Re: [ccp4bb] Trying to digest PISA results
Wasn't the original question directed to our (growing) feeling that many times PISA says No obvious oligomerization pattern but we already have evidence of dimer formation etc.. This should happen occasionally as the approach implied in the calculations is statistical in a sense. We should not be getting such contradictions on a regular basis. Possible I misunderstood the original point ... Jan On Thu, Sep 1, 2011 at 7:46 AM, Karthik S biokart...@gmail.com wrote: http://www.ebi.ac.uk/msd-srv/prot_int/pi_ilist_css.html so it depends on how many 'stable assemblies' pisa can find i suppose. more interfaces and especially if stable enough will make your fraction go down. i would have been more surprised or worried if that conservative mutation showed radically different CSS scores say one close to zero and the other one or close to it. so the exclamation marks here are really pointless (since both values are close to zero). hence i would ignore the CSS in these two cases. CSS is a statistical measure and does not imply biological meaning. in making me (us) assume the latter through this one singular value leads to all misconceptions. -- Karthik On Wed, Aug 31, 2011 at 7:52 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: I was playing around with PDBe PISA and came across the following: For pdb entry 1OYA. The most promising interface has an area bury of around 720A^2 and DeltaG of -10.6Kcal/mol. sym_op(y,x,-z+1) and CSS of 0.039! Assembly analysis says it has no strong indications that point to stable quaternary structure. This protein has been extensively studied and determined to be a dimer. Entry 3RND is the same protein with one single conservative mutation deep in the active site. They align with a RMSD of 0.3 A, 99.8% sequence identity. Superposition and inspection of the regions that contact the adjacent monomer shows they are basically identical. The interface here shows Area bury of 760 A^2 and DeltaG = -6.6Kcal/mol. sym_op (-y,-x,-z-1/2) CSS=0.00 ! Assembly analysis basically says no stable oligomers form. This enzyme also is dimer according to gel filtration. Could anyone ellaborate on this please, if they feel like they have the time... Cheers -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410
Re: [ccp4bb] Trying to digest PISA results
On Wednesday, 31 August 2011, Jan Dohnalek wrote: Wasn't the original question directed to our (growing) feeling that many times PISA says No obvious oligomerization pattern but we already have evidence of dimer formation etc.. This should happen occasionally as the approach implied in the calculations is statistical in a sense. We should not be getting such contradictions on a regular basis. I think there are at least two possibilities 1) the interface seen in the crystal is a real dimer interface, but the PISA score fails to rate it as significant 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I have no idea which, if either, of these might be the case for 1OYA. Ethan Possible I misunderstood the original point ... Jan On Thu, Sep 1, 2011 at 7:46 AM, Karthik S biokart...@gmail.com wrote: http://www.ebi.ac.uk/msd-srv/prot_int/pi_ilist_css.html so it depends on how many 'stable assemblies' pisa can find i suppose. more interfaces and especially if stable enough will make your fraction go down. i would have been more surprised or worried if that conservative mutation showed radically different CSS scores say one close to zero and the other one or close to it. so the exclamation marks here are really pointless (since both values are close to zero). hence i would ignore the CSS in these two cases. CSS is a statistical measure and does not imply biological meaning. in making me (us) assume the latter through this one singular value leads to all misconceptions. -- Karthik On Wed, Aug 31, 2011 at 7:52 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: I was playing around with PDBe PISA and came across the following: For pdb entry 1OYA. The most promising interface has an area bury of around 720A^2 and DeltaG of -10.6Kcal/mol. sym_op(y,x,-z+1) and CSS of 0.039! Assembly analysis says it has no strong indications that point to stable quaternary structure. This protein has been extensively studied and determined to be a dimer. Entry 3RND is the same protein with one single conservative mutation deep in the active site. They align with a RMSD of 0.3 A, 99.8% sequence identity. Superposition and inspection of the regions that contact the adjacent monomer shows they are basically identical. The interface here shows Area bury of 760 A^2 and DeltaG = -6.6Kcal/mol. sym_op (-y,-x,-z-1/2) CSS=0.00 ! Assembly analysis basically says no stable oligomers form. This enzyme also is dimer according to gel filtration. Could anyone ellaborate on this please, if they feel like they have the time... Cheers
Re: [ccp4bb] Trying to digest PISA results
I guess both of the mentioned possibilities occur and it is hard to judge which one it is for a particular case. PISA is extremely useful for clear-cut cases to judge them quick. In the borderline ones it remains to be the task of the research teams to prove what sort of oligomerisation state is biologically relevant. I wish we had a method that delivers a reliable answer regarding the real state of any protein studied... Jan On Thu, Sep 1, 2011 at 8:41 AM, Ethan Merritt merr...@u.washington.eduwrote: On Wednesday, 31 August 2011, Jan Dohnalek wrote: Wasn't the original question directed to our (growing) feeling that many times PISA says No obvious oligomerization pattern but we already have evidence of dimer formation etc.. This should happen occasionally as the approach implied in the calculations is statistical in a sense. We should not be getting such contradictions on a regular basis. I think there are at least two possibilities 1) the interface seen in the crystal is a real dimer interface, but the PISA score fails to rate it as significant 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I have no idea which, if either, of these might be the case for 1OYA. Ethan Possible I misunderstood the original point ... Jan On Thu, Sep 1, 2011 at 7:46 AM, Karthik S biokart...@gmail.com wrote: http://www.ebi.ac.uk/msd-srv/prot_int/pi_ilist_css.html so it depends on how many 'stable assemblies' pisa can find i suppose. more interfaces and especially if stable enough will make your fraction go down. i would have been more surprised or worried if that conservative mutation showed radically different CSS scores say one close to zero and the other one or close to it. so the exclamation marks here are really pointless (since both values are close to zero). hence i would ignore the CSS in these two cases. CSS is a statistical measure and does not imply biological meaning. in making me (us) assume the latter through this one singular value leads to all misconceptions. -- Karthik On Wed, Aug 31, 2011 at 7:52 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: I was playing around with PDBe PISA and came across the following: For pdb entry 1OYA. The most promising interface has an area bury of around 720A^2 and DeltaG of -10.6Kcal/mol. sym_op(y,x,-z+1) and CSS of 0.039! Assembly analysis says it has no strong indications that point to stable quaternary structure. This protein has been extensively studied and determined to be a dimer. Entry 3RND is the same protein with one single conservative mutation deep in the active site. They align with a RMSD of 0.3 A, 99.8% sequence identity. Superposition and inspection of the regions that contact the adjacent monomer shows they are basically identical. The interface here shows Area bury of 760 A^2 and DeltaG = -6.6Kcal/mol. sym_op (-y,-x,-z-1/2) CSS=0.00 ! Assembly analysis basically says no stable oligomers form. This enzyme also is dimer according to gel filtration. Could anyone ellaborate on this please, if they feel like they have the time... Cheers -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410
[ccp4bb] Protein Crystallography course via the web at Birkbeck College
Dear all, registration is currently open for the postgraduate certificate course in Protein Crystallography via the web at Birkbeck that starts on Monday October the 3rd. It is for the duration of 1 year during which all aspects of protein crystallography will be covered from the fundamentals of protein structure to validation. The emphasis is very much on techniques and the underlying principles (delivered in a mainly non-mathematical format) so is ideally suited to those currently enroled on PhD programs or those who wish to expand their skills in structural biology. Although a stand-alone course, it can also be taken as part of the MSc in Structural Biology (for more details see http://www.cryst.bbk.ac.uk/mscstructuralbiology.html) Information on registration and course content for the Postgraduate certificate can be found at: http://px.cryst.bbk.ac.uk/10/course/year.htm or contact the course director (Tracey Barrett) at p...@mail.cryst.bbk.ac.uk. Dr Tracey Barrett, School of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX Tel: 020 7631 6822 Fax: 020 7631 6803
Re: [ccp4bb] Trying to digest PISA results
This is regarding Ethan´s point, particularly: 2) the protein has crystallized as a monomer even though it [sometimes] exists in solution as a dimer. The interface seen in the crystal is not the real dimer interface and thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system)
Re: [ccp4bb] number of cycles in refmac
I noticed this kind of thing myself a long time ago, and wondered what refmac was doing to make things worse, so I let it keep going. And going and going. I was delighted to discover that although R and/or Rfree could rise over up to hundreds of cycles, it almost invariably turns around again, usually getting much better than that blip I saw after a measly 2-3 cycles. Leaving me to wonder why I was so greedy in the first place. Then I got even more greedy and let it keep running. There can be several hills and valleys when you do this, usually corresponding to larger-scale wiggle motions of backbone and the like, or even side chains like lysine flipping rotamers. It is perhaps understandable that these don't happen right away. After all, you are performing a search in a ~10,000-dimensional space. But eventually you get to the point where refmac no longer moves any of the atoms and the input and output files are essentially identical. (rmsd 0.002 A or so). This is what I call convergence, and this seems to be one of those rare occasions where Ian and I are in total agreement! After all, if you think about it, if you stop refinement just before R and Rfree go on a significant upswing, how are you supposed to interpret a significant upswing in your next round of refinement? Is it due to the rebuild you just did? Or is it something far away from the atoms you moved that was about to wiggle? The only way to really know is to let the whole thing settle down first. Then you can be sure that the subsequent change in R is due to something you just did, and not something you did a hundred cycles ago. Oh, and BTW, as far as I know, it is still a good idea to let refmac itself run for only ~5 cycles and just keep re-cycling the input file. In the old days, you wanted to do this because after the atoms moved a bit you had to re-run PROTIN to re-determine the restraints. If you didn't, you could get what I called water cannons where a poorly-modeled water would fly unrestrained through the unit cell, knocking side chains out of their density. Admittedly, this was a long time ago, and I'm sure it has been fixed by now. Garib can correct me if I'm wrong about that. Anyway, to answer the OP's question, I saw let refmac, phenix.refine, shelx or whatever refinement program you are using do it's thing. You wouldn't inject your protein until the column had a stable baseline would you? -James Holton MAD Scientist On Aug 24, 2011, at 5:24 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear all, especially at the beginning of model building and/or at low resolution both Rfree and -LL free as reported in the refmac logfile show a minimum at a some cycle before rising again. I am certainly not the only one tempted to first run refmac with a large number of refinement cycles, determine that minimum and rerun refmac with ncyc set to that minimum. Of course I want the resulting model and phases/map to be as close to the what's in the crystal as possible in order to facilitate model building. Is it therefore good practice to interrupt refmac wherever it finds a minimum (if so, the minimum w.r.t. which number reported in the log-file)? Thanks for everyone's opinion and experience, Tim - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOVReTUxlJ7aRr7hoRAqyzAKCZpMJPVSQJTDEoWGxZEymwvqfFcACeMNLL rvIDPlXiL5HQmoNm7yrTt6k= =UnKT -END PGP SIGNATURE-
Re: [ccp4bb] Phaser 2.3.0
Hi, Could you send me some representative logfiles (probably off-list)? This might give a hint. It must be something unusual, because we have a fairly wide range of test cases and none of them have any problems. Thanks and best wishes, Randy Read On 1 Sep 2011, at 14:58, alexander.schif...@sanofi-aventis.com wrote: Dear all, We just upgraded to ccp4 6.2.0 and with that upgrade switched from Phaser 2.1.4 to 2.3.0. During testing we ran into the problem that for some of our datasets phaser stops with a floating point exception . As it is not the case for all datasets I asume it must be linked to the input mtz or pdb file. Is there a way to check the integrity of those files and identify the specific problem? Best regards, Alexander Mit freundlichen Grüßen / Best regards / Cordialement Dr. Alexander Schiffer Sanofi-Aventis Deutschland GmbH RD LGCR/Struct.,Design Informatics FF Industriepark Hoechst Bldg. G877, Room 029 D-65926 Frankfurt am Main t: +49 69 305 24896 f: +49 69 305 80169 w:www.sanofi.de * Sanofi-Aventis Deutschland GmbH · Sitz der Gesellschaft: Frankfurt am Main · Handelsregister: Frankfurt am Main, Abt. B Nr. 40661 Vorsitzender des Aufsichtsrats: Hanspeter Spek - Geschäftsführer: Dr. Martin Siewert (Vorsitzender), Dr. Matthias Braun, Peter Guenter, Prof. Dr. Jochen Maas, Dr. Klaus Menken, Dr. Heinz Riederer, Dr. Emmanuel Siregar * -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
[ccp4bb] Low resolution structure determination advice
Dear all, We're looking for some advice about how to proceed with a structure we're working on. Our protein is 750 amino acids and naturally binds zinc. We have a SeMet data set that goes down to 3.7 angstroms. 4 of 8 selenium sites are ordered and visible in addition to our zincs and we've modeled about 450 residues of C-alpha backbone off a pure SAD density map (we've tried other phasing experiments such as Zn SAD, MAD, etc. and the best maps--clear density and visible secondary structures--we get are off Se SAD). We have one monomer per AU (and we have secondary structure coverage over our entire protein based on looking at conserved domains of our protein--unfortunately, MR is not working for this project). R/Rfree hits a minimum of 37/41 respectively in spacegroup P32 (we've tried P3 and P31, which haven't worked). We also have a native set down to 2.7 angstroms. We are able to place our working model into the native data set, but we are unable to further refine the structure in Refmac (density doesn’t improve and the stats creep up). Addition of side chains only makes our stats worse. The data sets are clean (no twinning, etc.). While we understand that we may need more phasing information (i.e. our initial model may still be quite inaccurate given resolution and size of the protein among other things--we are trying to improve this), we're wondering if anyone might have some other suggestions or insights about how we can move forward given the data that we currently have. Thanks in advance for any advice. Sincerely, Basu
[ccp4bb] 64-bit CCP4
I am almost sure this has been addressed before, so you can go after me for insufficient googling. However, 1. Is there any *significant* advantage in using 64-bit CCP4 binaries (primarily speed)? 2. IIUC, the standard CCP4 download results in 32-bit binaries being run on a 64-bit system. Works for me (except for the weird iMosflm issue), but given that 64-bit OS is becoming more and more common, isn't it time for 64-bit binaries option? The answer, of course, is no if you answered no to 1 above. And to make this post completely incomprehensible: It seems I have compiled 64-bit version on Ubuntu 11.04 just fine, but several examples fail, and all points to clipper libs. Could this be because I have clipper libs installed from ubuntu repositories and the solution is to use --disable-clipper option when configuring? I am trying this now but maybe someone can point me in the right direction. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] 64-bit CCP4
On Thursday, September 01, 2011 11:02:50 am Ed Pozharski wrote: I am almost sure this has been addressed before, so you can go after me for insufficient googling. However, 1. Is there any *significant* advantage in using 64-bit CCP4 binaries (primarily speed)? 2. IIUC, the standard CCP4 download results in 32-bit binaries being run on a 64-bit system. Works for me (except for the weird iMosflm issue), but given that 64-bit OS is becoming more and more common, isn't it time for 64-bit binaries option? The answer, of course, is no if you answered no to 1 above. The generic answer is that there is no intrinsic speed advantage to running a 64-bit binary rather than a 32-bit binary. In fact it may run slower due to larger pointer sizes and hence poorer cache performance. However, 32-bit binaries cannot access more than 4GB of address space. But the x64 architecture provides more registers and faster instructions than x86. So a 32-bit binary using the x64 instruction set can run faster than a 32-bit binary using only x86 instructions. Therefore you need to choose the right compiler options in order to get the benefit of the faster architecture. I do not know if there are specific CCP4 programs that fall outside of the generic case described above. Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Low resolution structure determination advice
Hi, Depending on how many zn sites you have, you may be able to do zn-mad for your native crystals. You don't mention if you've tried combining your various sources of phase information; if not, it's worth looking into. You may also want to look into various multi-crystal techniques (averaging, phasing and/or merging) - I've had decent luck with multi-crystal phasing off zn at low resolutions. Good luck, Pete Basudeb Bhattacharyya wrote: Dear all, We're looking for some advice about how to proceed with a structure we're working on. Our protein is 750 amino acids and naturally binds zinc. We have a SeMet data set that goes down to 3.7 angstroms. 4 of 8 selenium sites are ordered and visible in addition to our zincs and we've modeled about 450 residues of C-alpha backbone off a pure SAD density map (we've tried other phasing experiments such as Zn SAD, MAD, etc. and the best maps--clear density and visible secondary structures--we get are off Se SAD). We have one monomer per AU (and we have secondary structure coverage over our entire protein based on looking at conserved domains of our protein--unfortunately, MR is not working for this project). R/Rfree hits a minimum of 37/41 respectively in spacegroup P32 (we've tried P3 and P31, which haven't worked). We also have a native set down to 2.7 angstroms. We are able to place our working model into the native data set, but we are unable to further refine the structure in Refmac (density doesn’t improve and the stats creep up). Addition of side chains only makes our stats worse. The data sets are clean (no twinning, etc.). While we understand that we may need more phasing information (i.e. our initial model may still be quite inaccurate given resolution and size of the protein among other things--we are trying to improve this), we're wondering if anyone might have some other suggestions or insights about how we can move forward given the data that we currently have. Thanks in advance for any advice. Sincerely, Basu
Re: [ccp4bb] Low resolution structure determination advice
How about phase extension using DM, sure you say you only have one mol per asu but it might still be worth trying various approaches of solvent flattening/flipping. Don't know what you used to detect your sites and refine them, but it also might be worth sticking them into Sharp with your partial model and see if the phases improve. You say you have 450/750 residues what makes you believe that you placed them correctly ? Also do you have space from the crystal lattice packing for the additional 300 residues ? In other words are you certain that what you have crystalized is the full length version of your protein ? Adding side chains leading to worse Rfactors would suggest that you are most likely not in frame. Since you have a partial backbone you could try finding a homolog via EBI SSM to serve as a better starting model, aligning your sequence to it and replacing the side chains accordingly. Another suggestion keep changing programs don't stick to Refmac visit Phenix or the other way round, and as a last resort you could give GraphENT a try. Jürgen On Sep 1, 2011, at 3:00 PM, Pete Meyer wrote: Hi, Depending on how many zn sites you have, you may be able to do zn-mad for your native crystals. You don't mention if you've tried combining your various sources of phase information; if not, it's worth looking into. You may also want to look into various multi-crystal techniques (averaging, phasing and/or merging) - I've had decent luck with multi-crystal phasing off zn at low resolutions. Good luck, Pete Basudeb Bhattacharyya wrote: Dear all, We're looking for some advice about how to proceed with a structure we're working on. Our protein is 750 amino acids and naturally binds zinc. We have a SeMet data set that goes down to 3.7 angstroms. 4 of 8 selenium sites are ordered and visible in addition to our zincs and we've modeled about 450 residues of C-alpha backbone off a pure SAD density map (we've tried other phasing experiments such as Zn SAD, MAD, etc. and the best maps--clear density and visible secondary structures--we get are off Se SAD). We have one monomer per AU (and we have secondary structure coverage over our entire protein based on looking at conserved domains of our protein--unfortunately, MR is not working for this project). R/Rfree hits a minimum of 37/41 respectively in spacegroup P32 (we've tried P3 and P31, which haven't worked). We also have a native set down to 2.7 angstroms. We are able to place our working model into the native data set, but we are unable to further refine the structure in Refmac (density doesn’t improve and the stats creep up). Addition of side chains only makes our stats worse. The data sets are clean (no twinning, etc.). While we understand that we may need more phasing information (i.e. our initial model may still be quite inaccurate given resolution and size of the protein among other things--we are trying to improve this), we're wondering if anyone might have some other suggestions or insights about how we can move forward given the data that we currently have. Thanks in advance for any advice. Sincerely, Basu .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] 64-bit CCP4
In my (SHELX) experience, the difference in performance between 32bit and 64bit versions running on a 64bit OS scarcely justifies distributing two sets of binaries. The 64bit binaries are usually slightly faster (especially the multi-CPU SHELXD). As far as I know, there are no problems running the 32bit SHELX binaries on a 64bit system (zero dependencies again!). There is however one exception: for full-matrix SHELXL refinements with a large number of parameters, the matrix can be too large for 32bit addressing. George On Thu, Sep 01, 2011 at 11:36:21AM -0700, Ethan Merritt wrote: On Thursday, September 01, 2011 11:02:50 am Ed Pozharski wrote: I am almost sure this has been addressed before, so you can go after me for insufficient googling. However, 1. Is there any *significant* advantage in using 64-bit CCP4 binaries (primarily speed)? 2. IIUC, the standard CCP4 download results in 32-bit binaries being run on a 64-bit system. Works for me (except for the weird iMosflm issue), but given that 64-bit OS is becoming more and more common, isn't it time for 64-bit binaries option? The answer, of course, is no if you answered no to 1 above. The generic answer is that there is no intrinsic speed advantage to running a 64-bit binary rather than a 32-bit binary. In fact it may run slower due to larger pointer sizes and hence poorer cache performance. However, 32-bit binaries cannot access more than 4GB of address space. But the x64 architecture provides more registers and faster instructions than x86. So a 32-bit binary using the x64 instruction set can run faster than a 32-bit binary using only x86 instructions. Therefore you need to choose the right compiler options in order to get the benefit of the faster architecture. I do not know if there are specific CCP4 programs that fall outside of the generic case described above. Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742 -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
[ccp4bb] No Cl- or S Anomalous Signal
Dear Crystallographers, I recently have been working with a 2.5 Ang SeMet peak wavelength dataset which contains 2 cys's and also a couple of bona fide Cl ions (reasonable b-factor/site is semi-buried/water does not work). In the FFT anomalous difference map using PhiC from the refined model and Dano, I can see the MSE's at ~10 sigma, but no Cl ions, even though Cl should have f = ~0.3 versus Se's f = ~4, and no S's in the cys, despite f = 0.23e. There is really no anomalous peak at all--is it just the smallness of the signal, or are the Se's somehow swamping out the other signal? Perhaps the phases are tainted by the presence of semet in the model? Looking for suggestions, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] No Cl- or S Anomalous Signal
Where in refinement of your model are you ? At an early stage I wouldn't be surprised to only see SeMets but once you've refined your structure and go back to calculate an anomalous map with the improved phases you might double your signal for SeMet and start seeing sulfurs. An alternative explanation, you've blasted your crystals at the synchrotron and the remaining anomalous signal is too weak to show the sulfurs. Just two thoughts, Jürgen On Sep 1, 2011, at 4:03 PM, Jacob Keller wrote: Dear Crystallographers, I recently have been working with a 2.5 Ang SeMet peak wavelength dataset which contains 2 cys's and also a couple of bona fide Cl ions (reasonable b-factor/site is semi-buried/water does not work). In the FFT anomalous difference map using PhiC from the refined model and Dano, I can see the MSE's at ~10 sigma, but no Cl ions, even though Cl should have f = ~0.3 versus Se's f = ~4, and no S's in the cys, despite f = 0.23e. There is really no anomalous peak at all--is it just the smallness of the signal, or are the Se's somehow swamping out the other signal? Perhaps the phases are tainted by the presence of semet in the model? Looking for suggestions, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] No Cl- or S Anomalous Signal
Hi Jacob I agree with Juergen, and just add that your Cys and Cl might not be fully occupied. cheers Preben On 9/1/11 10:03 PM, Jacob Keller wrote: Dear Crystallographers, I recently have been working with a 2.5 Ang SeMet peak wavelength dataset which contains 2 cys's and also a couple of bona fide Cl ions (reasonable b-factor/site is semi-buried/water does not work). In the FFT anomalous difference map using PhiC from the refined model and Dano, I can see the MSE's at ~10 sigma, but no Cl ions, even though Cl should have f = ~0.3 versus Se's f = ~4, and no S's in the cys, despite f = 0.23e. There is really no anomalous peak at all--is it just the smallness of the signal, or are the Se's somehow swamping out the other signal? Perhaps the phases are tainted by the presence of semet in the model? Looking for suggestions, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
Re: [ccp4bb] Low resolution structure determination advice
Hi Basu, You mentioned molecular replacement was not successful for this project. Which model was used for this procedure? Have you tried your partially built structure as a model to obtain preliminary phases for your native (2.7A) data set? If there is any luck with that, you might be able to combine phases from this procedure with the Selenium SAD phases, as already suggested. Kianoush --- On Thu, 9/1/11, Basudeb Bhattacharyya bbhattach...@wisc.edu wrote: From: Basudeb Bhattacharyya bbhattach...@wisc.edu Subject: [ccp4bb] Low resolution structure determination advice To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, September 1, 2011, 9:31 AM Dear all, We're looking for some advice about how to proceed with a structure we're working on. Our protein is 750 amino acids and naturally binds zinc. We have a SeMet data set that goes down to 3.7 angstroms. 4 of 8 selenium sites are ordered and visible in addition to our zincs and we've modeled about 450 residues of C-alpha backbone off a pure SAD density map (we've tried other phasing experiments such as Zn SAD, MAD, etc. and the best maps--clear density and visible secondary structures--we get are off Se SAD). We have one monomer per AU (and we have secondary structure coverage over our entire protein based on looking at conserved domains of our protein--unfortunately, MR is not working for this project). R/Rfree hits a minimum of 37/41 respectively in spacegroup P32 (we've tried P3 and P31, which haven't worked). We also have a native set down to 2.7 angstroms. We are able to place our working model into the native data set, but we are unable to further refine the structure in Refmac (density doesn’t improve and the stats creep up). Addition of side chains only makes our stats worse. The data sets are clean (no twinning, etc.). While we understand that we may need more phasing information (i.e. our initial model may still be quite inaccurate given resolution and size of the protein among other things--we are trying to improve this), we're wondering if anyone might have some other suggestions or insights about how we can move forward given the data that we currently have. Thanks in advance for any advice. Sincerely, Basu
