Re: [ccp4bb] Phaser going into infinite loop in Ample
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Thanks for all the help! We will restart the job with the KILL option as Jens suggested. We will also send a copy of the Phaser log file to Randy. This is not a case of Phaser simply trying harder - it is doing the same search over and over again. After four days the log file is getting pretty long. I may have to use DropBox. We considered trying ARCIMBOLDO but the resolution of the data did not reach the 2.1 A limit usually suggested for that program. If we get AMPLE running, and it fails, we will give ARCIMBOLDO a try. Dale Tronrud Sarah Clark On 4/22/2015 2:06 AM, Thomas, Jens wrote: Dear Dale, This is a known issue with AMPLE and will be fixed with the next release. In the meantime you can tell AMPLE to pass the KILL option that Randy mentions to PHASER, by adding the following arguments to your script: -mr_keys PKEY KILL TIME 360 this will kill PHASER after 360 minutes (6 hours), which we've found if normally enough, although pick whatever time works for you, I should also point out that I think George is doing a disservice to SHELXE. In our last paper looking at coiled-coils, we saw successes at resolutions much lower than 2.1, in one case, AMPLE was able to solve a structure with a resolution of 2.9A: http://dx.doi.org/10.1107/S2052252515002080 If you have any issues getting the KILL command to work, please feel free to contact me off-list. Best wishes, Jens From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Randy Read [rj...@cam.ac.uk] Sent: 22 April 2015 09:04 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Phaser going into infinite loop in Ample Hi Dale, It must actually be AMPLE deciding how many copies to search for. Phaser will give you some information about how consistent the specified composition is with the Matthews volume, but it just searches for the number of copies that it's instructed to look for. We haven't put the intelligence into it to correlate the model(s) with the composition information and try out different possibilities. At the moment, we're leaving that level of analysis to pipelines like MRage. We're well aware of the tension between looking hard enough to find a solution in a difficult case and not throwing good CPU cycles after bad when it's hopeless. We're gradually introducing new features to make these decisions better, but we do tend to prefer wasting CPU time to missing solutions. However, we've introduced a couple of ways to limit the amount of time that Phaser spends pursuing very difficult or hopeless solutions, partly for the benefit of people such as the developers of Arcimboldo, AMPLE and the wide-search molecular replacement pipeline. One is the KILL command, which is a rather blunt instrument saying to give up if a solution isn't found in a certain length of time. In AMPLE, if you set quick mode, then the KILL time is set to 15 minutes. Another option (which I don't think AMPLE uses) is the PURGE command, where you can say (for instance) that Phaser should pursue a maximum of 25 partial solutions when adding the next component. If you're seeing an infinite loop, it would be handy if you could send me a copy of the logfile showing what is going on. There have been some bugs leading to such infinite loops under some circumstances, and if you're running into one of those there's a good chance that it has been fixed in a recent nightly build of Phaser available through Phenix. You can instruct CCP4 to use the Phaser executable from Phenix, and I think this should work fine in AMPLE, though I haven't tested it — I don't think any relevant syntax has changed. It's been a while since we've had a new stable release of Phaser in either CCP4 or Phenix, so we're aiming to get one out in the not too distant future. All the best, Randy On 22 Apr 2015, at 05:56, Dale Tronrud de...@daletronrud.com wrote: 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.
Re: [ccp4bb] relative domain motion calculation...
Dear Mintu, one possible option is the webserver DynDomhttp://fizz.cmp.uea.ac.uk/dyndom/. Kind regards, Matic On 21. 04. 2015 09:39, Mintu Chandra 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 attachment: matic_kisovec.vcf
Re: [ccp4bb] AW: [ccp4bb] Cleaved peptide density!
Once thing I don't fully understand: do you have a cleaved density or a cleaved peptide? If your peptide is sitting in a binding site, and that you can see only the part inside the pocket, with the rest supposably sitting in a 'more opened' part of the molecule, or else outside of the molecule, than the cleaved density could be due to non-specificity of this part of the peptide and high flexibility / non-recognition. This would obviously have great biological interest and implications. Leo Sent from my iPad (most likely in conference) On 21 Apr 2015, at 10:25, herman.schreu...@sanofi.com wrote: 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.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] Cleaved peptide density!
hello Dipankar, Your queries have generated much interest. It would be helpful if you told us which clan the cysteine peptidase you are working with is from. Also it would be very helpful if you could show us the electron density of the cleaved peptide in the active site. One presumes that you have a product complex with your peptidase and as has been pointed out this is very interesting. Serine peptidases often have products bound in the S1 to S4 subsites of the enzyme. They remain bound throughout the isolation, purification and crystallization steps of the structure determination. Regards, Michael James On Mon, Apr 20, 2015 at 2:47 PM, Dipankar Manna dipankar.biot...@gmail.com wrote: Thank you Rajesh, That could be a possibility though. I am planning to do some MS analysis from the extra gel bands, if I get any by running SDS-PAGE on the purified protein. Best, Dipankar On Mon, Apr 20, 2015 at 10:42 PM, rajesh ponnusamy ponnusam...@googlemail.com wrote: just to add up: Thought about any E.Coli protease as an impurity.. very small quantity? Cheers, Rajesh. On Mon, Apr 20, 2015 at 9:14 PM, Dipankar Manna dipankar.biot...@gmail.com wrote: Dear Barbel, Thank you! Yes you are right that I did the SDS-PAGE with bigger substrate. Regarding peptide, we did check the MS and HPLC profile for the peptides which clearly shows that there should not be any cleaved peptides! Best, Dipankar On Mon, Apr 20, 2015 at 9:46 PM, Bärbel Blaum baerbel.bl...@uni-tuebingen.de wrote: I suppose you do the SDS PAGE test not with the peptide but some bigger substrate. Are you sure your peptide is intact *before* soaking? I.e. have you checked the batch yourself with MS or NMR? We regularly get small compounds (sugars) that turn out not to be what the label says. Bärbel Zitat von Dipankar Manna dipankar.biot...@gmail.com: 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.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 -- Bärbel Blaum, Ph.D. Interfakultäres Institut für Biochemie (IFIB) Hoppe-Seyler-Strasse 4 D-72076 Tübingen Germany +49 70 71 29 75 359 http://www.ifib.uni-tuebingen.de/research/blaum.html -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway -- Dr. Rajesh Ponnusamy Macromolecular Crystallography Unit Instituto de Tecnologia Química e Biológica - ITQB-UNL Oeiras - Portugal phone: (+351) 21 446 96 63 fax: (+351) 21 443 36 44 email: raj...@itqb.unl.pt -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
Re: [ccp4bb] Cleaved peptide density!
We had a similar situation with a catalytically dead serine protease. Initially I was excited to think we might be seeing residual catalytic activity of the mutant enzyme on a highly specific substrate; however, the activity turned out to result from contamination with a very small amount of wt enzyme, likely as a result of using the same affinity column to purify both. When we incubated the enzyme preparation with PMSF (an inhibitor that covalently modifies the catalytic serine and would not have affected the mutant), we eliminated the residual activity of the enzyme preparation. However, in our case we never were able to get crystals with the intact substrate, which apparently was not compatible with our crystal form. Is there a covalent inhibitor of your cysteine protease that you could use to pre-treat your enzyme, to see if this eliminates the activity? If so this might help distinguish between residual activity of the mutant vs. contamination with wt enzyme. Good luck! Evette Evette S. Radisky, Ph.D. Associate Professor and Senior Associate Consultant Department of Cancer Biology Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 http://www.mayo.edu/research/labs/proteases-cancer/ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Monday, April 20, 2015 2:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cleaved peptide density! Dear Crystallographers, I am working with a cysteine protease. I co-crystallized the protease with some small chemically synthesised peptides of 7 amino acid residues. I mutated the active Cysteine residue with Alanine to avoid the peptide cleavage so that I can get the whole peptide bound with my protein. But interesting I got the density for a cleaved peptide with 4 amino acids instead of the whole peptide. The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred exactly at the same peptide bond where it should. But I do not know how! Now my question is why I am getting the cleaved peptide as I already mutated the active residue Cysteine with Alanine (this mutant did no show any activity when I checked with SDS-PAGE). If anybody has the same kind of experience please advice me. Thanks in advance. Best, Dipankar -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
Re: [ccp4bb] Cleaved peptide density!
Hi Dipkar, My understanding is that the majority of a protease's activity comes from the oxyanion hole activating the scissile bond. Mutating the nucleophile reduces activity, sometimes appreciably, but does not kill the enzyme. Were your SDS-PAGE experiments on the same time scale and buffer conditions as your crystallography experiments? Perhaps something in the crystallization buffer increases the activity of the enzyme, or all/some of the peptide is digested in the time it takes to attain crystals -or only crystals of the digested peptide complex can be attained. 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 Radisky, Evette S., Ph.D. Sent: Wednesday, April 22, 2015 3:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Cleaved peptide density! We had a similar situation with a catalytically dead serine protease. Initially I was excited to think we might be seeing residual catalytic activity of the mutant enzyme on a highly specific substrate; however, the activity turned out to result from contamination with a very small amount of wt enzyme, likely as a result of using the same affinity column to purify both. When we incubated the enzyme preparation with PMSF (an inhibitor that covalently modifies the catalytic serine and would not have affected the mutant), we eliminated the residual activity of the enzyme preparation. However, in our case we never were able to get crystals with the intact substrate, which apparently was not compatible with our crystal form. Is there a covalent inhibitor of your cysteine protease that you could use to pre-treat your enzyme, to see if this eliminates the activity? If so this might help distinguish between residual activity of the mutant vs. contamination with wt enzyme. Good luck! Evette Evette S. Radisky, Ph.D. Associate Professor and Senior Associate Consultant Department of Cancer Biology Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 http://www.mayo.edu/research/labs/proteases-cancer/ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Monday, April 20, 2015 2:42 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cleaved peptide density! Dear Crystallographers, I am working with a cysteine protease. I co-crystallized the protease with some small chemically synthesised peptides of 7 amino acid residues. I mutated the active Cysteine residue with Alanine to avoid the peptide cleavage so that I can get the whole peptide bound with my protein. But interesting I got the density for a cleaved peptide with 4 amino acids instead of the whole peptide. The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred exactly at the same peptide bond where it should. But I do not know how! Now my question is why I am getting the cleaved peptide as I already mutated the active residue Cysteine with Alanine (this mutant did no show any activity when I checked with SDS-PAGE). If anybody has the same kind of experience please advice me. Thanks in advance. Best, Dipankar -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
Re: [ccp4bb] Refinement at 4A resolution
Hi Appu, you start already with a fixed spacegroup (scaled merged data) according to your pointless log. So you can't get another possible solution from pointless. Cheers Am 22.04.2015 um 22:28 schrieb Appu kumar: Dear CCP4 Member, I seek your advice on the refinement issues at the low resolution 4A. I am trying to refine a membrane protein structure after getting the phases from MR using the PHASER. The soluble domain structure which comprises of 40% of protein has been used as template (sequence identity 80%) in MR search . The PHASER gave a good solution having TFZ value of about 14.3. I have then created the polyA model for the transmembrane domain from distant homolog which share 30% sequence identity for TM region and try to find the phases for whole TM domain keeping the soluble domain fixed. I got lucky in getting the phases for the whole protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork and Rfree got stalled at the 41 and 44 respectively after several cycle of the refinement in both refmac and phenix. I checked the spacegroup with pointless and it suggests C2221. I have attached the pointless and phenix.xtriage run file with this mail for your evaluation. Phenix.xtriage suggests no major pathologies with the data except the mild psuedomerohedral twining. There are two molecules of protein in ASU. Evaluation of the density maps, suggest reasonable map for the most of protein part. I am wondering why Rwork and Rfree are not coming down despite of the good MR solution and what i am doing wrong with refinement and if there is some pathologies associated with the data which needs to be answered before heading to refinement. Thanks for your help in advance. Appu
Re: [ccp4bb] Cleaved peptide density!
If your protease depends on an oxyanion hole for stabilizing the transition state, fluoride ions are known to be a potent inhibitor of these proteases. (It is a quasi-diagnostic test for serine-type proteases, and related cysteine proteases.) This might allow you to get reactant bound without cleavage. It's worth a shot. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 4/22/2015 3:40 PM, Radisky, Evette S., Ph.D. wrote: We had a similar situation with a catalytically dead serine protease. Initially I was excited to think we might be seeing residual catalytic activity of the mutant enzyme on a highly specific substrate; however, the activity turned out to result from contamination with a very small amount of wt enzyme, likely as a result of using the same affinity column to purify both. When we incubated the enzyme preparation with PMSF (an inhibitor that covalently modifies the catalytic serine and would not have affected the mutant), we eliminated the residual activity of the enzyme preparation. However, in our case we never were able to get crystals with the intact substrate, which apparently was not compatible with our crystal form. Is there a covalent inhibitor of your cysteine protease that you could use to pre-treat your enzyme, to see if this eliminates the activity? If so this might help distinguish between residual activity of the mutant vs. contamination with wt enzyme. Good luck! Evette Evette S. Radisky, Ph.D. Associate Professor and Senior Associate Consultant Department of Cancer Biology Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 http://www.mayo.edu/research/labs/proteases-cancer/ *From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Dipankar Manna *Sent:* Monday, April 20, 2015 2:42 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Cleaved peptide density! Dear Crystallographers, I am working with a cysteine protease. I co-crystallized the protease with some small chemically synthesised peptides of 7 amino acid residues. I mutated the active Cysteine residue with Alanine to avoid the peptide cleavage so that I can get the whole peptide bound with my protein. But interesting I got the density for a cleaved peptide with 4 amino acids instead of the whole peptide. The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred exactly at the same peptide bond where it should. But I do not know how! Now my question is why I am getting the cleaved peptide as I already mutated the active residue Cysteine with Alanine (this mutant did no show any activity when I checked with SDS-PAGE). If anybody has the same kind of experience please advice me. Thanks in advance. Best, Dipankar -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
[ccp4bb] Refinement at 4A resolution
Dear CCP4 Member, I seek your advice on the refinement issues at the low resolution 4A. I am trying to refine a membrane protein structure after getting the phases from MR using the PHASER. The soluble domain structure which comprises of 40% of protein has been used as template (sequence identity 80%) in MR search . The PHASER gave a good solution having TFZ value of about 14.3. I have then created the polyA model for the transmembrane domain from distant homolog which share 30% sequence identity for TM region and try to find the phases for whole TM domain keeping the soluble domain fixed. I got lucky in getting the phases for the whole protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork and Rfree got stalled at the 41 and 44 respectively after several cycle of the refinement in both refmac and phenix. I checked the spacegroup with pointless and it suggests C2221. I have attached the pointless and phenix.xtriage run file with this mail for your evaluation. Phenix.xtriage suggests no major pathologies with the data except the mild psuedomerohedral twining. There are two molecules of protein in ASU. Evaluation of the density maps, suggest reasonable map for the most of protein part. I am wondering why Rwork and Rfree are not coming down despite of the good MR solution and what i am doing wrong with refinement and if there is some pathologies associated with the data which needs to be answered before heading to refinement. Thanks for your help in advance. Appu 93_pointless.log Description: Binary data xtriage_50.log Description: Binary data
Re: [ccp4bb] Refinement at 4A resolution
The Xtriage Pointless logs don't show definitively that the space group is C2221 as they have been run on the merged data. You may need to check them with the unmerged data, and perhaps run molecular replacement in a lower symmetry such as C2 Phil On 22 Apr 2015, at 22:28, Appu kumar appu.kum...@gmail.com wrote: Dear CCP4 Member, I seek your advice on the refinement issues at the low resolution 4A. I am trying to refine a membrane protein structure after getting the phases from MR using the PHASER. The soluble domain structure which comprises of 40% of protein has been used as template (sequence identity 80%) in MR search . The PHASER gave a good solution having TFZ value of about 14.3. I have then created the polyA model for the transmembrane domain from distant homolog which share 30% sequence identity for TM region and try to find the phases for whole TM domain keeping the soluble domain fixed. I got lucky in getting the phases for the whole protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork and Rfree got stalled at the 41 and 44 respectively after several cycle of the refinement in both refmac and phenix. I checked the spacegroup with pointless and it suggests C2221. I have attached the pointless and phenix.xtriage run file with this mail for your evaluation. Phenix.xtriage suggests no major pathologies with the data except the mild psuedomerohedral twining. There are two molecules of protein in ASU. Evaluation of the density maps, suggest reasonable map for the most of protein part. I am wondering why Rwork and Rfree are not coming down despite of the good MR solution and what i am doing wrong with refinement and if there is some pathologies associated with the data which needs to be answered before heading to refinement. Thanks for your help in advance. Appu 93_pointless.logxtriage_50.log
Re: [ccp4bb] Refinement at 4A resolution
Dear All, Sorry for the wrong pointless file. With this mail i have attached the pointless run file from the unmerged data. This file also suggests the C2221 spacegroup. Appu On 22 April 2015 at 17:40, Christian Roth christian.r...@bbz.uni-leipzig.de wrote: Hi Appu, you start already with a fixed spacegroup (scaled merged data) according to your pointless log. So you can't get another possible solution from pointless. Cheers Am 22.04.2015 um 22:28 schrieb Appu kumar: Dear CCP4 Member, I seek your advice on the refinement issues at the low resolution 4A. I am trying to refine a membrane protein structure after getting the phases from MR using the PHASER. The soluble domain structure which comprises of 40% of protein has been used as template (sequence identity 80%) in MR search . The PHASER gave a good solution having TFZ value of about 14.3. I have then created the polyA model for the transmembrane domain from distant homolog which share 30% sequence identity for TM region and try to find the phases for whole TM domain keeping the soluble domain fixed. I got lucky in getting the phases for the whole protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork and Rfree got stalled at the 41 and 44 respectively after several cycle of the refinement in both refmac and phenix. I checked the spacegroup with pointless and it suggests C2221. I have attached the pointless and phenix.xtriage run file with this mail for your evaluation. Phenix.xtriage suggests no major pathologies with the data except the mild psuedomerohedral twining. There are two molecules of protein in ASU. Evaluation of the density maps, suggest reasonable map for the most of protein part. I am wondering why Rwork and Rfree are not coming down despite of the good MR solution and what i am doing wrong with refinement and if there is some pathologies associated with the data which needs to be answered before heading to refinement. Thanks for your help in advance. Appu #CCP4I VERSION CCP4Interface 2.2.1 #CCP4I SCRIPT LOG pointless #CCP4I DATE 22 Apr 2015 17:39:30 #CCP4I USER appu #CCP4I PROJECT AS015crv6ctd2nq #CCP4I JOB_ID 119 #CCP4I SCRATCH /tmp/appu #CCP4I HOSTNAME sasha #CCP4I PID 47982 ### ### ### ### CCP4 6.4: POINTLESS version 1.9.16 : 21/08/14## ### User: appu Run date: 22/ 4/2015 Run time: 17:39:30 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. Input command lines HKLIN /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/imosflm/AS115c_1_0001.mtz HKLOUT /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/refine/AS115c_1_0001_pointless1.mtz ## This script run with the command ## # /home/appu/Downloads/ccp4-6.4.0/bin/pointless End of input OS type: linux Release Date: 21st August2014 ** ** * POINTLESS * * 1.9.16 * ** * Determine Laue group from unmerged intensities * * Phil Evans MRC LMB, Cambridge * * Uses cctbx routines by Ralf Grosse-Kunstleve et al.* ** ** Spacegroup information obtained from library file: Logical Name: SYMINFO Filename: /home/appu/Downloads/ccp4-6.4.0/lib/data/syminfo.lib Reflection list generated from file: /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/imosflm/AS115c_1_0001.mtz Title: Untitled Space group from HKLIN file : C 2 2 21 Cell: 130.42 213.36 160.80 90.00 90.00 90.00 Resolution range in file: 65.213.75 Time for reading file(s):2.190 secs === * Summary of test data read in: Resolution range accepted:65.213.75 Number of reflections = 23371 Number of observations =150865 Number of parts=619082 Number of batches in file = 600 Number of datasets = 1 Project: New Crystal: New Dataset: New Run number: 1 consists of batches 1 to600 Resolution range for run:65.213.75 Phi range: 0.00 to 180.00 Time range: 0.00 to 180.00 Closest reciprocal axis to
Re: [ccp4bb] Phaser going into infinite loop in Ample
Hi Dale, It must actually be AMPLE deciding how many copies to search for. Phaser will give you some information about how consistent the specified composition is with the Matthews volume, but it just searches for the number of copies that it's instructed to look for. We haven't put the intelligence into it to correlate the model(s) with the composition information and try out different possibilities. At the moment, we're leaving that level of analysis to pipelines like MRage. We're well aware of the tension between looking hard enough to find a solution in a difficult case and not throwing good CPU cycles after bad when it's hopeless. We're gradually introducing new features to make these decisions better, but we do tend to prefer wasting CPU time to missing solutions. However, we've introduced a couple of ways to limit the amount of time that Phaser spends pursuing very difficult or hopeless solutions, partly for the benefit of people such as the developers of Arcimboldo, AMPLE and the wide-search molecular replacement pipeline. One is the KILL command, which is a rather blunt instrument saying to give up if a solution isn't found in a certain length of time. In AMPLE, if you set quick mode, then the KILL time is set to 15 minutes. Another option (which I don't think AMPLE uses) is the PURGE command, where you can say (for instance) that Phaser should pursue a maximum of 25 partial solutions when adding the next component. If you're seeing an infinite loop, it would be handy if you could send me a copy of the logfile showing what is going on. There have been some bugs leading to such infinite loops under some circumstances, and if you're running into one of those there's a good chance that it has been fixed in a recent nightly build of Phaser available through Phenix. You can instruct CCP4 to use the Phaser executable from Phenix, and I think this should work fine in AMPLE, though I haven't tested it — I don't think any relevant syntax has changed. It's been a while since we've had a new stable release of Phaser in either CCP4 or Phenix, so we're aiming to get one out in the not too distant future. All the best, Randy On 22 Apr 2015, at 05:56, Dale Tronrud de...@daletronrud.com wrote: -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- -- 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
Re: [ccp4bb] Phaser going into infinite loop in Ample
Dear Dale, Isabel Uson's ARCIMBOLDO-LITE works well for coiled coils and has the same resolution requirements (2.1A or better) as AMPLE because both use SHELXE to expand the solution. It also employs PHASER to place a small fragment but it is often sufficient to let it search for just two or three copies in the asymmetric unit. Best wishes, George On 04/22/2015 06:56 AM, Dale Tronrud wrote: -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- -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
Re: [ccp4bb] Phaser going into infinite loop in Ample
Dear Dale, as George points out, you may be interested in trying ARCIMBOLDO, as it has been successfully applied to coiled coil proteins recently, as in the case of Franke et al (2014) Open Biology, 4. p. 130172 or Sammito et al (2013) Nature Methods 10: 1099-1101 . Different models can be searched for, starting from single helices with or without sidechains, going to precomputed libraries of helices in parallel or antiparallel configurations. If you need help or support to use it, please feel free to ask any question. Best wishes, Claudia Claudia Millán 2015-04-22 6:56 GMT+02:00 Dale Tronrud de...@daletronrud.com: -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-
Re: [ccp4bb] Phaser going into infinite loop in Ample
Dear Dale, This is a known issue with AMPLE and will be fixed with the next release. In the meantime you can tell AMPLE to pass the KILL option that Randy mentions to PHASER, by adding the following arguments to your script: -mr_keys PKEY KILL TIME 360 this will kill PHASER after 360 minutes (6 hours), which we've found if normally enough, although pick whatever time works for you, I should also point out that I think George is doing a disservice to SHELXE. In our last paper looking at coiled-coils, we saw successes at resolutions much lower than 2.1, in one case, AMPLE was able to solve a structure with a resolution of 2.9A: http://dx.doi.org/10.1107/S2052252515002080 If you have any issues getting the KILL command to work, please feel free to contact me off-list. Best wishes, Jens From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Randy Read [rj...@cam.ac.uk] Sent: 22 April 2015 09:04 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Phaser going into infinite loop in Ample Hi Dale, It must actually be AMPLE deciding how many copies to search for. Phaser will give you some information about how consistent the specified composition is with the Matthews volume, but it just searches for the number of copies that it's instructed to look for. We haven't put the intelligence into it to correlate the model(s) with the composition information and try out different possibilities. At the moment, we're leaving that level of analysis to pipelines like MRage. We're well aware of the tension between looking hard enough to find a solution in a difficult case and not throwing good CPU cycles after bad when it's hopeless. We're gradually introducing new features to make these decisions better, but we do tend to prefer wasting CPU time to missing solutions. However, we've introduced a couple of ways to limit the amount of time that Phaser spends pursuing very difficult or hopeless solutions, partly for the benefit of people such as the developers of Arcimboldo, AMPLE and the wide-search molecular replacement pipeline. One is the KILL command, which is a rather blunt instrument saying to give up if a solution isn't found in a certain length of time. In AMPLE, if you set quick mode, then the KILL time is set to 15 minutes. Another option (which I don't think AMPLE uses) is the PURGE command, where you can say (for instance) that Phaser should pursue a maximum of 25 partial solutions when adding the next component. If you're seeing an infinite loop, it would be handy if you could send me a copy of the logfile showing what is going on. There have been some bugs leading to such infinite loops under some circumstances, and if you're running into one of those there's a good chance that it has been fixed in a recent nightly build of Phaser available through Phenix. You can instruct CCP4 to use the Phaser executable from Phenix, and I think this should work fine in AMPLE, though I haven't tested it — I don't think any relevant syntax has changed. It's been a while since we've had a new stable release of Phaser in either CCP4 or Phenix, so we're aiming to get one out in the not too distant future. All the best, Randy On 22 Apr 2015, at 05:56, Dale Tronrud de...@daletronrud.com wrote: -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 -
