feed them into Sharp. Sorry Gerard :-) Jürgen On May 20, 2010, at 10:02 PM, Jeremiah Farelli wrote: I second autoSHARP/SHARP. It makes great initial maps, and once you get it running, it is totally worth it. - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * ===
[ccp4bb] molecular replacement
Hi all I am trying to do molecular replacement with low resolution (4Å) using Molrep and Phaser. Overall R-factor is 11.3%, completeness 95.4%, I/sigma 2, and Chi^2 0.95. Identities between my protein and templates were more than 80%. I couldn’t get correct solution. Rotation function, translation score, and contrast were low, and they had no significance, though I changed the range of resolution. Molrep suggested solution coordinates clashed with symmetry molecules. I tried MR after remove clashed regions, but another clashes happened. In the case of phaser, there were many clashes, too. Please, give me any suggestion. Should I concern about any options when I run MR programs? Hope you guys will be interested to answer!! Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
Re: [ccp4bb] molecular replacement
Dear intekhab, a few suggestions: - are you sure of the space group or might there be alternatives? - is you protein globular or modular, i.e., is it worth running MR with stable subdomains one after the other? - try a poly-alanine model (e.g. chainsaw can do this for you, pdbset probably, too) - did you get overloads? If so, collect a low resolution pass to get correct intensities even at the poor resolution end. - what is your low resolution limit? Did you use the default or could you extend it? - did your crystal(s) suffer from radiation damage? It might be worth collection only a complete data set with not too high multiplicity. Good luck, Tim On Fri, May 21, 2010 at 08:01:10PM +0900, intekhab alam wrote: Hi all I am trying to do molecular replacement with low resolution (4Å) using Molrep and Phaser. Overall R-factor is 11.3%, completeness 95.4%, I/sigma 2, and Chi^2 0.95. Identities between my protein and templates were more than 80%. I couldn’t get correct solution. Rotation function, translation score, and contrast were low, and they had no significance, though I changed the range of resolution. Molrep suggested solution coordinates clashed with symmetry molecules. I tried MR after remove clashed regions, but another clashes happened. In the case of phaser, there were many clashes, too. Please, give me any suggestion. Should I concern about any options when I run MR programs? Hope you guys will be interested to answer!! Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Question: Refmac5 stats reported in pdb REMARK 3
Dear Phil, Your observation that the refinement details in PDB format REMARKs are difficult to interpret and compare is well taken. Each refinement package produces its own set of refinement results calculated in its own way. Both the calculation and presentation of this information in PDB format differs between programs, and even between program versions. The lack of standardization in how refinement information is reported is confusing to many PDB users. In the spirit of supporting innovation, the PDB has historically tried to accommodate this diversity by providing program- and version- specific REMARK 3 formats. However, the field of structural biology has matured considerably in the past few decades, and time-tested, consensus, and best-practice approaches can now be defined in many cases. In our view, adopting such approaches (rather than accomodating every variant ever implemented) would be the best way to serve the interests of both non-expert user communities and the experimental structural biology community. As an illustration, it is interesting to note that there are at least 20 different types of R-values reported in the current archive. The subtle differences in these quantities may be of interest in understanding the evolution of refinement methodology. However, we believe that a smaller, common set of well-defined data items describing refinement results would be more useful to the broader community of PDB users. To this end, the wwPDB maintains an Exchange Data Dictionary of community-vetted definitions and examples of each data item in the PDB archive. This is an extensible dictionary that grows with new technologies and science. For instance, wwPDB has used this extensibility to capture and define all the various R-values. While the dictionary technology provides a framework for definition and standardization, this only addresses part of the problem. Even though we have precise definitions for the wide range of R-value types, R-value comparisons between entries is still complicated because the values are not uniformly populated across the archive. To fully address the problem, we not only need the standardization provided by the dictionary technology but also the cooperation of the software package developers in producing a common set of statistics and diagnostics. This does not preclude reporting new and novel data items, but these should be provided in addition to a common core of data results. Further information about the PDB Exchange Data Dictionary can be found at our dictionary resource site, http://mmcif.pdb.org/ Correspondence information between our PDB Exchange Data Dictionary and items in the current PDB format is also available at http://mmcif.pdb.org/dictionaries/pdb-correspondence/pdb2mmcif-2010.html Sincerely, Christine Zardecki for the wwPDB From: Phil Jeffrey pjeff...@princeton.edu Date: May 19, 2010 4:02:22 PM EDT To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Question: Refmac5 stats reported in pdb REMARK 3 Reply-To: Phil Jeffrey pjeff...@princeton.edu Compare these two lines from phenix.refine: REMARK 3 NUMBER OF REFLECTIONS : 46001 REMARK 3 FREE R VALUE TEST SET COUNT : 2339 with those from refmac, ostensibly using the same data and start pdb: REMARK 3 NUMBER OF REFLECTIONS : 43672 REMARK 3 FREE R VALUE TEST SET COUNT : 2339 I know there are 46011 reflections with |F|0 in the files I used. phenix.refine removes 10 of these as outliers. The 46001 remaining reported in REMARK 3 *include* the test set. With REFMAC, 43672+2339=46011 so it appears that Refmac reports just the *working* set count in that first line, excluding the test set. Is this is a bug with one program or the other, or a bug in the PDB definition of REMARK 3 ? http://www.wwpdb.org/documentation/ format23/remark3.html This appears to be a source of inconsistency. phenix.refine 1.6-289 refmac5 5.4.0077 (I'm apparently a Luddite) Phil Jeffrey Princeton
[ccp4bb] off-topic--refrigerated shakers
Any recommendations for WELL-MADE refrigerated shakers? Our 1980-vintage New Brunswick Psycrotherm* is getting creaky, and the repair people claim that parts are not available. Hence, we're looking at the cost-effectiveness of replacing it with a new one. Probably best to reply off-list, and I can assemble a summary if appropriate. Thanks, Pat * yes, it's that lovely shade of harvest gold --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] molecular replacement
Also check if you have correctly estimated no. of molecules in asymmetric unit. On Fri, May 21, 2010 at 4:58 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear intekhab, a few suggestions: - are you sure of the space group or might there be alternatives? - is you protein globular or modular, i.e., is it worth running MR with stable subdomains one after the other? - try a poly-alanine model (e.g. chainsaw can do this for you, pdbset probably, too) - did you get overloads? If so, collect a low resolution pass to get correct intensities even at the poor resolution end. - what is your low resolution limit? Did you use the default or could you extend it? - did your crystal(s) suffer from radiation damage? It might be worth collection only a complete data set with not too high multiplicity. Good luck, Tim On Fri, May 21, 2010 at 08:01:10PM +0900, intekhab alam wrote: Hi all I am trying to do molecular replacement with low resolution (4Å) using Molrep and Phaser. Overall R-factor is 11.3%, completeness 95.4%, I/sigma 2, and Chi^2 0.95. Identities between my protein and templates were more than 80%. I couldn’t get correct solution. Rotation function, translation score, and contrast were low, and they had no significance, though I changed the range of resolution. Molrep suggested solution coordinates clashed with symmetry molecules. I tried MR after remove clashed regions, but another clashes happened. In the case of phaser, there were many clashes, too. Please, give me any suggestion. Should I concern about any options when I run MR programs? Hope you guys will be interested to answer!! Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.9 (GNU/Linux) iD8DBQFL9m5wUxlJ7aRr7hoRAnJRAJkBglzDPgqcJ07lOf8wLpGTornFyACeMxwB XCPdN9n9Km6Gdn2SK+jv8WY= =ncth -END PGP SIGNATURE-
[ccp4bb] Alignment software
Dear All, I'm trying to prepare an alignment figure of 2 proteins that highlight conserved and similar residues and probably secondary structures; I will greatly appreciate it if anybody can recommend a software that I can use. Thanks, Mohd
Re: [ccp4bb] For 24 well Netxal plates (non ccp4 question) == SUMMARY
Dear All, Thank you for the several responses. Please see the summary below. 1. Several of them: complain to Qiagen (two or three said: protests loudly). Unfortunately, my e-mail to the Qiagen marketing manager never got any reply. I e-mailed to him on 5-12-10. Let me know if you need his e-mail address. 2. Molecular dimensions has the following new item. a novel 96 well version of a screw top plate for hanging drop which is of standard 96 well plate size. Please see http://www.moleculardimensions.com/shopdisplayproducts.asp?id=54cat=Hanging +drop+ Kind regards, Mathews -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mathews, Irimpan I. Sent: Tuesday, May 11, 2010 12:41 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] For 24 well Netxal plates (non ccp4 question) Dear All, Qiagen has stopped selling the 24 well Netxal trays and changed to 15 well trays (almost same price) that doesn't fit with any screens. Do you know of some source for the 24 well screw cap trays? Thank you very much for your help, Mathews
Re: [ccp4bb] molecular replacement
I may be a pessimist, but I'd focus my efforts on getting experimental phases. It can be hard to tell a correct molecular replacement solution from garbage with that kind of data, and it can be a real time sink because its hard to know when to quit. If you can get an SeMet data set, even it it doesn't provide enough phasing power to crack the problem, you can use the positions of Se peaks in anomalous diff maps to check possible molecular replacement solutions. Good luck! Phoebe Rice Original message Date: Fri, 21 May 2010 20:23:23 +0530 From: Vineet Gaur vineetgaur1...@gmail.com Subject: Re: [ccp4bb] molecular replacement To: CCP4BB@JISCMAIL.AC.UK Also check if you have correctly estimated no. of molecules in asymmetric unit. On Fri, May 21, 2010 at 4:58 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear intekhab, a few suggestions: - are you sure of the space group or might there be alternatives? - is you protein globular or modular, i.e., is it worth running MR with stable subdomains one after the other? - try a poly-alanine model (e.g. chainsaw can do this for you, pdbset probably, too) - did you get overloads? If so, collect a low resolution pass to get correct intensities even at the poor resolution end. - what is your low resolution limit? Did you use the default or could you extend it? - did your crystal(s) suffer from radiation damage? It might be worth collection only a complete data set with not too high multiplicity. Good luck, Tim On Fri, May 21, 2010 at 08:01:10PM +0900, intekhab alam wrote: Hi all I am trying to do molecular replacement with low resolution (4Å) using Molrep and Phaser. Overall R-factor is 11.3%, completeness 95.4%, I/sigma 2, and Chi^2 0.95. Identities between my protein and templates were more than 80%. I couldn’t get correct solution. Rotation function, translation score, and contrast were low, and they had no significance, though I changed the range of resolution. Molrep suggested solution coordinates clashed with symmetry molecules. I tried MR after remove clashed regions, but another clashes happened. In the case of phaser, there were many clashes, too. Please, give me any suggestion. Should I concern about any options when I run MR programs? Hope you guys will be interested to answer!! Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.9 (GNU/Linux) iD8DBQFL9m5wUxlJ7aRr7hoRAnJRAJkBglzDPgqcJ07lOf8wLpGTornFyACeMxwB XCPdN9n9Km6Gdn2SK+jv8WY= =ncth -END PGP SIGNATURE- Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] Alignment software
Try ESPript.. Gauri
Re: [ccp4bb] electron microscopy: where open access fails
Dear Filip, CCP4BB, 3DEM, and PDB-L list members, Thank you for your enthusiastic showing of community support for mandatory deposition of cryoEM maps to EMDB and for their timely release. The two year hold is available to EMDB depositors for just one practical reason: it encourages deposition of maps that we would probably otherwise not be getting at all (and those experiments would thus never be falsifiable). Until journals and major funding agencies make deposition and immediate release mandatory, we have to compromise. A large number of petition signatures ( http://www.petitiononline.com/cryoEM/petition.html ) will certainly help us to solidify our case. It is easy to forget that it was not until 1989 (17 years after the PDB began collecting X-ray structures) that community-set guidelines were published for X-ray crystallography coordinate deposition, and several more years before the journals required deposition as a prerequisite for publication. Structure factor deposition only became mandatory in 2008. These policies emerged from cooperative action by the community of experimentalists and interestingly included a petition that was led by Fred Richards. The same process will be effective for the EM community. Sincerely, The EMDataBank.org team http://emdatabank.org
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
On May 20, 2010, at 1:39 PM, Lijun Liu wrote: I think this is somehow tortured, especially by a quick reading of Dale's explanation. If I understand what you are saying, I think it is too. You imply that asymmetry in the enzyme results in two isomerase pathways. This may be true, but it has no consequence on the prospects for irreversibility. To avoid confusion, let's call these pathways D and S. Both the D and S pathways would have their own kf and kr kinetic constants such that kf_D/kr_D = kr_S/kf_S = Keq, which reflects the dG of the reaction. When the dG is close to zero for the isomerase reaction (which I assume here), then you can't make it irreversible. James All natural epimerases, isomerase and racemases use a mechanism based on L-amino acids to deal with a mirror-symmetric (quasi-, sometimes) reaction. In another word, these enzymes use a non- mirror symmetric structure to deal with a mirror-symmetric reaction, which itself causes the asymmetric kinetics for different direction, though the dG is 0. The Arrhenius Law k = A*exp(-dE/RT) should be understood like this: a mutation's effect to dE will be symmetric as Dale pointed out. However, the effects on A are asymmetric. A is related to intramolecular diffusion, substrate- and product-binding affinity, etc. That is why with mutation these enzymes changed their kinetics on two directions differently. Please check glutamate racemase, alanine racemase, aspartate racemase, DAPE epimerase, if you are interested. Never a 1000 to 1000 relation! Thus, mutation is possible to make one direction more favored---the point is you need the correct hit. Of course, such an experiment is never a Maxwell's demon. Lijun On May 19, 2010, at 8:51 AM, Maia Cherney wrote: You absolutely right, I thought about it. Maia Marius Schmidt wrote: Interestingly, Maxwell's demon pops up here, wh... , don't do it. If you change the reaction rate in one direction 1000 times slower than in the other direction, then the reaction becomes practically irreversible. And the system might not be at equilibrium. Maia R. M. Garavito wrote: Vinson, As Dale and Randy pointed out, you cannot change the #916G of a reaction by mutation: enzyme, which is a catalyst, affects only the activation barrier (#916E double-dagger). You can just make it a better (or worse) catalyst which would allow the reaction to flow faster (or slower) towards equilibrium. Nature solves this problem very elegantly by taking a readily reversible enzyme, like an epimerase or isomerase, and coupling it to a much less reversible reaction which removes product quickly. Hence, the mass action is only in one direction. An example of such an arrangement is the triose phosphate isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction pair. TIM is readily reversible (DHA = G3P), but G3P is rapidly converted to 1,3-diphosphoglycerate by GAPDH. The oxidation and phosphorylation reactions of GAPDH now make TIM work in one direction. Since many epimerases are very optimized enzymes, why not consider making a fusion with a second enzyme (like a reductase) to make the system flow in one direction. Of course, this depends on what you want to do with the product. Cheers, Michael // /R. Michael Garavito, Ph.D./ /Professor of Biochemistry Molecular Biology/ /513 Biochemistry Bldg. / /Michigan State University / /East Lansing, MI 48824-1319/ /Office:// //(517) 355-9724 Lab: (517) 353-9125/ /FAX: (517) 353-9334Email: rmgarav...@gmail.com mailto:garav...@gmail.com/ // On May 18, 2010, at 11:54 AM, Dale Tronrud wrote: Hi, I'm more of a Fourier coefficient kind of guy, but I thought that a #916G of zero simply corresponded to an equilibrium constant of one. You can certainly have reversible reactions with other equilibrium constants. In fact I think irreversible reactions are simply ones where the equilibrium constant is so far to one side that, in practice, the reaction always goes all the way to product. As Randy pointed out the enzyme cannot change the #916G (or the equilibrium constant). You could drive a reaction out of equilibrium by coupling it to some other reaction which itself is way out of equilibrium (such as ATP hydrolysis in the cell) but I don't think that's a simple mutation of your enzyme. ;-) Dale Tronrud On 05/18/10 00:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the #916G of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the
Re: [ccp4bb] TLSANL total B factor question
Hi Shiva, Not directly related to the problem you reported, but I wanted to warn you that in refmac 5.5.0072 the default is for the keyword TLSD WATERS ADD to be applied. This keyword tells refmac to assign all of your water molecules to existing TLS groups. (It does this quietly without any notice as to which waters are assigned to which groups in the TLS header of the pdb, TLSOUT or to the log file and no listing of the default setting of this keyword in the logfile.) So unless you included the non-default keyword TLSD WATERS EXCLUDE then your waters have residual B's as well in your pdb file and TLSANL cannot convert them to full B's since it has no record of which waters belong to which TLS groups. Thus the output from TLSANL will be a file with mixed full (on protein atoms) and residual (on water atoms) B-factors. If you did not give the keyword TLSD WATERS EXCLUDE, then the only method I know to ensure that the B-factors on the waters are properly converted to full-B is to re-run your last refmac job and add the keyword TLSO ADDU which will give you full B's on your output. (Note that you should not use this file for input into another round of refmac refinement -- unless you do a B-factor reset first). Regards, Mitch -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Shiva Kumar Sent: Thursday, May 20, 2010 8:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] TLSANL total B factor question Dear Crystallographers I am trying to print out my total B factors using TLSANL (version: 6.1) in CCP4- 6.1.1. My TLSANL’s input file.pdb is coming from refmac (version: 5.5.0072) using the TLS restraint refinement option and isotropic B factors. The TLSANL’s output file.pdb contains the following ATOM and ANISOU records as an example. REMARK 3 TLS DETAILS REMARK 3 NUMBER OF TLS GROUPS :2 REMARK 3 ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS REMARK 3 ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS ATOM 88 C ASN A 14 0.748 -5.841 -6.258 1.00 35.84 C ANISOU 88 C ASN A 14 5335 4549 3734 0 0 0 C ATOM 89 O ASN A 14 0.807 -6.941 -6.845 1.00 35.04 O ANISOU 89 O ASN A 14 5229 4375 3709 0 0 0 O I am not able to understand why my ANISOU record contains ‘0 0 0’ for the anisotropic component. Something is not correct and I'm not sure why I am not able to print out my total B factors. I would appreciate it if someone could tell me what is going wrong and how can I print my total B factors. Thanks Regards Shiva Kumar
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
If I understand what you are saying, I think it is too. You imply that asymmetry in the enzyme results in two isomerase pathways. This may be true, but it has no consequence on the prospects for irreversibility. To avoid confusion, let's call these pathways D and S. Both the D and S pathways would have their own kf and kr kinetic constants such that kf_D/kr_D = kr_S/kf_S = Keq, which reflects the dG of the reaction. When the dG is close to zero for the isomerase reaction (which I assume here), then you can't make it irreversible. This is not the case, at least in part. Such kind of enzymes, if no cofactor-needed, use the identical intermediate for the mirror symmetric reaction. For the D -- Intermediate -- S reaction, the enzyme uses the same pathway. Enzymes, for example, glutamate racemase and aspartate racemase, use a kind of psudo-mirror symmetric alignment at the active site to adapt the binding of D or S isomer in the half A.S., respectively. Other 3 atoms associated to the chiral center keeps fixed relative conformation during the inversion. Standard dG(0) of such a reaction is 0. However, at the time when enzyme works (for example, cell needs D-ASP in an almost pure L-ASP environment), the racemase moves L-Asp to D-Asp, in this regard, the dG of the reaction (not standard) is not 0. Your last sentence means: for a reaction (assuming dG(0) = 0 like racemic reaction) almost reaches EQ (dG ~ 0), you cannot make it irreversiblethis is true. Just please do not forget: such kind of enzymes work when the D -- S EQ is highly broken by nature (dG 0) [not dG(0)]. Hopefully I explained clearly! Lijun James All natural epimerases, isomerase and racemases use a mechanism based on L-amino acids to deal with a mirror-symmetric (quasi-, sometimes) reaction. In another word, these enzymes use a non- mirror symmetric structure to deal with a mirror-symmetric reaction, which itself causes the asymmetric kinetics for different direction, though the dG is 0. The Arrhenius Law k = A*exp(-dE/RT) should be understood like this: a mutation's effect to dE will be symmetric as Dale pointed out. However, the effects on A are asymmetric. A is related to intramolecular diffusion, substrate- and product-binding affinity, etc. That is why with mutation these enzymes changed their kinetics on two directions differently. Please check glutamate racemase, alanine racemase, aspartate racemase, DAPE epimerase, if you are interested. Never a 1000 to 1000 relation! Thus, mutation is possible to make one direction more favored---the point is you need the correct hit. Of course, such an experiment is never a Maxwell's demon. Lijun On May 19, 2010, at 8:51 AM, Maia Cherney wrote: You absolutely right, I thought about it. Maia Marius Schmidt wrote: Interestingly, Maxwell's demon pops up here, wh... , don't do it. If you change the reaction rate in one direction 1000 times slower than in the other direction, then the reaction becomes practically irreversible. And the system might not be at equilibrium. Maia R. M. Garavito wrote: Vinson, As Dale and Randy pointed out, you cannot change the #916G of a reaction by mutation: enzyme, which is a catalyst, affects only the activation barrier (#916E double-dagger). You can just make it a better (or worse) catalyst which would allow the reaction to flow faster (or slower) towards equilibrium. Nature solves this problem very elegantly by taking a readily reversible enzyme, like an epimerase or isomerase, and coupling it to a much less reversible reaction which removes product quickly. Hence, the mass action is only in one direction. An example of such an arrangement is the triose phosphate isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction pair. TIM is readily reversible (DHA = G3P), but G3P is rapidly converted to 1,3-diphosphoglycerate by GAPDH. The oxidation and phosphorylation reactions of GAPDH now make TIM work in one direction. Since many epimerases are very optimized enzymes, why not consider making a fusion with a second enzyme (like a reductase) to make the system flow in one direction. Of course, this depends on what you want to do with the product. Cheers, Michael / / /R. Michael Garavito, Ph.D./ /Professor of Biochemistry Molecular Biology/ /513 Biochemistry Bldg. / /Michigan State University / /East Lansing, MI 48824-1319/ /Office:// //(517) 355-9724 Lab: (517) 353-9125/ /FAX: (517) 353-9334Email: rmgarav...@gmail.com mailto:garav...@gmail.com/ / / On May 18, 2010, at 11:54 AM, Dale Tronrud wrote: Hi, I'm more of a Fourier coefficient kind of guy, but I thought that a #916G of zero simply corresponded to an
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
I think we are having a problem with the definition of reversible and irreversible. By Lijun's definition the reaction is irreversible because it proceeds from far from equilibrium toward equilibrium. That situation is more a property of the system than the enzyme. If you make the enzyme 1000 times faster the reaction will proceed more quickly toward equilibrium despite the fact that the reverse reaction is also 1000 times faster. The reverse reaction doesn't matter when there is no product to act upon. The original question was about having a reversible epimerase and I want to mutate it into an inreversible(sic) one. Clearly the poster is talking about a property of the enzyme. I interpreted this question to be a request to differentially change the forward and backward reaction rates, but I could be mistaken. Maybe the original poster could clarify the question. Dale Tronrud On 05/21/10 13:53, Lijun Liu wrote: If I understand what you are saying, I think it is too. You imply that asymmetry in the enzyme results in two isomerase pathways. This may be true, but it has no consequence on the prospects for irreversibility. To avoid confusion, let's call these pathways D and S. Both the D and S pathways would have their own kf and kr kinetic constants such that kf_D/kr_D = kr_S/kf_S = Keq, which reflects the dG of the reaction. When the dG is close to zero for the isomerase reaction (which I assume here), then you can't make it irreversible. This is not the case, at least in part. Such kind of enzymes, if no cofactor-needed, use the identical intermediate for the mirror symmetric reaction. For the D -- Intermediate -- S reaction, the enzyme uses the same pathway. Enzymes, for example, glutamate racemase and aspartate racemase, use a kind of psudo-mirror symmetric alignment at the active site to adapt the binding of D or S isomer in the half A.S., respectively. Other 3 atoms associated to the chiral center keeps fixed relative conformation during the inversion. Standard dG(0) of such a reaction is 0. However, at the time when enzyme works (for example, cell needs D-ASP in an almost pure L-ASP environment), the racemase moves L-Asp to D-Asp, in this regard, the dG of the reaction (not standard) is not 0. Your last sentence means: for a reaction (assuming dG(0) = 0 like racemic reaction) almost reaches EQ (dG ~ 0), you cannot make it irreversiblethis is true. Just please do not forget: such kind of enzymes work when the D -- S EQ is highly broken by nature (dG 0) [not dG(0)]. Hopefully I explained clearly! Lijun James All natural epimerases, isomerase and racemases use a mechanism based on L-amino acids to deal with a mirror-symmetric (quasi-, sometimes) reaction. In another word, these enzymes use a non-mirror symmetric structure to deal with a mirror-symmetric reaction, which itself causes the asymmetric kinetics for different direction, though the dG is 0. The Arrhenius Law k = A*exp(-dE/RT) should be understood like this: a mutation's effect to dE will be symmetric as Dale pointed out. However, the effects on A are asymmetric. A is related to intramolecular diffusion, substrate- and product-binding affinity, etc. That is why with mutation these enzymes changed their kinetics on two directions differently. Please check glutamate racemase, alanine racemase, aspartate racemase, DAPE epimerase, if you are interested. Never a 1000 to 1000 relation! Thus, mutation is possible to make one direction more favored---the point is you need the correct hit. Of course, such an experiment is never a Maxwell's demon. Lijun On May 19, 2010, at 8:51 AM, Maia Cherney wrote: You absolutely right, I thought about it. Maia Marius Schmidt wrote: Interestingly, Maxwell's demon pops up here, wh... , don't do it. If you change the reaction rate in one direction 1000 times slower than in the other direction, then the reaction becomes practically irreversible. And the system might not be at equilibrium. Maia R. M. Garavito wrote: Vinson, As Dale and Randy pointed out, you cannot change the #916G of a reaction by mutation: enzyme, which is a catalyst, affects only the activation barrier (#916E double-dagger). You can just make it a better (or worse) catalyst which would allow the reaction to flow faster (or slower) towards equilibrium. Nature solves this problem very elegantly by taking a readily reversible enzyme, like an epimerase or isomerase, and coupling it to a much less reversible reaction which removes product quickly. Hence, the mass action is only in one direction. An example of such an arrangement is the triose phosphate isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction pair. TIM is readily reversible (DHA = G3P), but G3P is rapidly converted to 1,3-diphosphoglycerate by GAPDH. The oxidation and phosphorylation reactions of
[ccp4bb] Fwd: calculate the real space R factor using OVERLAPMAP
Hello all, I'm forwarding a question from my labmate Hailiang Zhang regarding OVERLAPMAP and real-space R factors. Thanks in advance for your suggestions. Nasos Dousis Rice University P.S. Can someone please add haili...@bcm.tmc.edu to the CCP4BB listserv? Original Message Subject:calculate the real space R factor using OVERLAPMAP Date: Fri, 21 May 2010 17:48:47 -0500 From: Zhang, Hailiang haili...@bcm.tmc.edu To: ndou...@rice.edu ndou...@rice.edu Hi, I wanted to calculate the real space R factor using OVERLAPMAP. According to the general definition of real space R value (http://reference.iucr.org/dictionary/Real-space_residual), everything should be absolute value in the formula. However, the results by OVERLAPMAP were giving massive negative values. I also checked OVERLAPMAP documentation and found that the absolute sign seems absent in the R factor formula )http://smb.slac.stanford.edu/facilities/software/ccp4/html/overlapmap.html). I was just wondering whether there is some other tools in eg CCP4, Phenix or CNS to calculate real-space R in the general way in absolute values. Thanks!
Re: [ccp4bb] Fwd: calculate the real space R factor using OVERLAPMAP
Hello,
I was just wondering whether there is some other tools in eg CCP4, Phenix or
CNS to calculate real-space R in the general way in absolute values.
related to your question: in PHENIX you can get a table of map CC
(computed between model and 2mFo-DFc maps), and the value of mFo-DFc and
2mFo-DFc maps at atomic centers (in sigmas), along with occupancies and
B-factors. This can be reported per residue or per atom. The real-space
R-factor is not computed - well, firstly, no-one asked for this feature
(I can add it if really wanted), secondly, it feels to me that the
triplet of values {map CC, 2mFo-DFc, mFo-DFc} is more than enough, and
also I'm not sure I know how to interpret the real-space R-factor -:)
Tools to get this:
- from PHENIX GUI: Comprehensive validation
- from the command line:
phenix.model_vs_data model.pdb data.mtz
or
phenix.real_space_correlation parameters.par
Let me know if you have any questions about the above programs.
Pavel.
[ccp4bb]
Dear all, Recently, I am working on a complex which includes two protein subunits. The interaction was based on the Zinc Finger motif of one protein. I co-purified the complex by nickel affinity column with one protein bearing a C terminal His tag and the other without any affinity tags. However, the complex was disassociated when applied to size exclusion chromatography. The buffer I use for SEC is 20mM Tris-HCl, 150mM NaCl, 1mM DTT, 5% Glycerol, pH 7.5, whearas the buffer I use for nickel affinity column is 50mM Na2HPO4, 10mM KH2PO4, 137mM NaCl, 2.7mM KCl, 10% Glycerol, pH7.4. So I am wondering is it possible for the Tris buffer to strip the Zn ions from the Zinc Finger motif of one protein that leads to the destruction of the complex? I will be very appreciated if anyone has some experience in such case and would like to share with me! Sincerely, Heng Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China _ 想知道明天天气如何？必应告诉你！ http://cn.bing.com/search?q=%E5%A4%A9%E6%B0%94%E9%A2%84%E6%8A%A5form=MICHJ2
[ccp4bb] Is it possible for the Tris buffer to strip the Zn ions from the Zinc Finger motif of a protein?
Date: Sat, 22 May 2010 11:17:41 +0800 From: rh_ibp2...@hotmail.com Subject: [ccp4bb] To: CCP4BB@JISCMAIL.AC.UK Dear all, Recently, I am working on a complex which includes two protein subunits. The interaction was based on the Zinc Finger motif of one protein. I co-purified the complex by nickel affinity column with one protein bearing a C terminal His tag and the other without any affinity tags. However, the complex was disassociated when applied to size exclusion chromatography. The buffer I use for SEC is 20mM Tris-HCl, 150mM NaCl, 1mM DTT, 5% Glycerol, pH 7.5, whearas the buffer I use for nickel affinity column is 50mM Na2HPO4, 10mM KH2PO4, 137mM NaCl, 2.7mM KCl, 10% Glycerol, pH7.4. So I am wondering is it possible for the Tris buffer to strip the Zn ions from the Zinc Finger motif of one protein that leads to the destruction of the complex? I will be very appreciated if anyone has some experience in such case and would like to share with me! Sincerely, Heng Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 搜索本应是彩色的,快来体验新一代搜索引擎-必应,精美图片每天换哦! 立即试用！ _ SkyDrive电子画册，带你领略精彩照片，分享“美”时“美”刻! http://www.windowslive.cn/campaigns/e-magazine/ngmchina/?a=c
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
Dear, Dale, Lijun, Michael and all, Thank you all very much for your comment and discussion, from which not only do I find the answer to my question, but also I have learned a lot. In my case, both substrates are in the same form, saying 'D'. And I don't expect extra energy to break the EQ of the reaction. So I think the best thing I could do is to take Mickael's suggestion and add another enzyme to change the concentration of one substrate. However, Lijun's comment do open my mind. I really appreciate all your help, Best Regards, Vinson Liang --- 10年5月22日，周六, Dale Tronrud det...@uoxray.uoregon.edu 写道： 发件人: Dale Tronrud det...@uoxray.uoregon.edu 主题: Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one? 收件人: CCP4BB@JISCMAIL.AC.UK 日期: 2010年5月22日,周六,上午6:38 I think we are having a problem with the definition of reversible and irreversible. By Lijun's definition the reaction is irreversible because it proceeds from far from equilibrium toward equilibrium. That situation is more a property of the system than the enzyme. If you make the enzyme 1000 times faster the reaction will proceed more quickly toward equilibrium despite the fact that the reverse reaction is also 1000 times faster. The reverse reaction doesn't matter when there is no product to act upon. The original question was about having a reversible epimerase and I want to mutate it into an inreversible(sic) one. Clearly the poster is talking about a property of the enzyme. I interpreted this question to be a request to differentially change the forward and backward reaction rates, but I could be mistaken. Maybe the original poster could clarify the question. Dale Tronrud On 05/21/10 13:53, Lijun Liu wrote: If I understand what you are saying, I think it is too. You imply that asymmetry in the enzyme results in two isomerase pathways. This may be true, but it has no consequence on the prospects for irreversibility. To avoid confusion, let's call these pathways D and S. Both the D and S pathways would have their own kf and kr kinetic constants such that kf_D/kr_D = kr_S/kf_S = Keq, which reflects the dG of the reaction. When the dG is close to zero for the isomerase reaction (which I assume here), then you can't make it irreversible. 锛濓紳锛濓紳锛濓紳锛濓紳锛濓紳锛濓紳锛濓紳锛濓紳 This is not the case, at least in part. Such kind of enzymes, if no cofactor-needed, use the identical intermediate for the mirror symmetric reaction. For the D -- Intermediate -- S reaction, the enzyme uses the same pathway. Enzymes, for example, glutamate racemase and aspartate racemase, use a kind of psudo-mirror symmetric alignment at the active site to adapt the binding of D or S isomer in the half A.S., respectively. Other 3 atoms associated to the chiral center keeps fixed relative conformation during the inversion. Standard dG(0) of such a reaction is 0. However, at the time when enzyme works (for example, cell needs D-ASP in an almost pure L-ASP environment), the racemase moves L-Asp to D-Asp, in this regard, the dG of the reaction (not standard) is not 0. Your last sentence means: for a reaction (assuming dG(0) = 0 like racemic reaction) almost reaches EQ (dG ~ 0), you cannot make it irreversiblethis is true. Just please do not forget: such kind of enzymes work when the D -- S EQ is highly broken by nature (dG 0) [not dG(0)]. Hopefully I explained clearly! Lijun James All natural epimerases, isomerase and racemases use a mechanism based on L-amino acids to deal with a mirror-symmetric (quasi-, sometimes) reaction. In another word, these enzymes use a non-mirror symmetric structure to deal with a mirror-symmetric reaction, which itself causes the asymmetric kinetics for different direction, though the dG is 0. The Arrhenius Law k = A*exp(-dE/RT) should be understood like this: a mutation's effect to dE will be symmetric as Dale pointed out. However, the effects on A are asymmetric. A is related to intramolecular diffusion, substrate- and product-binding affinity, etc. That is why with mutation these enzymes changed their kinetics on two directions differently. Please check glutamate racemase, alanine racemase, aspartate racemase, DAPE epimerase, if you are interested. Never a 1000 to 1000 relation! Thus, mutation is possible to make one direction more favored---the point is you need the correct hit. Of course, such an experiment is never a Maxwell's demon. Lijun On May 19, 2010, at 8:51 AM, Maia Cherney wrote: You absolutely right, I thought about it. Maia Marius Schmidt wrote: Interestingly, Maxwell's demon pops up here, wh... , don't do it. If you change the reaction rate in one direction 1000 times slower than in the other direction, then the reaction becomes practically irreversible. And the system might not be at equilibrium. Maia R. M. Garavito wrote: Vinson, As Dale and Randy pointed
