Re: [ccp4bb] str solving problem
Dear Eugene plz find the merging statics over this link https://www.dropbox.com/sh/3155bp0c8axo7tx/0P1RWTTD8z?n=21758536 I have tried different subset of images for indexing, only cell edges are changing very marginal ( 1 ) but no change in space group. Dear Manfred I have collected my data over mar345 detector, not present in detector type dropdown, how can I add. regards and thanks to all pramod On Mon, Jun 24, 2013 at 10:31 PM, Eugene Valkov eugene.val...@gmail.comwrote: Hi Pramod, Can you post your merging statistics in different space groups, not just log files from scaling? These are summarised nicely by Scala or Aimless. Also, have you tried indexing from different subsets of images? Perhaps there is a substantial contribution from a 'satellite' crystal in one orientation or crystal will be less split? I've had cases where I could not index properly if I had just used 0 and 90, but when I tried different subsets of images it worked. This is very easy to do in iMosflm. Andrew Leslie or other Mosflm developers, if they are reading this, might well be interested in looking at your images as they are currently interested in these kinds of problems with multiple lattices (see *Acta Cryst.* (2013). D*69*, 1195-1203) Eugene On 21 June 2013 21:58, Pramod Kumar pramod...@gmail.com wrote: Dear ... Francis Last I remember, HKL2000 bases its indexing on the 'strongest' spots on an image (though you could manually select spots). It could result in a misindex if the strongest spots come from separate lattices.. I have used both HKL2000 and mosflm giving the same results (although I have used manual selection of spots as a trial but results are identical). Try a program that uses all spots for indexing, across all images (XDS for example) and you might get the true space group.. I have given several efforts to the XDS but its giving error data image of particular no. does not exist (initially it was saying 11th image than i change image range then it says 21st and so on) *kindly check my data collection profile and XDS.INP* file in attachment' Or if the crystal is big enough, you could try shooting it in different areas and 'searching' for a better spot to collect data. Or 'grow a better crystal'. raising the crystals and struggle is on the peak... Dear Eugene plz find the attached scale log file, scaling table of mosflm When you index spots in Mosflm, do your predictions agree with the spots? plz see the snapshot of predicted spots.. Dear Eleanor Yes both the molecule are visible in the ASU. Dear Pozharski Balbes pipeline hitting extremely high marks when fed into Phaser while being complete nonsense (it's a 150kDa multi-domain protein and resulting domain arrangement made absolutely no sense). Refinement was stuck with high R-values and I sadly gave up on it for now. I suspected that refmac step included in the pipeline artificially shifts the model so that it conforms to Patterson map better, which results in high score in Phaser. My domain arrangement is as expected, two molecules in ASU. thanks and regards pramod On Thu, Jun 20, 2013 at 3:50 PM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: As others say - the Rfactors look pretty good for MR, mine usually start over 50% even with a better model and one hopes they then decrease.. But you say you took the Balbes model into phaser? and I think Balbes automatically runs cycles of refinement so any comment on R factors may not mean much. Have you found both molecules in the asymmetric unit? You only give LLG for one? Eleanor On 19 June 2013 17:44, Eugene Valkov eugene.val...@gmail.com wrote: Yes, I would agree with Francis that diffraction shows contribution from several lattices, which could lead to misindexing. However, it should be feasible to get a model that refines from this sort of data. Pramod - could you please post your data processing statistics from your scaling program? Better if you have several for different spacegroups. Also, I have no idea how HKL200 does this, but could you please provide an indexing solution table from Mosflm that shows penalties associated with each type of space group? Was there a sharp penalty drop at some point or was it more gradual? When you index spots in Mosflm, do your predictions agree with the spots? Or is there a substantial portion that are missed? I would consider altering thresholds in Mosflm for indexing (see the manual). Eugene On 19 June 2013 17:34, Francis E. Reyes francis.re...@colorado.eduwrote: On Jun 17, 2013, at 12:36 PM, Pramod Kumar pramod...@gmail.com wrote: I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO no ice ring is appeared,
Re: [ccp4bb] str solving problem
Hi Pramod, Can you post your merging statistics in different space groups, not just log files from scaling? These are summarised nicely by Scala or Aimless. Also, have you tried indexing from different subsets of images? Perhaps there is a substantial contribution from a 'satellite' crystal in one orientation or crystal will be less split? I've had cases where I could not index properly if I had just used 0 and 90, but when I tried different subsets of images it worked. This is very easy to do in iMosflm. Andrew Leslie or other Mosflm developers, if they are reading this, might well be interested in looking at your images as they are currently interested in these kinds of problems with multiple lattices (see *Acta Cryst.* (2013). D*69*, 1195-1203) Eugene On 21 June 2013 21:58, Pramod Kumar pramod...@gmail.com wrote: Dear ... Francis Last I remember, HKL2000 bases its indexing on the 'strongest' spots on an image (though you could manually select spots). It could result in a misindex if the strongest spots come from separate lattices.. I have used both HKL2000 and mosflm giving the same results (although I have used manual selection of spots as a trial but results are identical). Try a program that uses all spots for indexing, across all images (XDS for example) and you might get the true space group.. I have given several efforts to the XDS but its giving error data image of particular no. does not exist (initially it was saying 11th image than i change image range then it says 21st and so on) *kindly check my data collection profile and XDS.INP* file in attachment' Or if the crystal is big enough, you could try shooting it in different areas and 'searching' for a better spot to collect data. Or 'grow a better crystal'. raising the crystals and struggle is on the peak... Dear Eugene plz find the attached scale log file, scaling table of mosflm When you index spots in Mosflm, do your predictions agree with the spots? plz see the snapshot of predicted spots.. Dear Eleanor Yes both the molecule are visible in the ASU. Dear Pozharski Balbes pipeline hitting extremely high marks when fed into Phaser while being complete nonsense (it's a 150kDa multi-domain protein and resulting domain arrangement made absolutely no sense). Refinement was stuck with high R-values and I sadly gave up on it for now. I suspected that refmac step included in the pipeline artificially shifts the model so that it conforms to Patterson map better, which results in high score in Phaser. My domain arrangement is as expected, two molecules in ASU. thanks and regards pramod On Thu, Jun 20, 2013 at 3:50 PM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: As others say - the Rfactors look pretty good for MR, mine usually start over 50% even with a better model and one hopes they then decrease.. But you say you took the Balbes model into phaser? and I think Balbes automatically runs cycles of refinement so any comment on R factors may not mean much. Have you found both molecules in the asymmetric unit? You only give LLG for one? Eleanor On 19 June 2013 17:44, Eugene Valkov eugene.val...@gmail.com wrote: Yes, I would agree with Francis that diffraction shows contribution from several lattices, which could lead to misindexing. However, it should be feasible to get a model that refines from this sort of data. Pramod - could you please post your data processing statistics from your scaling program? Better if you have several for different spacegroups. Also, I have no idea how HKL200 does this, but could you please provide an indexing solution table from Mosflm that shows penalties associated with each type of space group? Was there a sharp penalty drop at some point or was it more gradual? When you index spots in Mosflm, do your predictions agree with the spots? Or is there a substantial portion that are missed? I would consider altering thresholds in Mosflm for indexing (see the manual). Eugene On 19 June 2013 17:34, Francis E. Reyes francis.re...@colorado.eduwrote: On Jun 17, 2013, at 12:36 PM, Pramod Kumar pramod...@gmail.com wrote: I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO no ice ring is appeared, diffraction pattern looks ok, misindexing in any direction is not conclusive to me (plz see the imj attachment) The diffraction does not look ok... there's hints of multiple lattices... which is not a problem if the two lattice orientations do not perfectly overlap (i.e. their spots are separable). Last I remember, HKL2000 bases its indexing on the 'strongest' spots on an image (though you could manually select spots). It could result in a misindex if the strongest spots come from separate lattices
Re: [ccp4bb] str solving problem
Pramod: [1] Please refrain from posting excessively large (1MB) attachments to the ccp4bb. Either use a compression technique or use another means of transmitting large files to your recipients without spamming the entire group. [2] Your predictions are not overlaying well with the spots . Well it may be overlaying well with a subset of spots (in this case, the strongest spots that obey the symmetry *you* chose), but your are leaving out a large number of spots. As I said before, your diffraction pattern is exhibiting strong reflections with weak reflections at a given resolution. It looks as if there are multiple lattices probably due to bad crystal morphology ( a cracked crystal, twinning, pseudosymmetry) or multiple crystals in the beam, the list goes on I've experienced your case many, many times you'll get a good MR solution, but then the refinement becomes 'stuck'. I think the idiom... 'garbage in, garbage out' applies here.. [3] I have given several efforts to the XDS but its giving error data image of particular no. does not exist (initially it was saying 11th image than i change image range then it says 21st and so on) kindly check my data collection profile and XDS.INP file in attachment' Sounds like a file name problem. [4] Or if the crystal is big enough, you could try shooting it in different areas and 'searching' for a better spot to collect data. Or 'grow a better crystal'. raising the crystals and struggle is on the peak... There are a number of ccp4bb postings about crystal reproducibility or crystal diffraction improvement. Search the archives for these. F - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] str solving problem
As others say - the Rfactors look pretty good for MR, mine usually start over 50% even with a better model and one hopes they then decrease.. But you say you took the Balbes model into phaser? and I think Balbes automatically runs cycles of refinement so any comment on R factors may not mean much. Have you found both molecules in the asymmetric unit? You only give LLG for one? Eleanor On 19 June 2013 17:44, Eugene Valkov eugene.val...@gmail.com wrote: Yes, I would agree with Francis that diffraction shows contribution from several lattices, which could lead to misindexing. However, it should be feasible to get a model that refines from this sort of data. Pramod - could you please post your data processing statistics from your scaling program? Better if you have several for different spacegroups. Also, I have no idea how HKL200 does this, but could you please provide an indexing solution table from Mosflm that shows penalties associated with each type of space group? Was there a sharp penalty drop at some point or was it more gradual? When you index spots in Mosflm, do your predictions agree with the spots? Or is there a substantial portion that are missed? I would consider altering thresholds in Mosflm for indexing (see the manual). Eugene On 19 June 2013 17:34, Francis E. Reyes francis.re...@colorado.eduwrote: On Jun 17, 2013, at 12:36 PM, Pramod Kumar pramod...@gmail.com wrote: I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO no ice ring is appeared, diffraction pattern looks ok, misindexing in any direction is not conclusive to me (plz see the imj attachment) The diffraction does not look ok... there's hints of multiple lattices... which is not a problem if the two lattice orientations do not perfectly overlap (i.e. their spots are separable). Last I remember, HKL2000 bases its indexing on the 'strongest' spots on an image (though you could manually select spots). It could result in a misindex if the strongest spots come from separate lattices (and even worse if you have twinning/pseudosymmetry issues). Try a program that uses all spots for indexing, across all images (XDS for example) and you might get the true space group. Or if the crystal is big enough, you could try shooting it in different areas and 'searching' for a better spot to collect data. Or 'grow a better crystal'. F - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder -- Dr Eugene Valkov MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 407840
Re: [ccp4bb] str solving problem
Dear Robert I have cross checked by running the gel and silver stain that confirms it is only the protein i am targeting. no exact hit was obtained for this cell parameter and SG, as it previously checked during balbes run... thanks and regards pramod On Wed, Jun 19, 2013 at 2:28 PM, Robert Esnouf rob...@strubi.ox.ac.ukwrote: Hi Pramod, You mention this is the limitation of my data that came from three month old crystal. If the crystal took that long to grow (did it?) then a crystallized (1) proteolytic fragment or (2) low level contaminant is quite likely. 1) If you have the crystal still you could try to redissolve it and run a gel 2) If it is the second case then the unit cell is probably already in the PDB. You can check with this tool... http://www.strubi.ox.ac.uk/nearest-cell/nearest-cell.cgi Good luck, Robert -- Dr. Robert Esnouf, University Research Lecturer and Head of Research Computing, Wellcome Trust Centre for Human Genetics, Old Road Campus, Roosevelt Drive, Oxford OX3 7BN, UK Emails: rob...@strubi.ox.ac.uk Tel: (+44) - 1865 - 287783 and rob...@well.ox.ac.uk Fax: (+44) - 1865 - 287547 Original message Date: Wed, 19 Jun 2013 03:20:57 +0530 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Pramod Kumar pramod...@gmail.com) Subject: Re: [ccp4bb] str solving problem To: CCP4BB@JISCMAIL.AC.UK Apology for delay Dear Abhinav.. Kept the ratio 2 with correlation of completeness above 75 r factor reduces with under -5 to 6 lees than r free. I am trying different models.. Dear Herman -Is the protein that crystallized the protein you think it is? Plasmids do get mixed-up and sometimes a more abundant natural protein with about the same molecular weight as the protein of interest gets purified. Yes, I have done silver stain that confirms exact MW, further related literature and biological properties supports, -How do the diffraction images look like: are there strong ice-rings, multiple diffraction patterns or other problems? Could there have been misindexing in one of the directions? no ice ring is appeared, diffraction pattern looks ok, misindexing in any direction is not conclusive to me (plz see the imj attachment) Dear Eugene the phaser .sol file states... SOLU SET RFZ=6.6 TFZ=8.8 PAK=0 LLG=102 TFZ==8.2 LLG=1804 TFZ==22.9 SOLU SPAC C 2 2 21 SOLU 6DIM ENSE ensemble1 EULER 180.061 0.000 0.000 FRAC -0.49950 -0.50021 -0.6 BFAC 0.0 SOLU ENSE ensemble1 VRMS 0.877 for building the model I used the 'autorikshaw pipeline' and it has given rfact/rfree 0.4645 / 0.4888 but back to refmac again starts rising Dear Edward Patterson analysis obtained from xtrige... Xtrige summary Twinning and intensity statistics summary (acentric data): Statistics independent of twin laws I^2/I^2 : 2.078 F^2/F^2 : 0.777 |E^2-1| : 0.758 |L|, L^2: 0.507, 0.340 Multivariate Z score L-test: 2.003 The multivariate Z score is a quality measure of the given spread in intensities. Good to reasonable data are expected to have a Z score lower than 3.5. Large values can indicate twinning, but small values do not necessarily exclude it. No (pseudo)merohedral twin laws were found. Patterson analyses - Largest peak height : 4.674 (corresponding p value : 0.95872) The largest off-origin peak in the Patterson function is 4.67% of the height of the origin peak. No significant pseudotranslation is detected. The results of the L-test indicate that the intensity statistics behave as expected. No twinning is suspected. integration in P1 and attempt to carry out molecular replacement in P1 from this Zanuda concludes as follow ^ |5 | C 2 2 21 | 0.5985 | 0.4265 | 0.3918 | 0.4880 | - | 1 | P 1| 0.5308 | 0.4236 | 0.3970 | 0.5024 | | 2 | P 1 21 1 | 0.6302 |--| 0.3912 | 0.4989 | | 5 | C 2 2 21 | 0.7318 |--| 0.3804 | 0.5009 | - |5 | C 2 2 21 | 0.7318 |--| 0.3804 | 0.5009 | - R-factor in the original subgroup is NOT the best. The original spacegroup assignment seems to be incorrect. thanks and regards pramod On Tue, Jun 18, 2013 at 3:32 PM, Edward Lowe edward.l...@bioch.ox.ac.uk wrote: Dear Pramod, It's difficult to be certain based on what you have posted but my best guess would be problems
Re: [ccp4bb] str solving problem
Yes, I would agree with Francis that diffraction shows contribution from several lattices, which could lead to misindexing. However, it should be feasible to get a model that refines from this sort of data. Pramod - could you please post your data processing statistics from your scaling program? Better if you have several for different spacegroups. Also, I have no idea how HKL200 does this, but could you please provide an indexing solution table from Mosflm that shows penalties associated with each type of space group? Was there a sharp penalty drop at some point or was it more gradual? When you index spots in Mosflm, do your predictions agree with the spots? Or is there a substantial portion that are missed? I would consider altering thresholds in Mosflm for indexing (see the manual). Eugene On 19 June 2013 17:34, Francis E. Reyes francis.re...@colorado.edu wrote: On Jun 17, 2013, at 12:36 PM, Pramod Kumar pramod...@gmail.com wrote: I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO no ice ring is appeared, diffraction pattern looks ok, misindexing in any direction is not conclusive to me (plz see the imj attachment) The diffraction does not look ok... there's hints of multiple lattices... which is not a problem if the two lattice orientations do not perfectly overlap (i.e. their spots are separable). Last I remember, HKL2000 bases its indexing on the 'strongest' spots on an image (though you could manually select spots). It could result in a misindex if the strongest spots come from separate lattices (and even worse if you have twinning/pseudosymmetry issues). Try a program that uses all spots for indexing, across all images (XDS for example) and you might get the true space group. Or if the crystal is big enough, you could try shooting it in different areas and 'searching' for a better spot to collect data. Or 'grow a better crystal'. F - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder -- Dr Eugene Valkov MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. Email: eval...@mrc-lmb.cam.ac.uk Tel: +44 (0) 1223 407840
Re: [ccp4bb] str solving problem
Hi Pramod, Refined LLG value of 1800 and R-factor of 46% at the end of the Phaser run does indicate a good MR solution. Your maps also look quite good and some positive density is visible near side chains. It would be helpful to see your translational Z-scores for both molecules placed, which can be found in the .sol file that Phaser outputs. It is puzzling that your model does not refine. Have your tried feeding your MR model and phases into an automated building program like ArpWarp, Buccaneer or AutoBuild (in Phenix suite)? Hope this helps. Eugene On 17 June 2013 22:28, Pramod Kumar pramod...@gmail.com wrote: Dear Abhinav * I would suggest you integrate in p1 and run pointless.* after integrating in p1 and running the pointless its still concluding the SG c2221. *How do you determine the resolution cut off of 2.9A?* the basis of resolution at 2.9A* was the completeness and I/Sigma value. Two molecule in ASU further supported by Matthews coefficient and solvent percentage content. *Is this a homology model? or at least side chains replaced by correct ones, like with chainsaw? Have you trimmed loops and floppy regions in the model?* its not homology model, I have used chainsaw to make the model for molecular replacement, and trimmed the model for floppy, loopy ambiguousness regions. plz see the snap shot of the model. thanks and regards... pramod On Tue, Jun 18, 2013 at 1:16 AM, Abhinav Kumar abhin...@slac.stanford.edu wrote: Hi Pramod, I would suggest you integrate in p1 and run pointless. If your Rfree is above 0.45, I wouldn't trust the model. How do you determine the resolution cut off of 2.9A? In MR, the score should improve when the program places the second copy to the first molecule. Is this a homology model? or at least side chains replaced by correct ones, like with chainsaw? Have you trimmed loops and floppy regions in the model? Thanks, Abhinav JCSG@SSRL, SLAC (650) 926-2992 On 06/17/2013 12:36 PM, Pramod Kumar wrote: *Dear Abhinav Kumar * *thanks for kind suggestions * * I have tried as follow. 1. You should try to identify the correct space group first. *integration in p21 given the following statics in pointless * * Alternative reindexing Lklhd CC R(E^2) Number Cell_deviation* * [h,k,l] 0.5660.9570.094 25580 0.00* * [l,-k,h] 0.4340.8530.195 25580 0.20* * *integration in c2221 given the following statics in pointless * * Spacegroup TotProb SysAbsProb Reindex Conditions* ** * ( 20)0.997 0.997 00l: l=2n (zone 1) * * moreover Zanuda and molrep's chekall space group option also suggests c2221, 2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success.Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser? * * * this is the limitation of my data that came from three month old crystal, there are two molecules in ASU (what is the extant to say reasonably good please ?), initially phaser was failed, with the balbes output as ensemble it worked with following profile .. * *** Phaser Module: AUTOMATED MOLECULAR REPLACEMENT 2.5.2 *** ** * Current is Best Solution (first)* * New Best LLG : 101.7 (8.70)* * Best Search Component so far: ensemble1 * ** *** Phaser Module: MOLECULAR REPLACEMENT REFINEMENT AND PHASING 2.5.2 *** ** * Resolution of All Data (Number):2.91 47.64 (15877)* * Resolution of Selected Data (Number): 2.91 47.64 (15877)* ** ** * Refinement Table (Unsorted)* * ---* * #+ = input number#* = results number* * #+ #* (Initial LLG Rval) (Refined LLG Rval) Unique =# Tmplt SpaceGroup * * 11 908.3 48.8 1804.3 46.4YESC 2 2 21 * ** ** * Refinement Table (Sorted)* * -* * #+ = input number#* = results number* * #+ #* (Initial LLG Rval) (Refined LLG Rval) Unique =# Tmplt SpaceGroup * * 11 908.3 48.8 1804.3 46.4YESC 2 2 21 **but again the refmac is showing * * *R factor 0.4404 0.4203 R free 0.4955 0.5027 Rms BondLength 0.0254 0.0093 Rms BondAngle 3.1234 1.4581 Rms ChirVolume 0.1741 0.0706 * * * * * * *3. Did you check for twinning? * *TWINNING SUMMARY* * * *Twinning fraction from H-test: 0.00* *L-statistic from L-Test: 0.48* * * *
Re: [ccp4bb] str solving problem
Dear Pramod, It's difficult to be certain based on what you have posted but my best guess would be problems with symmetry determination it's quite possible for pointless to be fooled into assigning higher symmetry if some form of pseudo-symmetry is present. What does the native Patterson look like? Any large off-origin peaks might give you a clue. The safest thing to do would be to integrate in P1 and attempt to carry out molecular replacement in P1 and see how that refines. Hopefully be able to determine the correct symmetry from this (Zanuda may well be able to help at this point!). Ed. -- Dr. E.D. Lowe Department of Biochemistry University of Oxford South Parks Road Oxford, UK OX1 3QU e:edward.l...@bioch.ox.ac.uk t: +44 (0) 1865 613288 f: +44 (0) 1865 613201 From: Pramod Kumar pramod...@gmail.commailto:pramod...@gmail.com Reply-To: Pramod Kumar pramod...@gmail.commailto:pramod...@gmail.com Date: Tue, 18 Jun 2013 02:58:36 +0530 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] str solving problem Dear Abhinav I would suggest you integrate in p1 and run pointless. after integrating in p1 and running the pointless its still concluding the SG c2221. How do you determine the resolution cut off of 2.9A? the basis of resolution at 2.9A* was the completeness and I/Sigma value. Two molecule in ASU further supported by Matthews coefficient and solvent percentage content. Is this a homology model? or at least side chains replaced by correct ones, like with chainsaw? Have you trimmed loops and floppy regions in the model? its not homology model, I have used chainsaw to make the model for molecular replacement, and trimmed the model for floppy, loopy ambiguousness regions. plz see the snap shot of the model. thanks and regards... pramod On Tue, Jun 18, 2013 at 1:16 AM, Abhinav Kumar abhin...@slac.stanford.edumailto:abhin...@slac.stanford.edu wrote: Hi Pramod, I would suggest you integrate in p1 and run pointless. If your Rfree is above 0.45, I wouldn't trust the model. How do you determine the resolution cut off of 2.9A? In MR, the score should improve when the program places the second copy to the first molecule. Is this a homology model? or at least side chains replaced by correct ones, like with chainsaw? Have you trimmed loops and floppy regions in the model? Thanks, Abhinav JCSG@SSRL, SLAC (650) 926-2992 On 06/17/2013 12:36 PM, Pramod Kumar wrote: Dear Abhinav Kumar thanks for kind suggestions I have tried as follow. 1. You should try to identify the correct space group first. *integration in p21 given the following statics in pointless Alternative reindexing Lklhd CC R(E^2) Number Cell_deviation [h,k,l] 0.5660.9570.094 25580 0.00 [l,-k,h] 0.4340.8530.195 25580 0.20 *integration in c2221 given the following statics in pointless Spacegroup TotProb SysAbsProb Reindex Conditions ( 20)0.997 0.997 00l: l=2n (zone 1) moreover Zanuda and molrep's chekall space group option also suggests c2221, 2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success.Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser? * this is the limitation of my data that came from three month old crystal, there are two molecules in ASU (what is the extant to say reasonably good please ?), initially phaser was failed, with the balbes output as ensemble it worked with following profile .. ** Phaser Module: AUTOMATED MOLECULAR REPLACEMENT 2.5.2 *** * Current is Best Solution (first) New Best LLG : 101.7 (8.70) Best Search Component so far: ensemble1 * *** Phaser Module: MOLECULAR REPLACEMENT REFINEMENT AND PHASING 2.5.2 *** * Resolution of All Data (Number):2.91 47.64 (15877) Resolution of Selected Data (Number): 2.91 47.64 (15877) Refinement Table (Unsorted) --- #+ = input number#* = results number #+ #* (Initial LLG Rval) (Refined LLG Rval) Unique =# Tmplt SpaceGroup 11 908.3 48.8 1804.3 46.4YESC 2 2 21 Refinement Table (Sorted) - #+ = input number#* = results number #+ #* (Initial LLG Rval) (Refined LLG Rval) Unique =# Tmplt SpaceGroup 11 908.3 48.8 1804.3 46.4YESC 2 2 21 but again the refmac is showing R factor 0.4404 0.4203 R free 0.4955 0.5027 Rms BondLength 0.0254 0.0093 Rms
Re: [ccp4bb] str solving problem
Hi Pramod, 1. You should try to identify the correct space group first. Did you integrate in p1 and run pointless? 2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success. Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser? 3. Did you check for twinning? Thanks, Abhinav JCSG@SSRL, SLAC (650) 926-2992 On 06/17/2013 09:35 AM, Pramod Kumar wrote: Dear group I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO, now the model for molecular replacement with closest identity of 31 given a contrast of 2, score 0.30 and wrfac 0.60. but balbes uses different models with lesser identity, no matter which way I am going the rFree keep on increasing during refinement in refmac, when I build the model in coot with deletion and addition of residue it starts with relatively low but gradually rises through almost all cycles although model fits to the density well and residue are building, coot validation parameters are also reasonable OK for geometry, rotamer, density fit,.. now my question * where should i first check for possible correction? * In molecular replacement what should be the red line for identity and related criteria? * if initial rFree starts around 50, how likely that its not the right way? * my rms bond angle is close to 1 while the bond length is 0.01 and chiral 0.1 concludes what is serious? *sincere apology for amateur query* if any... thanks in advance pramod -- Pramod Kumar. Graduate Student. Crystallography lab. Department Of Biotechnology. Indian Institute Of Technology Roorkee Uttranchal.247667 India +919359189657.
Re: [ccp4bb] str solving problem
*Dear Abhinav Kumar * *thanks for kind suggestions * * I have tried as follow. 1. You should try to identify the correct space group first. *integration in p21 given the following statics in pointless * * Alternative reindexing Lklhd CC R(E^2) Number Cell_deviation* * [h,k,l] 0.5660.9570.094 25580 0.00* * [l,-k,h] 0.4340.8530.195 25580 0.20 * * *integration in c2221 given the following statics in pointless * * Spacegroup TotProb SysAbsProb Reindex Conditions* * * * ( 20)0.997 0.997 00l: l=2n (zone 1) * * moreover Zanuda and molrep's chekall space group option also suggests c2221, 2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success.Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser? * * * this is the limitation of my data that came from three month old crystal, there are two molecules in ASU (what is the extant to say reasonably good please ?), initially phaser was failed, with the balbes output as ensemble it worked with following profile .. * *** Phaser Module: AUTOMATED MOLECULAR REPLACEMENT 2.5.2 *** * * * Current is Best Solution (first)* * New Best LLG : 101.7 (8.70)* * Best Search Component so far: ensemble1 * * * *** Phaser Module: MOLECULAR REPLACEMENT REFINEMENT AND PHASING 2.5.2 *** * * * Resolution of All Data (Number):2.91 47.64 (15877)* * Resolution of Selected Data (Number): 2.91 47.64 (15877)* * * * * * Refinement Table (Unsorted)* * ---* * #+ = input number#* = results number* * #+ #* (Initial LLG Rval) (Refined LLG Rval) Unique =# Tmplt SpaceGroup * * 11 908.3 48.8 1804.3 46.4YES C 2 2 21 * * * * * * Refinement Table (Sorted)* * -* * #+ = input number#* = results number* * #+ #* (Initial LLG Rval) (Refined LLG Rval) Unique =# Tmplt SpaceGroup * * 11 908.3 48.8 1804.3 46.4YES C 2 2 21 **but again the refmac is showing * * *R factor 0.4404 0.4203 R free 0.4955 0.5027 Rms BondLength 0.0254 0.0093 Rms BondAngle 3.1234 1.4581 Rms ChirVolume 0.1741 0.0706 * * * * * * *3. Did you check for twinning? * *TWINNING SUMMARY* * * *Twinning fraction from H-test: 0.00* *L-statistic from L-Test: 0.48* * * * Relation between L statistics and twinning fraction:* * Twinning fraction = 0.000 L statistics = 0.500:* * Twinning fraction = 0.100 L statistics = 0.440:* * Twinning fraction = 0.500 L statistics = 0.375:* *NO Twinning detected* * * * * *thanks and regards * * pramod... * * * * * * * * * *On Mon, Jun 17, 2013 at 10:15 PM, Abhinav Kumar abhin...@slac.stanford.edu wrote: * *Hi Pramod, 1. You should try to identify the correct space group first. Did you integrate in p1 and run pointless? ** 2. A template with 31% identity is not a great model. The number of molecules in ASU will affect your chances of success. Hopefully it's not large. wrfac of 0.6 and Rfree of 0.5 suggest the solution may be incorrect. Did you try phaser? 3. Did you check for twinning? * * Thanks, Abhinav * * JCSG@SSRL, SLAC (650) 926-2992 * * On 06/17/2013 09:35 AM, Pramod Kumar wrote: * *Dear group * * I have a crystal data diffracted around 2.9 A*, during the data reduction HKL2000 not convincingly showed the space group (indexed in lower symmetry p1), while the mosflm given C-centered Orthorhombic, and again with little play around HKL2000 given CO, now the model for molecular replacement with closest identity of 31 given a contrast of 2, score 0.30 and wrfac 0.60. but balbes uses different models with lesser identity, no matter which way I am going the rFree keep on increasing during refinement in refmac, when I build the model in coot with deletion and addition of residue it starts with relatively low but gradually rises through almost all cycles although model fits to the density well and residue are building, coot validation parameters are also reasonable OK for geometry, rotamer, density fit,.. ** * *now my question * ** where should i first check for possible correction? * ** In molecular replacement what should be the red line for identity and related criteria? * ** if initial rFree starts around 50, how likely that its not the right way? * ** my rms bond angle is close to 1 while the bond length is 0.01 and chiral 0.1 concludes what is serious? * * *
