Re: [ccp4bb] relative domain motion calculation...
I am not sure about a web server, but if you use gesant to align one domain of the two structures, then fit the second domain of structure 1 to the second domain of the aligned structure 2; the polar angle Kappa will give you the amount of rotation between the second domains. Eleanor On 21 April 2015 at 08:39, Mintu Chandra mi...@iiserb.ac.in wrote: Dear All, I am working with a protein containing tandem domains, connected with 3-4 residue linker. I want to calculate the relative motion/orientation of one domain with respect to the hinge region as well as with respect to the other domain. Both the domains have very less seq. identity (20%). Please suggest some web server to calculate the same. Thanks, Mintu
Re: [ccp4bb] on NCS restraint
All: I strongly disagree with Reza's suggestion that one should abandon NCS at better than 2.5 Å resolution (or even Herman's suggestion at better than 2.0 Å resolution). Either of these may be true in any particular case, BUT one should do the experiment - Run parallel refinements from the same starting model with and without NCS restraints and compare R-free, the gap between R-free and R-work, and particular places where one knows or suspects that the local geometry is different, before deciding to abandon NCS restraints. This was certainly true in the bad old days when loose (as opposed to strict) NCS restraints were used and bound each chain more-or-less to a single chain's geometry, even though one was refining all extant chains. Using so-called loose NCS restraints, I once had a loop pulled out of electron density during refinement and the tip moved ~6 Å! However, for the last 5 years or so, BUSTER, which I use, and REFMAC (and presumably PHENIX) have used LSSR (local secondary structure restraints) where the maximum pull to uniformity tops out at a certain value. I have not rigorously followed my own advice above to run parallel refinements, but I have yet to find a case where LSSR-type NCS restraints have hurt the refinement down to at least ~1.5 Å resolution. To give credit where it is due, Oliver Smart, who implemented LSSR in BUSTER, attributed the concept to George Sheldrick. Steven -- Date:Mon, 20 Apr 2015 10:38:27 + From:Reza Khayat rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu Subject: Re: [ccp4bb] on NCS restraint Hi, The purpose of NCS is to reduce the degrees of freedom in order to avoid over refinement -not only to expedite refinement. Strict or restrained NCS should be applied at lower resolutions (2.7Å) or data completeness. Forgo NCS If you have a complete and better than 2.5Å dataset. Also, you can define the regions where NCS is applied and thus avoid loops/regions where the NCS is violated. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070 rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu%3cmailto:rkha...@ccny.cuny.edu -- On Apr 20, 2015, at 4:01 AM, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com%3cmailto:herman.schreu...@sanofi.com wrote: Dear Smith, There used to be something called strict NCS which meant that instead of many identical subunits, only one average subunit was refined, which would speed up the refinement significantly, at the expense of requiring that all subunits are exactly identical. I do not think that this option is used anymore and most refinement programs would require NCS related subunits to be similar, but not identical to each other. As Robbie Joosten pointed at, this can help a lot, especially when you do not have high resolution data. So for data with better than 2.0 Å resolution, including NCS restraints would probably not make a big difference, but otherwise I would switch them on. Best, Herman -- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu Gesendet: Freitag, 17. April 2015 06:02 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK%3cmailto:CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] on NCS restraint Dear Jurgen, My understanding is that NCS restraint can significantly enhance the speed of calculation, but considering the subunits even with the eactly same sequence may not be identical, to have NCS restraint may be not necessary or may be not good for the refinement, am I right? Smith At 2015-04-17 09:09:05, Jurgen Bosch jbos...@jhu.edumailto:jbos...@jhu.edumailto:jbos...@jhu.edu%3cmailto:jbos...@jhu.edu wrote: yes. Have two sets of NCS operators one that describe the four subunits and one describing the two subunits. If during the refinement of your structure you should find out that the subunits are not identical to each other you can relax the NCS weights. Jürgen .. 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: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.eduhttp://lupo.jhsph.edu/http://lupo.jhsph.edu%3chttp:/lupo.jhsph.edu/ -- On Apr 16, 2015, at 9:02 PM, Smith Lee 0459ef8548d5-dmarc-requ...@jiscmail.ac.ukmailto:0459ef8548d5-dmarc-requ...@jiscmail.ac.ukmailto:0459ef8548d5-dmarc-requ...@jiscmail.ac.uk%3cmailto:0459ef8548d5-dmarc-requ...@jiscmail.ac.uk
Re: [ccp4bb] on NCS restraint
Hi all, We have carried out a large-scale test of the use of Refmac's NCS-restraints during model building with ARP/wARP. We have found advantageous to have such restraints turned on with data resolution extending to as high as 1.5 A. Victor On 21/04/2015 14:46, Sheriff, Steven wrote: All: I strongly disagree with Reza’s suggestion that one should abandon NCS at better than 2.5 Å resolution (or even Herman’s suggestion at better than 2.0 Å resolution). Either of these may be true in any particular case, BUT one should do the experiment – Run parallel refinements from the same starting model with and without NCS restraints and compare R-free, the gap between R-free and R-work, and particular places where one knows or suspects that the local geometry is different, before deciding to abandon NCS restraints. This was certainly true in the bad old days when “loose” (as opposed to strict) NCS restraints were used and “bound” each chain more-or-less to a single chain’s geometry, even though one was refining all extant chains. Using so-called “loose” NCS restraints, I once had a loop pulled out of electron density during refinement and the tip moved ~6 Å! However, for the last 5 years or so, BUSTER, which I use, and REFMAC (and presumably PHENIX) have used LSSR (local secondary structure restraints) where the maximum “pull” to uniformity tops out at a certain value. I have not rigorously followed my own advice above to run parallel refinements, but I have yet to find a case where LSSR-type NCS restraints have “hurt” the refinement down to at least ~1.5 Å resolution. To give credit where it is due, Oliver Smart, who implemented LSSR in BUSTER, attributed the concept to George Sheldrick. Steven -- Date: Mon, 20 Apr 2015 10:38:27 + From: Reza Khayat rkha...@ccny.cuny.edu Subject: Re: [ccp4bb] on NCS restraint Hi, The purpose of NCS is to reduce the degrees of freedom in order to avoid over refinement -not only to expedite refinement. Strict or restrained NCS should be applied at lower resolutions (2.7Å) or data completeness. Forgo NCS If you have a complete and better than 2.5Å dataset. Also, you can define the regions where NCS is applied and thus avoid loops/regions where the NCS is violated. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070 rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu -- On Apr 20, 2015, at 4:01 AM, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote: Dear Smith, There used to be something called “strict NCS“ which meant that instead of many identical subunits, only one “average” subunit was refined, which would speed up the refinement significantly, at the expense of requiring that all subunits are exactly identical. I do not think that this option is used anymore and most refinement programs would require NCS related subunits to be similar, but not identical to each other. As Robbie Joosten pointed at, this can help a lot, especially when you do not have high resolution data. So for data with better than 2.0 Å resolution, including NCS restraints would probably not make a big difference, but otherwise I would switch them on. Best, Herman -- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu Gesendet: Freitag, 17. April 2015 06:02 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] on NCS restraint Dear Jurgen, My understanding is that NCS restraint can significantly enhance the speed of calculation, but considering the subunits even with the eactly same sequence may not be identical, to have NCS restraint may be not necessary or may be not good for the refinement, am I right? Smith At 2015-04-17 09:09:05, "Jurgen
[ccp4bb] relative domain motion calculation...
Dear All, I am working with a protein containing tandem domains, connected with 3-4 residue linker. I want to calculate the relative motion/orientation of one domain with respect to the hinge region as well as with respect to the other domain. Both the domains have very less seq. identity (20%). Please suggest some web server to calculate the same. Thanks, Mintu
Re: [ccp4bb] relative domain motion calculation...
Hi Mintu You can use Chimera to calculate rotation angel. http://www.cgl.ucsf.edu/chimera/videodoc/AlignDomains/ Veerendra On 21/4/15 3:39 PM, Mintu Chandra mi...@iiserb.ac.in wrote: Dear All, I am working with a protein containing tandem domains, connected with 3-4 residue linker. I want to calculate the relative motion/orientation of one domain with respect to the hinge region as well as with respect to the other domain. Both the domains have very less seq. identity (20%). Please suggest some web server to calculate the same. Thanks, Mintu Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.
[ccp4bb] AW: [ccp4bb] Cleaved peptide density!
Dear Dipankar, as Bonsor mentioned, 2-3 weeks is something completely different from the few hours normally used for enzyme assays. A lot may happen during the long crystallization that does not happen in the assay. Also, your enzyme will still have the remainder of the catalytic machinery (oxy-anion hole, Hisp-Asp proton shuttle) intact and a water molecule in the cavity left by the removed sulfur may function as nucleophile. One question: do you incubate overnight and then set up crystallizations, or do you grow apo-crystals and incubate fully grown crystals with peptide? In the first case, I would freeze the crystals after overnight incubation with fresh peptide. In the second case, I would incubate only 30 minutes and then freeze the crystals. In both cases, I would add as much intact peptide as I can, since it gets cleaved during incubation. One other question: do you seen both ends of the peptide, or only half of it. If you see only half of the peptide, it might be that the full peptide is not compatible with the crystal packing and only protein molecules with half a peptide bound may enter the crystal lattice. In this case, you may have to screen for completely new crystallization conditions in the presence of some protease inhibitor(s) like PMSF to see if you can get a different crystal form. You may also want to scan the literature to see if some non-cleavable substrate-analog (inhibitor) is available. On the positive side: your structure with the cleaved peptide is also interesting, since it represents the product complex! Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dipankar Manna Gesendet: Montag, 20. April 2015 21:22 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Cleaved peptide density! Dear Bonsor, Thanks for your suggestions! It need 2-3 weeks to get the fully grown crystals. And harvest, freeze and data collection just take usually next 3-5 days. I usually incubated substrate overnight. Initially I was purifying with the same column as the WT but in the next batch I used new beads to purify the mutant as you categorically pointed out. But results are the same, I mean I got the same cleaved peptide density! I tried soaking with different time frames and with different peptide concentrations as well but in this case I can't see any peptide density at all. Best, Dipankar On Mon, Apr 20, 2015 at 9:06 PM, D Bonsor dbon...@ihv.umaryland.edumailto:dbon...@ihv.umaryland.edu wrote: First of all, you don't say how long it took to first set up crystals, for them to grow, harvest, freeze and collect data on. Secondly how long did leave the peptide/substrate for your SDS PAGE experiment? If they are of a different time scale e.g. 6 hours v.s. 30 days, it may be that your enzyme is not totally dead. Also how did you purify the alanine mutant? If you purified it on the same columns/beads as the WT protein you may have a residual amount of active protein which could cleave your peptide over the course of crystallization. You may want to use fresh beads, or treat columns with pepsin or sodium hydroxide. Not real answers I am afraid, more like suggestions. -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
Re: [ccp4bb] visualizing .mrc maps in coot and model building
Dear onetwo, Coot will open .mrc files. However, different programs often modify the header of the mrc file, which can cause problems. This recent paper gives more information about the standard format for mrc files and proposed extensions to it: http://www.ncbi.nlm.nih.gov/pubmed/25882513 The sigma level you need to work at is very much map-dependent, and can change considerably within the map depending on local resolution differences. The following website should give you more information about model building and refinement into EM maps using CCP4 tools, and a tutorial is forthcoming: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/em_fitting/em_fitting.html. The ccp-em mailing list (http://www.ccpem.ac.uk/) is also a good place to ask for advice. Best wishes, Alan On 9 Apr 2015, at 19:48, onetwo twoon...@rediffmail.com wrote: Dear All, This is regarding the protein structure model building using the density files or the .mrc file obtained by cryo-electron microscopy data. I want to seek some help on how to visualize these files in COOT. I am working on mac system and have recently installed CCP4 which has Coot- Dec 2014 version. When I load mrc file in coot via open map it gives error: map failed to open. Then using the em2em program, i have been successful in converting it to .ccp4 which I am able to open it in the coot. Please let me know if this the way I am supposed to do it. I have one more query that may be very naive but at which sigma level one should work towards these maps in coot ? I have read the paper by Brown et al, in ACTA D, 2015, they have mentioned the tools for macromolecular model building and refinement into electron cryo-microscopy and mentioned many useful tools in COOT like jiggle fit etc for this. So, if I can use ccp4 also in the same way like we do for crystallography and do refinement after model building in coot.For information, the protein I am working on has around 8A resolution. Looking forward for the help and guidance. Regards Get your own FREE website, FREE domain FREE mobile app with Company email. Know More
Re: [ccp4bb] advice on anomoulous data collection strategy---low resolution, radiation damage, high mosaicity
On Mon, 20 Apr 2015 23:15:17 +, Keller, Jacob kell...@janelia.hhmi.org wrote: I have processed test data sets (measured at the SLS) which have only a few non-zero pixels on each frame. Can be nicely integrated - but it was insulin. What were the values of the non-zero pixels, I wonder? How many total counts per image? And this was, I assume, Pilatus? Sounds pretty amazing, even for insulin... JPK Pilatus 2M. This is the histogram of a random frame I picked: counts number 0 2211457 thus 97% of all pixels have zero counts 1 65154that's 2.8% of all pixels 22107 3 96 4 20 5 12 6 3 7 5 9 2 10 2 11 3 12 4 14 2 15 1 29 1 all other counts are zero. The average count is 0.03. Processing was done with the March-2015 version of XDS; older versions do not work well with average counts below about 0.5 . best, Kay
Re: [ccp4bb] on the electron microscopy function of refmac5
Dear Smith, The easiest way to access the EM functions of REFMAC are to use the scripts provided here: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/em_fitting/em_fitting.html. Best wishes, Alan On 21 Apr 2015, at 04:20, Smith Liu smith_liu...@163.com wrote: Dear All, The CCP4 document says refmac can process electron microscopy map for refinement. But I cannot localize that function in the refmac5 of the CCP4. Will you please advise how to have the refmac process the electron microscopy map? Smith
Re: [ccp4bb] on NCS restraint
All: Oops, someone pointed out to me off line that I slipped-up earlier today when I wrote that LSSR is an abbreviation for Local Secondary Structure Restraints. Rather it is an abbreviation for Local Structure Similarity Restraints. I know better, but I guess my unconscious got the better of me. Steven From: Victor Lamzin [mailto:vic...@embl-hamburg.de] Sent: Tuesday, April 21, 2015 9:07 AM To: Sheriff, Steven; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] on NCS restraint Hi all, We have carried out a large-scale test of the use of Refmac's NCS-restraints during model building with ARP/wARP. We have found advantageous to have such restraints turned on with data resolution extending to as high as 1.5 A. Victor On 21/04/2015 14:46, Sheriff, Steven wrote: All: I strongly disagree with Reza's suggestion that one should abandon NCS at better than 2.5 Å resolution (or even Herman's suggestion at better than 2.0 Å resolution). Either of these may be true in any particular case, BUT one should do the experiment - Run parallel refinements from the same starting model with and without NCS restraints and compare R-free, the gap between R-free and R-work, and particular places where one knows or suspects that the local geometry is different, before deciding to abandon NCS restraints. This was certainly true in the bad old days when loose (as opposed to strict) NCS restraints were used and bound each chain more-or-less to a single chain's geometry, even though one was refining all extant chains. Using so-called loose NCS restraints, I once had a loop pulled out of electron density during refinement and the tip moved ~6 Å! However, for the last 5 years or so, BUSTER, which I use, and REFMAC (and presumably PHENIX) have used LSSR (local secondary structure restraints) where the maximum pull to uniformity tops out at a certain value. I have not rigorously followed my own advice above to run parallel refinements, but I have yet to find a case where LSSR-type NCS restraints have hurt the refinement down to at least ~1.5 Å resolution. To give credit where it is due, Oliver Smart, who implemented LSSR in BUSTER, attributed the concept to George Sheldrick. Steven -- Date:Mon, 20 Apr 2015 10:38:27 + From:Reza Khayat rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu Subject: Re: [ccp4bb] on NCS restraint Hi, The purpose of NCS is to reduce the degrees of freedom in order to avoid over refinement -not only to expedite refinement. Strict or restrained NCS should be applied at lower resolutions (2.7Å) or data completeness. Forgo NCS If you have a complete and better than 2.5Å dataset. Also, you can define the regions where NCS is applied and thus avoid loops/regions where the NCS is violated. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070 rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu%3cmailto:rkha...@ccny.cuny.edu -- On Apr 20, 2015, at 4:01 AM, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com%3cmailto:herman.schreu...@sanofi.com wrote: Dear Smith, There used to be something called strict NCS which meant that instead of many identical subunits, only one average subunit was refined, which would speed up the refinement significantly, at the expense of requiring that all subunits are exactly identical. I do not think that this option is used anymore and most refinement programs would require NCS related subunits to be similar, but not identical to each other. As Robbie Joosten pointed at, this can help a lot, especially when you do not have high resolution data. So for data with better than 2.0 Å resolution, including NCS restraints would probably not make a big difference, but otherwise I would switch them on. Best, Herman -- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu Gesendet: Freitag, 17. April 2015 06:02 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK%3cmailto:CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] on NCS restraint Dear Jurgen, My understanding is that NCS restraint can significantly enhance the speed of calculation, but considering the subunits even with the eactly same sequence may not be identical, to have NCS restraint may be not necessary or may be not good for the refinement, am I right? Smith At 2015-04-17 09:09:05, Jurgen Bosch jbos...@jhu.edumailto:jbos...@jhu.edumailto:jbos...@jhu.edu%3cmailto:jbos...@jhu.edu wrote: yes. Have two sets of NCS operators one that describe the four subunits and one describing the two subunits. If during the refinement of your structure you should find out that the subunits are not identical to each other you can relax
Re: [ccp4bb] on NCS restraint
Dear Reza, have the results been published? Not all of them. The first implementation of NCS-restraints in ARP/wARP's model building at a resolution of 2.3 A and lower was published: Wiegels T, Lamzin VS. Use of noncrystallographic symmetry for automated model building at medium to low resolution. (2012) Acta Crystallogr D Biol Crystallogr. 68, 446-453. PMID: 22505265 Subsequent advances and applicability to data up to 1.5 A is still to be published; however it is now the default setting of ARP/wARP. Victor On 21/04/2015 15:32, Reza Khayat wrote: Hi, I have to agree with Steven. I was too haste in my reply to the initial e-mail. As Steven put it, it is best to run multiple experiments at the same time and identify which produces the best result. On a follow up to Victor Lamin’s reply, have the results been published? Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Victor Lamzin Sent: Tuesday, April 21, 2015 9:07 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] on NCS restraint Hi all, We have carried out a large-scale test of the use of Refmac's NCS-restraints during model building with ARP/wARP. We have found advantageous to have such restraints turned on with data resolution extending to as high as 1.5 A. Victor On 21/04/2015 14:46, Sheriff, Steven wrote: All: I strongly disagree with Reza’s suggestion that one should abandon NCS at better than 2.5 Å resolution (or even Herman’s suggestion at better than 2.0 Å resolution). Either of these may be true in any particular case, BUT one should do the experiment – Run parallel refinements from the same starting model with and without NCS restraints and compare R-free, the gap between R-free and R-work, and particular places where one knows or suspects that the local geometry is different, before deciding to abandon NCS restraints. This was certainly true in the bad old days when “loose” (as opposed to strict) NCS restraints were used and “bound” each chain more-or-less to a single chain’s geometry, even though one was refining all extant chains. Using so-called “loose” NCS restraints, I once had a loop pulled out of electron density during refinement and the tip moved ~6 Å! However, for the last 5 years or so, BUSTER, which I use, and REFMAC (and presumably PHENIX) have used LSSR (local secondary structure restraints) where the maximum “pull” to uniformity tops out at a certain value. I have not rigorously followed my own advice above to run parallel refinements, but I have yet to find a case where LSSR-type NCS restraints have “hurt” the refinement down to at least ~1.5 Å resolution. To give credit where it is due, Oliver Smart, who implemented LSSR in BUSTER, attributed the concept to George Sheldrick. Steven -- Date: Mon, 20 Apr 2015 10:38:27 + From: Reza Khayat rkha...@ccny.cuny.edu Subject: Re: [ccp4bb] on NCS restraint Hi, The purpose of NCS is to reduce the degrees of freedom in order to avoid over refinement -not only to expedite refinement. Strict or restrained NCS should be applied at lower resolutions (2.7Å) or data completeness. Forgo NCS If you have a complete and better than 2.5Å dataset. Also, you can define the regions where NCS is applied and thus avoid loops/regions where the NCS is violated. Best wishes, Reza
Re: [ccp4bb] on NCS restraint
Hi, I have to agree with Steven. I was too haste in my reply to the initial e-mail. As Steven put it, it is best to run multiple experiments at the same time and identify which produces the best result. On a follow up to Victor Lamin's reply, have the results been published? Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Victor Lamzin Sent: Tuesday, April 21, 2015 9:07 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] on NCS restraint Hi all, We have carried out a large-scale test of the use of Refmac's NCS-restraints during model building with ARP/wARP. We have found advantageous to have such restraints turned on with data resolution extending to as high as 1.5 A. Victor On 21/04/2015 14:46, Sheriff, Steven wrote: All: I strongly disagree with Reza's suggestion that one should abandon NCS at better than 2.5 Å resolution (or even Herman's suggestion at better than 2.0 Å resolution). Either of these may be true in any particular case, BUT one should do the experiment - Run parallel refinements from the same starting model with and without NCS restraints and compare R-free, the gap between R-free and R-work, and particular places where one knows or suspects that the local geometry is different, before deciding to abandon NCS restraints. This was certainly true in the bad old days when loose (as opposed to strict) NCS restraints were used and bound each chain more-or-less to a single chain's geometry, even though one was refining all extant chains. Using so-called loose NCS restraints, I once had a loop pulled out of electron density during refinement and the tip moved ~6 Å! However, for the last 5 years or so, BUSTER, which I use, and REFMAC (and presumably PHENIX) have used LSSR (local secondary structure restraints) where the maximum pull to uniformity tops out at a certain value. I have not rigorously followed my own advice above to run parallel refinements, but I have yet to find a case where LSSR-type NCS restraints have hurt the refinement down to at least ~1.5 Å resolution. To give credit where it is due, Oliver Smart, who implemented LSSR in BUSTER, attributed the concept to George Sheldrick. Steven -- Date:Mon, 20 Apr 2015 10:38:27 + From:Reza Khayat rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu Subject: Re: [ccp4bb] on NCS restraint Hi, The purpose of NCS is to reduce the degrees of freedom in order to avoid over refinement -not only to expedite refinement. Strict or restrained NCS should be applied at lower resolutions (2.7Å) or data completeness. Forgo NCS If you have a complete and better than 2.5Å dataset. Also, you can define the regions where NCS is applied and thus avoid loops/regions where the NCS is violated. Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York 160 Convent Ave, MR-1135 New York, NY 10031 (212) 650-6070 rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edumailto:rkha...@ccny.cuny.edu%3cmailto:rkha...@ccny.cuny.edu -- On Apr 20, 2015, at 4:01 AM, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com%3cmailto:herman.schreu...@sanofi.com wrote: Dear Smith, There used to be something called strict NCS which meant that instead of many identical subunits, only one average subunit was refined, which would speed up the refinement significantly, at the expense of requiring that all subunits are exactly identical. I do not think that this option is used anymore and most refinement programs would require NCS related subunits to be similar, but not identical to each other. As Robbie Joosten pointed at, this can help a lot, especially when you do not have high resolution data. So for data with better than 2.0 Å resolution, including NCS restraints would probably not make a big difference, but otherwise I would switch them on. Best, Herman -- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Smith Liu Gesendet: Freitag, 17. April 2015 06:02 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK%3cmailto:CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] on NCS restraint Dear Jurgen, My understanding is that NCS restraint can significantly enhance the speed of calculation, but considering the subunits even with the eactly same sequence may not be identical, to have NCS restraint may be not necessary or may be not good for the refinement, am I right? Smith At 2015-04-17 09:09:05, Jurgen Bosch jbos...@jhu.edumailto:jbos...@jhu.edumailto:jbos...@jhu.edu%3cmailto:jbos...@jhu.edu wrote: yes. Have two sets of NCS operators one that describe the four subunits and one describing
[ccp4bb] Phaser going into infinite loop in Ample
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 We are having a problem with AMPLE and hope someone can help. The protein is about 70 amino acids long and we suspect it forms a coiled-coil. Our previous attempts at molecular replacement have failed so we hoped that AMPLE, with its ability to generate a variety of potential models, would do the trick. Our problem is that all of our CPU cores are consumed by Phaser jobs that are not making progress. With this protein Phaser decides that it will look for 11 copies in the asymmetric unit. For a few of the possible ensembles it fails to find even one copy and gives up. That's fine with us. For other ensembles it finds a handful of possible first positions, goes on to look for a second and fails, then goes back to try to place a second copy again. We presume that the intent is to lower the acceptance criteria in the second pass, but in actuality Phaser simply repeats the same search that failed before and fails again. The leads to an infinite loop. Once all the cores are occupied in this futile endeavor AMPLE makes no further progress. How can we get Phaser to either try harder to place a molecule or to give up? We are using CCP4 6.5.008 and the copy of Phaser that came with it. We used CCP4i to create a script which we modified slightly and ran using the at command. The command is: /usr/local/ccp4-6.5/bin/ccp4-python -u /usr/local/ccp4-6.5/bin/ample - -mtz /user/sarah/xray/1Apr_Athena/SD6004_2_001_mergedunique14.mtz - -fasta /user/sarah/xray/1Apr_Athena/swaseq.fa -mr_sequence /user/sarah/xray/1Apr_Athena/swaseq.fa -nmodels 500 -name MVD0 - -run_dir /home/sarah/AMPLE -nproc 6 -make_models True -rosetta_dir /usr/local/rosetta-3.5 -frags_3mers /user/sarah/xray/1Apr_Athena/aat000_03_05.200_v1_3 -frags_9mers /user/sarah/xray/1Apr_Athena/aat000_09_05.200_v1_3 -make_frags False - -F F -SIGF SIGF -FREE FreeR_flag -early_terminate True -use_shelxe True -shelx_cycles 15 -use_arpwarp False Any help is appreciated, Dale Tronrud Sarah Clark -BEGIN PGP SIGNATURE- Version: GnuPG v2.0.22 (MingW32) iEYEARECAAYFAlU3KhEACgkQU5C0gGfAG117JACfXahEX8z1k3ev043a7V2SzhNh p6UAnAmKkUZe46zbiXckdDTNgUQ0dgWq =A/Sj -END PGP SIGNATURE-
[ccp4bb] PDBe workshop - Making the most of PDB and EMDB data
Dear All, The PDBe team will be running a satellite workshop at ECM29 this summer - http://ecm29.ecanews.org/programme/satellite-meetings/ Registration is through the usual ECM registration process. We look forward to seeing you there and answer your queries about PDB and EMDB. Best wishes, Sameer Velankar / When/?August 22nd, 2015 (Saturday), 10:00 AM – 5:00 PM/ Where/? Rovinj, Hotel “Park”, Cissa conference hall / Registration fee/:EUR 20,00 (Registration through the registration system of the ECM29. Number of participants is limited to 40 and they will be accepted on a first-come-first-served basis. Applicants are kindly asked to book their own accommodation in Rovinj. Registration fee includes lunch box and refreshments./ Website: pdbe.org http://www.ebi.ac.uk/pdbe/// A few words from the organizers/: In this workshop we will show how macromolecular structure data are deposited in the PDB and EMDB archives and how these data are annotated, analysed and validated. We will demonstrate how users of PDB and EMDB data can make optimal use of PDBe’s improved search systems to select and analyse a structure best suited to their needs. The Protein Data Bank in Europe (PDBe; pdbe.org) is a founding member of the worldwide collaborations that manage the PDB and EMDB archives of macromolecular structure data. To achieve its mission of “Bringing structure to biology”, PDBe develops easy to use data access and analysis tools. The team is also responsible for developing the new wwPDB validation pipelines.
Re: [ccp4bb] advice on anomoulous data collection strategy---low resolution, radiation damage, high mosaicity
Burning/frying the crystal is never the right strategy, at least not if you use a Pilatus detector. Burning/frying is a strategy that results from the widespread misunderstanding of using Rsym as a criterion of data quality. I apologise if I didn't make myself clear. The point I tried to make is that it is ok to strive to obtain higher resolution with native data sets ... and yes, fry is a poor choice of a word on my side as it implies damage to the crystal, which is not what I meant. I simply tried to emphasise that going for high resolution is ok when collecting native data sets but not when collecting anomalous ones. The rest of my message made a point of collecting data with high attenuation and fast shutter speed at a lower resolution to keep the anomalous signal high in as many images as possible before X-ray damage sets in lowers it. Tony -- Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE
Re: [ccp4bb] off topic: DLS instrument
Adam, We had a trial of the Avid nano W130i DLS system. I liked the small size and the disposable cuvettes. At present we have a Dynapro which is an old machine now but works well. Best wishes Clare From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tianyu Wang Sent: 17 April 2015 19:37 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic: DLS instrument Sorry for an off-topic question. We are seeking a new DLS(Dynamic Light Scattering) instrument which is best for our needs. Any suggestion to a traditional structural biology lab? The make, model #? Any suggestion will be very appreciated. Thank you! Best, Adam
