Re: [ccp4bb] calculation of active site area
Fpocket and CAVER are good. Yarrow On Friday, September 19, 2014, Faisal Tarique faisaltari...@gmail.com wrote: Dear all Please tell me the names of good servers / tools which calculate the size and surface area of the active site pocket of a protein.. -- Regards Faisal School of Life Sciences JNU
[ccp4bb] wilson B in xds vs. truncate.
Hello CCP4 users. I noticed a discrepancy between the estimated wilson B reported by XDS and that reported by truncate. As far as I know XDSconv and truncate use the French and Wilson (K.S. Acta. Cryst. (1978), A34, 517-525.) method for converting to structure factors. However, the result below is from XDS before XDSconv. Does anyone know exactly how XDS is calculating the Wilson-B compared to truncate? XDS: WILSON LINE (using all data) : A= 7.372 B= 20.624 CORRELATION= 0.98 Truncate: Results Wilson plot: B = 11.375 intercept = 5.959 siga = 0.264 sigb = 0.053 scale factor on intensity = 387.0343
Re: [ccp4bb] Solvent channels
You can use CAVER but you would have to make all the symmetry mates as one chain in order to fool it. Still better to just do the experiment I think. Either it will work or it won't, regardless of what any software tells you. Just a wild idea : ) On Fri, Jun 27, 2014 at 5:06 PM, Yarrow Madrona amadr...@uci.edu wrote: Hi Reza, CAVER is a great tool for this. There is a web version. You can also download it to customize and run it in the command line. There is also a Pymol CAVER plug in that works very well. I have even used it to analyze MD trajectories. You can find it here: http://www.caver.cz/ -Yarrow On Fri, Jun 27, 2014 at 4:00 AM, Reza Khayat rkha...@ccny.cuny.edu wrote: Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the hypothesized binding site. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org
Re: [ccp4bb] solvent exposed
Hey Jeff, Why not try the command line version of CAVER. It is easily adjustable and provides nice figures of solvent accessibility for Pymol. It also prints out a ton of stats in the log files if you want numbers. -Yarrow On Wed, Jun 25, 2014 at 1:10 PM, Jeff Holden hold...@uci.edu wrote: Experts, I would like to compare the substrate binding site of two homologous proteins. Based on crystal structures it is clear that the substrate binding site of protein A is more solvent exposed then protein B. Is there a way to measure the solvent exposure of the substrate in protein A and B? Or perhaps you have an additional suggestion for making a structural comparison (besides what seems obvious...noting the differences in non-covalent interactions)? Thanks, Jeff
[ccp4bb] stalled refinement after MR solution
Hello CCP4 community, I am stumped and would love some help. I have a molecular replacement solution that has Rfree stuck around 40% while Rwork is aorund 30%. The model is actually the same enzyme with a similar inhibitor bound. Relevant information is below. -Yarrow I have solved a structure in a P21 spacegroup: 51.53 88.91 89.65, beta = 97.1. Processing stats (XDS) are very good with low Rmerge (~5% overall) and good completeness. I don't think twinning is an option with these unit cell dimensions. My data was highly aniosotropic. I ran the data through the UCLA anisotropic server to scale in the B- direction ( http://services.mbi.ucla.edu/anisoscale/) I get a small (a little over 5) patterson peak suggesting there is not much t-NCS to worry about. However, the output structure does have 2 fold symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS in a P21 space group with two monomers related by a 2-fold axis. I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32. rota_matrix -0.9860 -0.1636 -0.0309 rota_matrix -0.16590.95110.2605 rota_matrix -0.01320.2620 -0.9650 tran_orth 34.3310 -24.0033 107.0457 center_orth 15.76077.2426 77.7512 *Phaser stats:* * SOLU SET RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745 LLG=4947*
Re: [ccp4bb] stalled refinement after MR solution
Sorry Dale. I left that out. I thought that it was almost parallel or very close. I guess that is not enough. This is after all an exact science. My fault. On Thu, May 8, 2014 at 10:54 AM, Dale Tronrud d...@daletronrud.com wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 By the way... I did not say what you claim I said. I said there is always translational ncs in P21 when there is a ncs 2-fold PARALLEL to the crystallographic screw. Your 2-fold appears to be off-set from the screw axis enough that Phaser is not detecting tNCS, which means it is not strong enough for it to be a problem for Phaser. Whatever your problem is, it is not translational NCS. Is there anyone in your area that could help you? In situations like this you need to go back to square one and reevaluate every decision you made starting from the images. This is nearly impossible to do remotely. We do not know your protein. We do not know how your crystal relates to the crystal which you are using as your MR probe. All of the thousand things that can go wrong are unknown to us. Unless you are willing to dump your entire project onto the BB there is little we can do for you. Dale On 05/08/2014 10:11 AM, Yarrow Madrona wrote: Hello CCP4 community, I am stumped and would love some help. I have a molecular replacement solution that has Rfree stuck around 40% while Rwork is aorund 30%. The model is actually the same enzyme with a similar inhibitor bound. Relevant information is below. -Yarrow I have solved a structure in a P21 spacegroup: 51.53 88.91 89.65, beta = 97.1. Processing stats (XDS) are very good with low Rmerge (~5% overall) and good completeness. I don't think twinning is an option with these unit cell dimensions. My data was highly aniosotropic. I ran the data through the UCLA anisotropic server to scale in the B- direction (http://services.mbi.ucla.edu/anisoscale/) I get a small (a little over 5) patterson peak suggesting there is not much t-NCS to worry about. However, the output structure does have 2 fold symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS in a P21 space group with two monomers related by a 2-fold axis. I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32. rota_matrix -0.9860 -0.1636 -0.0309 rota_matrix -0.1659 0.95110.2605 rota_matrix -0.01320.2620 -0.9650 tran_orth 34.3310 -24.0033 107.0457 center_orth 15.76077.2426 77.7512 * * *Phaser stats:* * SOLU SET RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745 LLG=4947* * * -BEGIN PGP SIGNATURE- Version: GnuPG v2.0.14 (GNU/Linux) iEYEARECAAYFAlNrxPAACgkQU5C0gGfAG11ivgCglp//CaOwgKyP3j73GRwH0tX8 n28AnijNVEjm/wXz19Hn8EEdyatTpedw =aOOF -END PGP SIGNATURE-
Re: [ccp4bb] stalled refinement after MR solution
Hi Randy, Again, sorry about the mis-quote. From a quick look I thought the NCS 2-fold was parallel or close enough. I didn't know that 10 degrees would change the patterson that much. I have tried refinement with both sets of data and get the same results. I will investigate the space group assignment again. On Thu, May 8, 2014 at 12:40 PM, Randy Read rj...@cam.ac.uk wrote: Hi Yarrow, If Dale said that, he probably wasn’t saying what he meant clearly enough! The NCS 2-fold axis has to be parallel to the crystallographic 2-fold (screw) axis to generate tNCS. In your case, the NCS is a 2-fold approximately parallel to the y-axis, but it’s nearly 9 degrees away from being parallel to y. That explains why the Patterson peak is so small, and there will be very little disruption from the statistical effects of tNCS. The anisotropy could be an issue. It might be interesting to look at the R-factors for the stronger subset of the data. It can make sense to apply an elliptical cutoff of the data using the anisotropy server (though Garib says that having systematically incomplete data can create problems for Refmac), but I hope you’re not using the anisotropically scaled data for refinement. The determination of the anisotropic B-factors by Phaser without a model (underlying the anisotropy server) will not be as accurate as what Refmac or phenix.refine can do with a model. Finally, as Phil Evans always says, the space group is just a hypothesis, so you should always be willing to go back and look at the evidence for the space group if something doesn’t work as expected. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 8 May 2014, at 18:11, Yarrow Madrona amadr...@uci.edu wrote: Hello CCP4 community, I am stumped and would love some help. I have a molecular replacement solution that has Rfree stuck around 40% while Rwork is aorund 30%. The model is actually the same enzyme with a similar inhibitor bound. Relevant information is below. -Yarrow I have solved a structure in a P21 spacegroup: 51.53 88.91 89.65, beta = 97.1. Processing stats (XDS) are very good with low Rmerge (~5% overall) and good completeness. I don't think twinning is an option with these unit cell dimensions. My data was highly aniosotropic. I ran the data through the UCLA anisotropic server to scale in the B- direction ( http://services.mbi.ucla.edu/anisoscale/) I get a small (a little over 5) patterson peak suggesting there is not much t-NCS to worry about. However, the output structure does have 2 fold symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS in a P21 space group with two monomers related by a 2-fold axis. I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32. rota_matrix -0.9860 -0.1636 -0.0309 rota_matrix -0.16590.95110.2605 rota_matrix -0.01320.2620 -0.9650 tran_orth 34.3310 -24.0033 107.0457 center_orth 15.76077.2426 77.7512 Phaser stats: SOLU SET RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745 LLG=4947
Re: [ccp4bb] stalled refinement after MR solution
Hi Jacob. I am worried that I would dramatically suffer in data completeness. I am not sure how reliable the data is when you are have 50% completeness. These crystals are also pretty much impossible to reproduce at the moment. On Thu, May 8, 2014 at 1:30 PM, Keller, Jacob kell...@janelia.hhmi.orgwrote: Since your search model is so good, why not go down to p1 to see what’s going on, then re-merge if necessary? JPK *From:* yarrowmadr...@gmail.com [mailto:yarrowmadr...@gmail.com] *On Behalf Of *Yarrow Madrona *Sent:* Thursday, May 08, 2014 4:29 PM *To:* Keller, Jacob *Subject:* Re: [ccp4bb] stalled refinement after MR solution I have had problems in the past with a and c cell being equal and having pseudo-merhohedral twining where the space group looked like C2221 but the true space group was P21 (near perfect 2fold NCS). But I didn't think twining was possible in this case. On Thu, May 8, 2014 at 12:43 PM, Keller, Jacob kell...@janelia.hhmi.org wrote: The b and c cell constants look remarkably similar JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Randy Read Sent: Thursday, May 08, 2014 3:41 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] stalled refinement after MR solution Hi Yarrow, If Dale said that, he probably wasn't saying what he meant clearly enough! The NCS 2-fold axis has to be parallel to the crystallographic 2-fold (screw) axis to generate tNCS. In your case, the NCS is a 2-fold approximately parallel to the y-axis, but it's nearly 9 degrees away from being parallel to y. That explains why the Patterson peak is so small, and there will be very little disruption from the statistical effects of tNCS. The anisotropy could be an issue. It might be interesting to look at the R-factors for the stronger subset of the data. It can make sense to apply an elliptical cutoff of the data using the anisotropy server (though Garib says that having systematically incomplete data can create problems for Refmac), but I hope you're not using the anisotropically scaled data for refinement. The determination of the anisotropic B-factors by Phaser without a model (underlying the anisotropy server) will not be as accurate as what Refmac or phenix.refine can do with a model. Finally, as Phil Evans always says, the space group is just a hypothesis, so you should always be willing to go back and look at the evidence for the space group if something doesn't work as expected. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 8 May 2014, at 18:11, Yarrow Madrona amadr...@uci.edu wrote: Hello CCP4 community, I am stumped and would love some help. I have a molecular replacement solution that has Rfree stuck around 40% while Rwork is aorund 30%. The model is actually the same enzyme with a similar inhibitor bound. Relevant information is below. -Yarrow I have solved a structure in a P21 spacegroup: 51.53 88.91 89.65, beta = 97.1. Processing stats (XDS) are very good with low Rmerge (~5% overall) and good completeness. I don't think twinning is an option with these unit cell dimensions. My data was highly aniosotropic. I ran the data through the UCLA anisotropic server to scale in the B- direction ( http://services.mbi.ucla.edu/anisoscale/) I get a small (a little over 5) patterson peak suggesting there is not much t-NCS to worry about. However, the output structure does have 2 fold symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS in a P21 space group with two monomers related by a 2-fold axis. I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32. rota_matrix -0.9860 -0.1636 -0.0309 rota_matrix -0.16590.95110.2605 rota_matrix -0.01320.2620 -0.9650 tran_orth 34.3310 -24.0033 107.0457 center_orth 15.76077.2426 77.7512 Phaser stats: SOLU SET RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745 LLG=4947
Re: [ccp4bb] stalled refinement after MR solution
Hi Brent, I forgot to mention the resolution and other statistics. Here they are (XDS -unmerged data P2 below). Overall completeness is 93.4(85.2)% for 2.2A. I do have some anisotropy in the b-direction. I have tried running phaser with data scaled by the UCLA anisotropy server and obtained the same results. -Yarrow SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 6.4047881487 1718 86.6% 2.7% 2.9% 4675 33.78 3.2%99.8*20* 0.895 873 4.6086442667 2852 93.5% 3.0% 3.1% 8511 30.77 3.6%99.8* 90.8561461 3.78 106593301 3598 91.7% 3.0% 3.1% 10559 31.05 3.6%99.8* 00.7891747 3.28 123983874 4261 90.9% 3.7% 3.7% 12319 25.46 4.4%99.7*-40.7791981 2.94 149704587 4766 96.2% 4.8% 4.9% 14886 20.15 5.8%99.7*-20.7792476 2.69 167185129 5292 96.9% 6.7% 6.9% 16638 15.44 8.1%99.4*-20.7672681 2.49 178425536 5722 96.7% 9.3% 9.4% 17734 11.89 11.1%98.9*-20.7552779 2.33 197436017 6133 98.1% 12.0% 12.6% 196239.27 14.3%98.4*-40.7373132 2.20 167775566 6533 85.2% 17.4% 16.9% 164206.77 21.2%96.8*-80.7492334 total 122539 38164 40875 93.4% 5.3% 5.4% 121365 17.41 6.4%99.7*-20.776 19464 NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES 134095 NUMBER OF REJECTED MISFITS 11536 NUMBER OF SYSTEMATIC ABSENT REFLECTIONS 0 NUMBER OF ACCEPTED OBSERVATIONS 122559 NUMBER OF UNIQUE ACCEPTED REFLECTIONS38170 On Thu, May 8, 2014 at 10:48 AM, Segelke, Brent W. segel...@llnl.govwrote: You didn’t report your resolution and completeness. Also, is there anisotropy in your data? Brent *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Yarrow Madrona *Sent:* Thursday, May 08, 2014 10:12 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] stalled refinement after MR solution Hello CCP4 community, I am stumped and would love some help. I have a molecular replacement solution that has Rfree stuck around 40% while Rwork is aorund 30%. The model is actually the same enzyme with a similar inhibitor bound. Relevant information is below. -Yarrow I have solved a structure in a P21 spacegroup: 51.53 88.91 89.65, beta = 97.1. Processing stats (XDS) are very good with low Rmerge (~5% overall) and good completeness. I don't think twinning is an option with these unit cell dimensions. My data was highly aniosotropic. I ran the data through the UCLA anisotropic server to scale in the B- direction ( http://services.mbi.ucla.edu/anisoscale/) I get a small (a little over 5) patterson peak suggesting there is not much t-NCS to worry about. However, the output structure does have 2 fold symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS in a P21 space group with two monomers related by a 2-fold axis. I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32. rota_matrix -0.9860 -0.1636 -0.0309 rota_matrix -0.16590.95110.2605 rota_matrix -0.01320.2620 -0.9650 tran_orth 34.3310 -24.0033 107.0457 center_orth 15.76077.2426 77.7512 *Phaser stats:* * SOLU SET RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745 LLG=4947*
Re: [ccp4bb] stalled refinement after MR solution
Thank you for the tips Brent. I will try what you suggest. There are actually multiple lattices. And I actually only processed the dominant one in XDS. On May 8, 2014 2:26 PM, Segelke, Brent W. segel...@llnl.gov wrote: Completeness looks pretty good and you have a good enough resolution that you should be able to get this eventually. A couple of things to look at to start. First, suspect your data processing. As others have already said, double check your space group. You could even do an initial sanity check by expanding your MR solution into P1 and refining with strong NCS in P1 reduced data. You will automatically get a lower R because you are introducing more parameters, but if you use strong NCS and both R and Rfree are better behaved, then something is going on with the merging (e.g., wrong space group). Look in your log and see if there are an unusual number of reflection or frames thrown out, it should be only a few percentage. If you have more than 10-15% rejected, something probably went awry. Tweak your error model and/or summing box, etc. You could also try refining to a less ambitious resolution to start, assuming anisotropy will affect the higher resolution to a greater degree. Hope this helps. Brent *From:* yarrowmadr...@gmail.com [mailto:yarrowmadr...@gmail.com] *On Behalf Of *Yarrow Madrona *Sent:* Thursday, May 08, 2014 1:50 PM *To:* Segelke, Brent W.; CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] stalled refinement after MR solution Hi Brent, I forgot to mention the resolution and other statistics. Here they are (XDS -unmerged data P2 below). Overall completeness is 93.4(85.2)% for 2.2A. I do have some anisotropy in the b-direction. I have tried running phaser with data scaled by the UCLA anisotropy server and obtained the same results. -Yarrow SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas CC(1/2) Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 6.4047881487 1718 86.6% 2.7% 2.9% 4675 33.78 3.2%99.8*20* 0.895 873 4.6086442667 2852 93.5% 3.0% 3.1% 8511 30.77 3.6%99.8* 90.8561461 3.78 106593301 3598 91.7% 3.0% 3.1% 10559 31.05 3.6%99.8* 00.7891747 3.28 123983874 4261 90.9% 3.7% 3.7% 12319 25.46 4.4%99.7*-40.7791981 2.94 149704587 4766 96.2% 4.8% 4.9% 14886 20.15 5.8%99.7*-20.7792476 2.69 167185129 5292 96.9% 6.7% 6.9% 16638 15.44 8.1%99.4*-20.7672681 2.49 178425536 5722 96.7% 9.3% 9.4% 17734 11.89 11.1%98.9*-20.7552779 2.33 197436017 6133 98.1% 12.0% 12.6% 196239.27 14.3%98.4*-40.7373132 2.20 167775566 6533 85.2% 17.4% 16.9% 164206.77 21.2%96.8*-80.7492334 total 122539 38164 40875 93.4% 5.3% 5.4% 121365 17.41 6.4%99.7*-20.776 19464 NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES 134095 NUMBER OF REJECTED MISFITS 11536 NUMBER OF SYSTEMATIC ABSENT REFLECTIONS 0 NUMBER OF ACCEPTED OBSERVATIONS 122559 NUMBER OF UNIQUE ACCEPTED REFLECTIONS38170 On Thu, May 8, 2014 at 10:48 AM, Segelke, Brent W. segel...@llnl.gov wrote: You didn’t report your resolution and completeness. Also, is there anisotropy in your data? Brent *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Yarrow Madrona *Sent:* Thursday, May 08, 2014 10:12 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] stalled refinement after MR solution Hello CCP4 community, I am stumped and would love some help. I have a molecular replacement solution that has Rfree stuck around 40% while Rwork is aorund 30%. The model is actually the same enzyme with a similar inhibitor bound. Relevant information is below. -Yarrow I have solved a structure in a P21 spacegroup: 51.53 88.91 89.65, beta = 97.1. Processing stats (XDS) are very good with low Rmerge (~5% overall) and good completeness. I don't think twinning is an option with these unit cell dimensions. My data was highly aniosotropic. I ran the data through the UCLA anisotropic server to scale in the B- direction ( http://services.mbi.ucla.edu/anisoscale/) I get a small (a little over 5) patterson peak suggesting there is not much t-NCS to worry
Re: [ccp4bb] regarding TLS records
Thanks Pavel. I was a little tired from an overnight Synchrotron run when I wrote that. Lesson: Stay off the internet when you are tired. Haha. I forgot that the TLS groups would be assigned per chain not per molecule. Thanks for the correction. I didn't know you could run find TLS groups as part of the refinement strategy. Very helpful. -Yarrow On Tue, Apr 15, 2014 at 8:36 PM, Pavel Afonine pafon...@gmail.com wrote: Hello Yarrow, in some refinement software (phenix.refine), if you run TLS refinement and you don't specify the TLS groups, the entire structure is considered one TLS group if you use TLS parameterization and do not specify TLS groups, two scenarios are possible: a) each chain will be treated as one TLS group, b) if you define tls.find_automatically=true (or check appropriate box in the GUI) it will find TLS groups as part of refinement similarly to TLSMD but ~100 times faster. This may be why the annotators could not find the TLS groups. In any case, if TLS refinement was used, then TLS matrices defining refined TLS parameters are always present in PDB file header (in REMARK 3 records). In case of just one group there will be just one set of T,L and S. Finally, if you've done TLS refinement and decided not to use TLS in the next refinement you do not need to remove ANISOU records manually - the program will figure this out. All the best, Pavel
Re: [ccp4bb] regarding TLS records
Hi Vipin, I'm not sure what software you are running or your refinement strategy. However, in some refinement software (phenix.refine), if you run TLS refinement and you don't specify the TLS groups, the entire structure is considered one TLS group which is not helpful. This may be why the annotators could not find the TLS groups. Also, If at any point you refined using TLS groups or anisotropically, you will have ANISOU records in your PDB. However in some refinement software (phenix.refine), if you decide not to refine using either of these strategies after having done so you need to remove the ANISOU records (This may be done for you in new versions). -Yarrow On Sunday, April 13, 2014, vipin kashyap vipinpatel@gmail.com wrote: Dear All I have deposited a PDB and received a mail for the data annotation staff regarding TLS records. The message is ANISOU records exists but no TLS records (resolution=3.30A). Please send us the TLS records. I want to know how to generate this TLS records. Thanks in advance.
[ccp4bb]
Hi Bjorn, Thanks for your example. Those are some big gaps to be sure. On Wed, Mar 19, 2014 at 1:38 PM, Bjørn Panyella Pedersen bj...@msg.ucsf.edu wrote: Hi Yarrow, That solution looks very reasonable to me. If you are worried about the size of your doughnut-hole, look at the packing of the latest structure we solved. :) https://dl.dropboxusercontent.com/u/5116503/4J05.png best -Bjørn -- Bjørn Panyella Pedersen Macromolecular Structure Group Dept. of Biochemistry and Biophysics University of California, San Francisco On 03/19/2014 08:58 AM, Yarrow Madrona wrote: Thank you to everyone for their input. I am posting a picture to some of the symmetry related molecules shortly. There are six dimers related by symmetry (60 degrees) with a donut hole in the middle. This was troubling to me as I have solved mostly tighter packing structures (monoclinic or orthorhombic) in the past. If expanded further there are a bunch of tightly packed donut holes (though I didn't show these). I want to know if this is really a viable solution. The crystals are huge (300microns X 300microns) and this would maybe explain why they are only diffracting to 3.2 angstroms. Thank you! https://www.dropbox.com/s/r01u37owbkz9pon/donut.png -Yarrow On Wed, Mar 19, 2014 at 6:59 AM, Yarrow Madrona amadr...@uci.edu mailto:amadr...@uci.edu wrote: Yes in the first couple of rounds of refinement it refines very well for a 3.2 angstrom structure (Now at 30%/24% Rfree/R). Everything Packs contiguously except for a donut hole in between six dimers that are related by symmetry. Trying to put a molecule there disrupts the symmetry and leads to clashes. I have a synchrotron trip next week, hopefully this should help clear things up a bit. On Wed, Mar 19, 2014 at 12:17 AM, Eleanor Dodson eleanor.dod...@york.ac.uk mailto:eleanor.dod...@york.ac.uk wrote: I think you have solved it! That is an excellent LLG and if you can't see anything else in the map, then there s prob. not another molecule. Does it refine? If you look at the maps following refinement any missing features should become more obvious. Solvent content of 65% is not uncommon. Eleanor On 19 Mar 2014, at 03:46, Yarrow Madrona wrote: Hello CCP4 Users, I recently collected data in-house on an Raxis IV and am trying to solve a 3.2 angstrom structure. I have obtained only partial solutions using Phaser and would like some help. I believe I only have two molecules in the ASU instead of three as suggested by the mathew's calculation. I believe I have two molecules in the ASU with a space group of P312 despite a high solvent content. I have outlined by line of reasoning below. 1. Indexes as primitive hexagonal 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis in the P312 space group. See this link, https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 4 molecules. 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 2 molecules. Mathews calculation for data scaled in P312: *For estimated molecular weight 44000.* *Nmol/asym Matthews Coeff %solvent P(3.20) P(tot)* ** * 1 6.8482.03 0.00 0.00* * 2 3.4264.07 0.18 0.13* * 3 2.2846.10 0.81 0.86* * 4 1.7128.13 0.01 0.01* * 5 1.3710.17 0.00 0.00* ** *Phaser Stats:* ** Partial Solution for data scaled in P312: RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 TFZ==30.3 6. No peaks in patterson map (No translational symmetry). 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 bromo-phenyl Immidazole) despite not including them in the search model. 6. There is only one black hole where it would be possible place another subunit but there is not much interpretable density and the symmetry of the space group would be broken if this was done. Six Dimers are arranged around this hole. I can post a picture if anyone wants to see it. 6. Early refinement of the partial solution gives
[ccp4bb]
Hi Savvas, Native PAGE and DLS suggest a monodisperse monomer in solution. On Wed, Mar 19, 2014 at 12:12 PM, Savvas Savvides savvas.savvi...@ugent.bewrote: Dear Yarrow your toroidal structure suggests that the protein may actually have the propensity to assemble as such in solution, hinting a connection to a biologically relevant state. Do you have any experimental information that it does so? e.g. via SAXS, MALS, native PAGE etc.? best regards Savvas Savvas Savvides Unit for Structural Biology, L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html On 19 Mar 2014, at 16:58, Yarrow Madrona amadr...@uci.edu wrote: Thank you to everyone for their input. I am posting a picture to some of the symmetry related molecules shortly. There are six dimers related by symmetry (60 degrees) with a donut hole in the middle. This was troubling to me as I have solved mostly tighter packing structures (monoclinic or orthorhombic) in the past. If expanded further there are a bunch of tightly packed donut holes (though I didn't show these). I want to know if this is really a viable solution. The crystals are huge (300microns X 300microns) and this would maybe explain why they are only diffracting to 3.2 angstroms. Thank you! https://www.dropbox.com/s/r01u37owbkz9pon/donut.png -Yarrow On Wed, Mar 19, 2014 at 6:59 AM, Yarrow Madrona amadr...@uci.edu wrote: Yes in the first couple of rounds of refinement it refines very well for a 3.2 angstrom structure (Now at 30%/24% Rfree/R). Everything Packs contiguously except for a donut hole in between six dimers that are related by symmetry. Trying to put a molecule there disrupts the symmetry and leads to clashes. I have a synchrotron trip next week, hopefully this should help clear things up a bit. On Wed, Mar 19, 2014 at 12:17 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I think you have solved it! That is an excellent LLG and if you can't see anything else in the map, then there s prob. not another molecule. Does it refine? If you look at the maps following refinement any missing features should become more obvious. Solvent content of 65% is not uncommon. Eleanor On 19 Mar 2014, at 03:46, Yarrow Madrona wrote: Hello CCP4 Users, I recently collected data in-house on an Raxis IV and am trying to solve a 3.2 angstrom structure. I have obtained only partial solutions using Phaser and would like some help. I believe I only have two molecules in the ASU instead of three as suggested by the mathew's calculation. I believe I have two molecules in the ASU with a space group of P312 despite a high solvent content. I have outlined by line of reasoning below. 1. Indexes as primitive hexagonal 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis in the P312 space group. See this link, https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 4 molecules. 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 2 molecules. Mathews calculation for data scaled in P312: *For estimated molecular weight 44000.* *Nmol/asym Matthews Coeff %solvent P(3.20) P(tot)* ** * 1 6.8482.03 0.00 0.00* * 2 3.4264.07 0.18 0.13* * 3 2.2846.10 0.81 0.86* * 4 1.7128.13 0.01 0.01* * 5 1.3710.17 0.00 0.00* ** *Phaser Stats:* Partial Solution for data scaled in P312: RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 TFZ==30.3 6. No peaks in patterson map (No translational symmetry). 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 bromo-phenyl Immidazole) despite not including them in the search model. 6. There is only one black hole where it would be possible place another subunit but there is not much interpretable density and the symmetry of the space group would be broken if this was done. Six Dimers are arranged around this hole. I can post a picture if anyone wants to see it. 6. Early refinement of the partial solution gives an Rwork/Rfee ~ 24%/31% for a 3.2 angstrom data set. Probably over parameterized judging by the gap in R/Rfree but still better than I would guess if I had only 2/3 of the ASU composition. *My belief is that there really is only two molecules in the ASU and that there just happens to be a very large solvent channel giving a 65
[ccp4bb]
Yes, thats correct. I didn't show it but the donuts move in the direction perpendicular to the paper. On Wed, Mar 19, 2014 at 12:07 PM, Felix Frolow mbfro...@post.tau.ac.ilwrote: Very reasonable structure, I guess in the direction perpendicular to your picture you will have next donut etc. You can call this - nan-pore ;-) Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Mar 19, 2014, at 18:37 , Eleanor Dodson eleanor.dod...@york.ac.uk wrote: How pretty - I love circular molecules! Eleanor On 19 March 2014 15:58, Yarrow Madrona amadr...@uci.edu wrote: Thank you to everyone for their input. I am posting a picture to some of the symmetry related molecules shortly. There are six dimers related by symmetry (60 degrees) with a donut hole in the middle. This was troubling to me as I have solved mostly tighter packing structures (monoclinic or orthorhombic) in the past. If expanded further there are a bunch of tightly packed donut holes (though I didn't show these). I want to know if this is really a viable solution. The crystals are huge (300microns X 300microns) and this would maybe explain why they are only diffracting to 3.2 angstroms. Thank you! https://www.dropbox.com/s/r01u37owbkz9pon/donut.png -Yarrow On Wed, Mar 19, 2014 at 6:59 AM, Yarrow Madrona amadr...@uci.edu wrote: Yes in the first couple of rounds of refinement it refines very well for a 3.2 angstrom structure (Now at 30%/24% Rfree/R). Everything Packs contiguously except for a donut hole in between six dimers that are related by symmetry. Trying to put a molecule there disrupts the symmetry and leads to clashes. I have a synchrotron trip next week, hopefully this should help clear things up a bit. On Wed, Mar 19, 2014 at 12:17 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I think you have solved it! That is an excellent LLG and if you can't see anything else in the map, then there s prob. not another molecule. Does it refine? If you look at the maps following refinement any missing features should become more obvious. Solvent content of 65% is not uncommon. Eleanor On 19 Mar 2014, at 03:46, Yarrow Madrona wrote: Hello CCP4 Users, I recently collected data in-house on an Raxis IV and am trying to solve a 3.2 angstrom structure. I have obtained only partial solutions using Phaser and would like some help. I believe I only have two molecules in the ASU instead of three as suggested by the mathew's calculation. I believe I have two molecules in the ASU with a space group of P312 despite a high solvent content. I have outlined by line of reasoning below. 1. Indexes as primitive hexagonal 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis in the P312 space group. See this link, https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 4 molecules. 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 2 molecules. Mathews calculation for data scaled in P312: *For estimated molecular weight 44000.* *Nmol/asym Matthews Coeff %solvent P(3.20) P(tot)* ** * 1 6.8482.03 0.00 0.00* * 2 3.4264.07 0.18 0.13* * 3 2.2846.10 0.81 0.86* * 4 1.7128.13 0.01 0.01* * 5 1.3710.17 0.00 0.00* ** *Phaser Stats:* Partial Solution for data scaled in P312: RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 TFZ==30.3 6. No peaks in patterson map (No translational symmetry). 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 bromo-phenyl Immidazole) despite not including them in the search model. 6. There is only one black hole where it would be possible place another subunit but there is not much interpretable density and the symmetry of the space group would be broken if this was done. Six Dimers are arranged around this hole. I can post a picture if anyone wants to see it. 6. Early refinement of the partial solution gives an Rwork/Rfee ~ 24%/31% for a 3.2 angstrom data set. Probably over parameterized judging by the gap in R/Rfree but still better than I would guess if I had only 2/3 of the ASU composition. *My belief is that there really is only two molecules in the ASU and that there just
[ccp4bb]
Yes in the first couple of rounds of refinement it refines very well for a 3.2 angstrom structure (Now at 30%/24% Rfree/R). Everything Packs contiguously except for a donut hole in between six dimers that are related by symmetry. Trying to put a molecule there disrupts the symmetry and leads to clashes. I have a synchrotron trip next week, hopefully this should help clear things up a bit. On Wed, Mar 19, 2014 at 12:17 AM, Eleanor Dodson eleanor.dod...@york.ac.ukwrote: I think you have solved it! That is an excellent LLG and if you can't see anything else in the map, then there s prob. not another molecule. Does it refine? If you look at the maps following refinement any missing features should become more obvious. Solvent content of 65% is not uncommon. Eleanor On 19 Mar 2014, at 03:46, Yarrow Madrona wrote: Hello CCP4 Users, I recently collected data in-house on an Raxis IV and am trying to solve a 3.2 angstrom structure. I have obtained only partial solutions using Phaser and would like some help. I believe I only have two molecules in the ASU instead of three as suggested by the mathew's calculation. I believe I have two molecules in the ASU with a space group of P312 despite a high solvent content. I have outlined by line of reasoning below. 1. Indexes as primitive hexagonal 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis in the P312 space group. See this link, https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 4 molecules. 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 2 molecules. Mathews calculation for data scaled in P312: *For estimated molecular weight 44000.* *Nmol/asym Matthews Coeff %solvent P(3.20) P(tot)* ** * 1 6.8482.03 0.00 0.00* * 2 3.4264.07 0.18 0.13* * 3 2.2846.10 0.81 0.86* * 4 1.7128.13 0.01 0.01* * 5 1.3710.17 0.00 0.00* ** *Phaser Stats:* Partial Solution for data scaled in P312: RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 TFZ==30.3 6. No peaks in patterson map (No translational symmetry). 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 bromo-phenyl Immidazole) despite not including them in the search model. 6. There is only one black hole where it would be possible place another subunit but there is not much interpretable density and the symmetry of the space group would be broken if this was done. Six Dimers are arranged around this hole. I can post a picture if anyone wants to see it. 6. Early refinement of the partial solution gives an Rwork/Rfee ~ 24%/31% for a 3.2 angstrom data set. Probably over parameterized judging by the gap in R/Rfree but still better than I would guess if I had only 2/3 of the ASU composition. *My belief is that there really is only two molecules in the ASU and that there just happens to be a very large solvent channel giving a 65% solvent content.* *I would like help in determining whether this is likely or if I have missed something. Thank you for your help in advance!* *-Yarrow* Post Doctoral Scholar UCSF Genentech Hall, Rm N551 600 16th St., San Francisco, CA 94158-2517
[ccp4bb]
Thank you to everyone for their input. I am posting a picture to some of the symmetry related molecules shortly. There are six dimers related by symmetry (60 degrees) with a donut hole in the middle. This was troubling to me as I have solved mostly tighter packing structures (monoclinic or orthorhombic) in the past. If expanded further there are a bunch of tightly packed donut holes (though I didn't show these). I want to know if this is really a viable solution. The crystals are huge (300microns X 300microns) and this would maybe explain why they are only diffracting to 3.2 angstroms. Thank you! https://www.dropbox.com/s/r01u37owbkz9pon/donut.png -Yarrow On Wed, Mar 19, 2014 at 6:59 AM, Yarrow Madrona amadr...@uci.edu wrote: Yes in the first couple of rounds of refinement it refines very well for a 3.2 angstrom structure (Now at 30%/24% Rfree/R). Everything Packs contiguously except for a donut hole in between six dimers that are related by symmetry. Trying to put a molecule there disrupts the symmetry and leads to clashes. I have a synchrotron trip next week, hopefully this should help clear things up a bit. On Wed, Mar 19, 2014 at 12:17 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I think you have solved it! That is an excellent LLG and if you can't see anything else in the map, then there s prob. not another molecule. Does it refine? If you look at the maps following refinement any missing features should become more obvious. Solvent content of 65% is not uncommon. Eleanor On 19 Mar 2014, at 03:46, Yarrow Madrona wrote: Hello CCP4 Users, I recently collected data in-house on an Raxis IV and am trying to solve a 3.2 angstrom structure. I have obtained only partial solutions using Phaser and would like some help. I believe I only have two molecules in the ASU instead of three as suggested by the mathew's calculation. I believe I have two molecules in the ASU with a space group of P312 despite a high solvent content. I have outlined by line of reasoning below. 1. Indexes as primitive hexagonal 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis in the P312 space group. See this link, https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 4 molecules. 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 2 molecules. Mathews calculation for data scaled in P312: *For estimated molecular weight 44000.* *Nmol/asym Matthews Coeff %solvent P(3.20) P(tot)* ** * 1 6.8482.03 0.00 0.00* * 2 3.4264.07 0.18 0.13* * 3 2.2846.10 0.81 0.86* * 4 1.7128.13 0.01 0.01* * 5 1.3710.17 0.00 0.00* ** *Phaser Stats:* Partial Solution for data scaled in P312: RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 TFZ==30.3 6. No peaks in patterson map (No translational symmetry). 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 bromo-phenyl Immidazole) despite not including them in the search model. 6. There is only one black hole where it would be possible place another subunit but there is not much interpretable density and the symmetry of the space group would be broken if this was done. Six Dimers are arranged around this hole. I can post a picture if anyone wants to see it. 6. Early refinement of the partial solution gives an Rwork/Rfee ~ 24%/31% for a 3.2 angstrom data set. Probably over parameterized judging by the gap in R/Rfree but still better than I would guess if I had only 2/3 of the ASU composition. *My belief is that there really is only two molecules in the ASU and that there just happens to be a very large solvent channel giving a 65% solvent content.* *I would like help in determining whether this is likely or if I have missed something. Thank you for your help in advance!* *-Yarrow* Post Doctoral Scholar UCSF Genentech Hall, Rm N551 600 16th St., San Francisco, CA 94158-2517
[ccp4bb]
Hello CCP4 Users, I recently collected data in-house on an Raxis IV and am trying to solve a 3.2 angstrom structure. I have obtained only partial solutions using Phaser and would like some help. I believe I only have two molecules in the ASU instead of three as suggested by the mathew's calculation. I believe I have two molecules in the ASU with a space group of P312 despite a high solvent content. I have outlined by line of reasoning below. 1. Indexes as primitive hexagonal 2. Self rotation function (MolRep) gives six peaks for chi = 180. (I'm assuming chi is equivalent to kappa for Molrep) supporting the 2 fold axis in the P312 space group. See this link, https://www.dropbox.com/s/sj7c28pvuz7fei2/031414_21_rf.pdf 3. Scaling in P3 gives 6 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 4 molecules. 4. Scaling in P312 gives 3 molecules/ASU predicted by Mathew's calculation. Phaser gives solutions for only 2 molecules. Mathews calculation for data scaled in P312: *For estimated molecular weight 44000.* *Nmol/asym Matthews Coeff %solvent P(3.20) P(tot)* ** * 1 6.8482.03 0.00 0.00* * 2 3.4264.07 0.18 0.13* * 3 2.2846.10 0.81 0.86* * 4 1.7128.13 0.01 0.01* * 5 1.3710.17 0.00 0.00* ** *Phaser Stats:* Partial Solution for data scaled in P312: RFZ=11.7 TFZ=27.4 PAK=0 LLG=528 TFZ==18.8 RF++ TFZ=52.1 PAK=0 LLG=2374 TFZ==30.3 6. No peaks in patterson map (No translational symmetry). 5. Very strong 2fo-fc density for two ligands in each monomer (Heme and 4 bromo-phenyl Immidazole) despite not including them in the search model. 6. There is only one black hole where it would be possible place another subunit but there is not much interpretable density and the symmetry of the space group would be broken if this was done. Six Dimers are arranged around this hole. I can post a picture if anyone wants to see it. 6. Early refinement of the partial solution gives an Rwork/Rfee ~ 24%/31% for a 3.2 angstrom data set. Probably over parameterized judging by the gap in R/Rfree but still better than I would guess if I had only 2/3 of the ASU composition. *My belief is that there really is only two molecules in the ASU and that there just happens to be a very large solvent channel giving a 65% solvent content.* *I would like help in determining whether this is likely or if I have missed something. Thank you for your help in advance!* *-Yarrow* Post Doctoral Scholar UCSF Genentech Hall, Rm N551 600 16th St., San Francisco, CA 94158-2517
[ccp4bb] [CCP4] Converting ShelX .phs to mtz
Hello CCP4 users, I seem to be loosing data (50%) when converting shelX .phs reflelection file to mtz format using F2MTZ. I think it has something to do with loosing my anamolous data. Is it possible to merge the anamolous data before conversion or to tell F2MTZ to include it. These are my labels: H K L I FOM PHI SIGI Thank you Yarrow Madrona
[ccp4bb] shelx anamalous data
I'm sorry, I have not used shelx before and didn't realize in my last post that the anamolous data is kept separate. I am planning on converting both the mysad.phs and mysad.pha to mtz files and then merge them. However, I am not sure of the column lables in mysad.pha. Does anyone know how to get this info? -Yarrow -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] [CCP4] Converting ShelX .phs to mtz
Hi Yury, Your idea seemed to work well, however I now have 50% completeness in phi and 100% completeness in Intensities. I am not sure where I am going wrong. H K L IMEAN FOM PHI SIGIMEAN Hi Yarrow, You may consider producing mtz file from your original .sca or .hkl file first. Then take the .mtz file you have created from a .phs file and merge the two using CAD in CCP4. I assume you need only phase information from you .phs file, so select only two sets of parameters - FOM and PHIB - from there and take the rest from the first file. The result will be a complete merge.mtz with all original data plus phase information. This will assure you do not loose anything on the way. Good luck, Yury From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yarrow Madrona [amadr...@uci.edu] Sent: Thursday, November 14, 2013 3:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] [CCP4] Converting ShelX .phs to mtz Hello CCP4 users, I seem to be loosing data (50%) when converting shelX .phs reflelection file to mtz format using F2MTZ. I think it has something to do with loosing my anamolous data. Is it possible to merge the anamolous data before conversion or to tell F2MTZ to include it. These are my labels: H K L I FOM PHI SIGI Thank you Yarrow Madrona -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Thanks Randy, The a cell edge is 56.118, so not exactly half of 30.86. I am currently refining using NCS cartesian restraints as Phil suggested. Then I will visually inspect the model as well as compare b-factors. Thanks for your suggestions, I will look into them. -Yarrow Hi, It's not uncommon for pseudosymmetry to be found together with twinning, and the presence of pseudosymmetry perturbs the statistics used to test for twinning. In that circumstance, as Phil suggests, a really good way to see what is going on is to take the lower symmetry solution and see if it really obeys higher symmetry, but you can do that either with coordinates or calculated structure factors. Your NCS matrix specifies a 2-fold rotation around an axis that is about 1 degree off the x axis. Whether that 1 degree matters or not depends on how precisely the molecules are placed in the MR solution. If 30.8649 is precisely half of the a-cell edge, then this corresponds to a 2(1) screw axis, but whether or not that is crystallographic depends on whether the origin of that axis is in the right place relative to the 2(1) you're assuming is correct. Working all that out from coordinates can be a bit of a challenge, which will really have you hitting the books! The other way we've approached this kind of problem is to take the Fcalcs from an MR model (usually solved in P1 if possible to avoid making any assumptions about which symmetry operators are correct) and then use either pointless or xtriage to see if those Fcalcs obey higher symmetry. Another good approach is to use the zanuda program in the CCP4 suite, which is designed to answer questions about pseudosymmetry and other related problems. Good luck! Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 15 Oct 2013, at 22:31, Yarrow Madrona amadr...@uci.edu wrote: Thank you Dale, I will hit-the-books to better the rotation matrices. I am concluding from all of this that the space group is indeed P212121. So I still wonder why I have some outliers in the intensity stats for the two additional screw axis and why R and Rfree both drop by 5% when I apply a twin law to refinement in P21. Thanks for your help. -Yarrow Since Phil is no doubt in bed, I'll answer the easier part. Your second matrix is nearly the equivalent position (x,-y,-z). This is a two-fold rotation about the x axis. You also have a translation of about 31 A along x so if your A cell edge is about 62 A you have a 2_1 screw. Dale Tronrud On 10/15/2013 12:29 PM, Yarrow Madrona wrote: Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron
[ccp4bb] A case of perfect pseudomerehedral twinning?
1.798 0.1302E+03 0.8407E+02 1.55 1 0 600 1.768 0.5786E+03 0.8353E+02 6.93 1 0 610 1.739 0.1614E+02 0.7909E+02 0.20 1 30018.729 0.4087E+01 0.4877E+01 0.84 2 40014.047 0.4620E+04 0.1563E+0329.57 1 50011.238 0.2628E+02 0.7980E+01 3.29 2 700 8.027 0.1388E+03 0.1199E+0211.57 2 800 7.023 0.4997E+04 0.1207E+0341.41 2 900 6.243 -0.3491E+01 0.1404E+02-0.25 2 1000 5.619 0.7205E+03 0.2461E+0229.28 2 1600 3.512 0.7500E+03 0.3599E+0220.84 2 1700 3.305 0.2017E+03 0.2954E+02 6.83 2 1800 3.122 0.8955E+04 0.2177E+0341.14 2 1900 2.957 0.2619E+02 0.3592E+02 0.73 1 2000 2.809 0.5455E+03 0.4308E+0212.66 1 2100 2.676 0.4300E+01 0.3632E+02 0.12 1 2200 2.554 0.6695E+03 0.4925E+0213.59 1 2300 2.443 0.1916E+02 0.4766E+02 0.40 1 2400 2.341 0.7016E+03 0.6038E+0211.62 1 2500 2.248 -0.6989E+02 0.5147E+02-1.36 1 2600 2.161 0.9047E+03 0.6482E+0213.96 1 2700 2.081 0.6900E+02 0.5447E+02 1.27 1 2800 2.007 0.4734E+03 0.5668E+02 8.35 1 2900 1.938 0.1150E+03 0.5947E+02 1.93 1 Res OBS UNIQUE POS COMP R(obs) R(exp)I/Sig Rmeas CC1/2 4.96 13512 3348 3568 93.8% 2.5% 2.5% 13461 47.52 2.8% 99.9* 2 0.8592641 3.56 24267 5885 5938 99.1% 2.4% 2.5% 24249 48.26 2.7% 99.9* -20.8094704 2.93 22330 7401 7551 98.0% 2.8% 2.7% 22018 34.01 3.4% 99.8* 30.8732097 2.54 19890 8751 8971 97.5% 3.5% 3.5% 19338 22.53 4.7% 99.6* 20.968 232 2.28 20645 9773 10080 97.0% 4.6% 4.7% 19775 16.89 6.2% 99.4* -990.919 2 2.08 21352 10332 11093 93.1% 6.1% 6.5% 20047 12.69 8.3% 99.0* 00.000 0 1.93 21331 10559 12116 87.1% 9.4% 10.2% 19606 8.51 12.7% 97.7* 00.000 0 1.80 19417 9965 12977 76.8% 15.5% 17.2% 17229 5.19 21.1% 94.5* 00.000 0 1.70 11478 6792 13746 49.4% 22.2% 24.3% 8608 3.24 30.2% 88.2* 00.000 0 total174222 72806 86040 84.6% 3.5% 3.7% 164331 18.57 4.4% 99.8* 10.8409676 Systematic absences suggest one 2(1) fold rotation symetry axis Almost 2 more but there are a number of violations. Very low res data complete to ~82% (10-20A) twinning analysis Accentric I**2/I**2 = 1.97 (untwinned 2.0, pefect twin 1.5) F**2/F**2 =0.792 (0.785 untwinned, 0.885 perfect twin) |L| = 0.490 (0.5 expected, 0.375, perfect twin) L**2 = 0.324 (0.33 expected, perfect twin .20) possible twin law h, -k, -l Wilson plot - estimated B factor = 10.6 2 molec /asu 1 off origin patterson peak Height: 4.974 Molecular Replacement (Phaser, automated) and refinement Solution in P21 when processed in P2 Refinement without twin law: Rwork = 0.3387 Rfree = 0.3650 ~85% Ramachandran favored (+ missing part of helix) Refinement with twin law: Rwork = 0.2994 Rfree = 0.3214 -- Yarrow Madrona Postdoctoral Scholar Ortiz De Montellano Lab Pharmeceutical Chemistry Dept. University of California, San Francisco Genentech Hall, Rm N556
[ccp4bb] A case of perfect pseudomerehedral twinning?
7.023 0.4997E+04 0.1207E+03 41.41 2 900 6.243 -0.3491E+01 0.1404E+02 -0.25 2 1000 5.619 0.7205E+03 0.2461E+0229.28 2 1600 3.512 0.7500E+03 0.3599E+0220.84 2 1700 3.305 0.2017E+03 0.2954E+02 6.83 2 1800 3.122 0.8955E+04 0.2177E+0341.14 2 1900 2.957 0.2619E+02 0.3592E+02 0.73 1 2000 2.809 0.5455E+03 0.4308E+0212.66 1 2100 2.676 0.4300E+01 0.3632E+02 0.12 1 2200 2.554 0.6695E+03 0.4925E+0213.59 1 2300 2.443 0.1916E+02 0.4766E+02 0.40 1 2400 2.341 0.7016E+03 0.6038E+0211.62 1 2500 2.248 -0.6989E+02 0.5147E+02-1.36 1 2600 2.161 0.9047E+03 0.6482E+02 13.96 1 2700 2.081 0.6900E+02 0.5447E+02 1.27 1 2800 2.007 0.4734E+03 0.5668E+02 8.35 1 2900 1.938 0.1150E+03 0.5947E+02 1.93 1 Res OBS UNIQUE POS COMP R(obs) R(exp)I/Sig Rmeas CC1/2 4.96 13512 3348 3568 93.8% 2.5% 2.5% 13461 47.52 2.8% 99.9* 2 0.8592641 3.56 24267 5885 5938 99.1% 2.4% 2.5% 24249 48.26 2.7% 99.9* -20.8094704 2.93 22330 7401 7551 98.0% 2.8% 2.7% 22018 34.01 3.4% 99.8* 30.8732097 2.54 19890 8751 8971 97.5% 3.5% 3.5% 19338 22.53 4.7% 99.6* 20.968 232 2.28 20645 9773 10080 97.0% 4.6% 4.7% 19775 16.89 6.2% 99.4* -990.919 2 2.08 21352 10332 11093 93.1% 6.1% 6.5% 20047 12.69 8.3% 99.0* 00.000 0 1.93 21331 10559 12116 87.1% 9.4% 10.2% 19606 8.51 12.7% 97.7* 00.000 0 1.80 19417 9965 12977 76.8% 15.5% 17.2% 17229 5.19 21.1% 94.5* 00.000 0 1.70 11478 6792 13746 49.4% 22.2% 24.3% 8608 3.24 30.2% 88.2* 00.000 0 total174222 72806 86040 84.6% 3.5% 3.7% 164331 18.57 4.4% 99.8* 10.8409676 Systematic absences suggest one 2(1) fold rotation symetry axis Almost 2 more but there are a number of violations. Very low res data complete to ~82% (10-20A) twinning analysis Accentric I**2/I**2 = 1.97 (untwinned 2.0, pefect twin 1.5) F**2/F**2 =0.792 (0.785 untwinned, 0.885 perfect twin) |L| = 0.490 (0.5 expected, 0.375, perfect twin) L**2 = 0.324 (0.33 expected, perfect twin .20) possible twin law h, -k, -l Wilson plot - estimated B factor = 10.6 2 molec /asu 1 off origin patterson peak Height: 4.974 Molecular Replacement (Phaser, automated) and refinement Solution in P21 when processed in P2 Refinement without twin law: Rwork = 0.3387 Rfree = 0.3650 ~85% Ramachandran favored (+ missing part of helix) Refinement with twin law: Rwork = 0.2994 Rfree = 0.3214 -- Yarrow Madrona Postdoctoral Scholar Ortiz De Montellano Lab Pharmeceutical Chemistry Dept. University of California, San Francisco Genentech Hall, Rm N556 -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
[ccp4bb] A case of perfect pseudomerehedral twinning?
0.2619E+02 0.3592E+02 0.73 1 2000 2.809 0.5455E+03 0.4308E+0212.66 1 2100 2.676 0.4300E+01 0.3632E+02 0.12 1 2200 2.554 0.6695E+03 0.4925E+0213.59 1 2300 2.443 0.1916E+02 0.4766E+02 0.40 1 2400 2.341 0.7016E+03 0.6038E+0211.62 1 2500 2.248 -0.6989E+02 0.5147E+02-1.36 1 2600 2.161 0.9047E+03 0.6482E+02 13.96 1 2700 2.081 0.6900E+02 0.5447E+02 1.27 1 2800 2.007 0.4734E+03 0.5668E+02 8.35 1 2900 1.938 0.1150E+03 0.5947E+02 1.93 1 Res OBS UNIQUE POS COMP R(obs) R(exp)I/Sig Rmeas CC1/2 4.96 13512 3348 3568 93.8% 2.5% 2.5% 13461 47.52 2.8% 99.9* 2 0.8592641 3.56 24267 5885 5938 99.1% 2.4% 2.5% 24249 48.26 2.7% 99.9* -20.8094704 2.93 22330 7401 7551 98.0% 2.8% 2.7% 22018 34.01 3.4% 99.8* 30.8732097 2.54 19890 8751 8971 97.5% 3.5% 3.5% 19338 22.53 4.7% 99.6* 20.968 232 2.28 20645 9773 10080 97.0% 4.6% 4.7% 19775 16.89 6.2% 99.4* -990.919 2 2.08 21352 10332 11093 93.1% 6.1% 6.5% 20047 12.69 8.3% 99.0* 00.000 0 1.93 21331 10559 12116 87.1% 9.4% 10.2% 19606 8.51 12.7% 97.7* 00.000 0 1.80 19417 9965 12977 76.8% 15.5% 17.2% 17229 5.19 21.1% 94.5* 00.000 0 1.70 11478 6792 13746 49.4% 22.2% 24.3% 8608 3.24 30.2% 88.2* 00.000 0 total174222 72806 86040 84.6% 3.5% 3.7% 164331 18.57 4.4% 99.8* 10.8409676 Systematic absences suggest one 2(1) fold rotation symetry axis Almost 2 more but there are a number of violations. Very low res data complete to ~82% (10-20A) twinning analysis Accentric I**2/I**2 = 1.97 (untwinned 2.0, pefect twin 1.5) F**2/F**2 =0.792 (0.785 untwinned, 0.885 perfect twin) |L| = 0.490 (0.5 expected, 0.375, perfect twin) L**2 = 0.324 (0.33 expected, perfect twin .20) possible twin law h, -k, -l Wilson plot - estimated B factor = 10.6 2 molec /asu 1 off origin patterson peak Height: 4.974 Molecular Replacement (Phaser, automated) and refinement Solution in P21 when processed in P2 Refinement without twin law: Rwork = 0.3387 Rfree = 0.3650 ~85% Ramachandran favored (+ missing part of helix) Refinement with twin law: Rwork = 0.2994 Rfree = 0.3214 -- Yarrow Madrona Postdoctoral Scholar Ortiz De Montellano Lab Pharmeceutical Chemistry Dept. University of California, San Francisco Genentech Hall, Rm N556 -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697 -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron density between the two halves of the asymmetric unit in P21 then that's a basis for describing it as P21 rather than P212121. However if the two halves look nearly identical, down to equivalent water molecule densities, then you've got no experimental evidence that P21 with 2x molecules generates a better model than P212121 than 1x molecules. An averaging program would show very high correlation between the two halves of the P21 asymmetric unit if it was really P212121 and you could overlap the maps corresponding to the different monomers using those programs. Phil Jeffrey Princeton -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Thank you Dale, I will hit-the-books to better the rotation matrices. I am concluding from all of this that the space group is indeed P212121. So I still wonder why I have some outliers in the intensity stats for the two additional screw axis and why R and Rfree both drop by 5% when I apply a twin law to refinement in P21. Thanks for your help. -Yarrow Since Phil is no doubt in bed, I'll answer the easier part. Your second matrix is nearly the equivalent position (x,-y,-z). This is a two-fold rotation about the x axis. You also have a translation of about 31 A along x so if your A cell edge is about 62 A you have a 2_1 screw. Dale Tronrud On 10/15/2013 12:29 PM, Yarrow Madrona wrote: Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron density between the two halves of the asymmetric unit in P21 then that's a basis for describing it as P21 rather than P212121. However if the two halves look nearly identical, down to equivalent water molecule densities, then you've got no experimental evidence that P21 with 2x molecules generates a better model than P212121 than 1x molecules. An averaging program would show very high correlation between the two halves of the P21 asymmetric unit if it was really P212121 and you could overlap the maps corresponding to the different monomers using those programs. Phil Jeffrey Princeton -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] How to compare B-factors between structures?
James, Thank you for your help. I appreciate the very thorough explanation. I have never heard of noise although I have produced a 'kicked' map in phenix and refined the structure using those maps..though I guess this is different. I will try it. Thanks. -Yarrow Formally, the best way to compare B factors in two structures with different average B is to add a constant to all the B factors in the low-B structure until the average B factor is the same in both structures. Then you can compare apples to apples as it were. The extra B being added is equivalent to blurring the more well-ordered map to make it match the less-ordered one. Subtracting a B factor from the less-ordered structure is sharpening, and the reason why you shouldn't do that here is because you'd be assuming that a sharpened map has just as much structural information as the better diffracting crystal, and that's obviously no true (not as many spots). In reality, your comparison will always be limited by the worst-resolution data you have. Another reason to add rather than subtract a B factor is because B factors are not really linear with anything sensible. Yes, B=50 is more disordered than B=25, but is it twice as disordered? That depends on what you mean by disorder, but no matter how you look at it, the answer is generally no. One way to define the degree of disorder is the volume swept out by the atom's nucleus as it vibrates (or otherwise varies from cell to cell). This is NOT proportional to the B-factor, but rather the 3/2 power of the B factor. Yes, 3/2 power. The value of B, is proportional to the SQUARE of the width of the probability distribution of the nucleus, so to get the volume of space swept out by it you have to take the square root to get something proportional the the width and then you take the 3rd power to get something proportional to the volume. An then, of course, if you want to talk about the electron cloud (which is what x-rays see) and not the nuclear position (which you can only see if you are a neutron person), then you have to add a B factor of about 8 to every atom to account for the intrinsic width of the electron cloud. Formally, the B factor is convoluted with the intrinsic atomic form factor, but a native B factor of 8 is pretty close for most atoms. For those of you who are interested in something more exact than proportional the equation for the nuclear probability distribution generated by a given B factor is: kernel_B(r) = (4*pi/B)^1.5*exp(-4*pi^2/B*r^2) where r is the distance from the average position (aka the x-y-z coordinates in the PDB file). Note that the width of this distribution of atomic positions is not really an error bar, it is a range. There's a difference between an atom actually being located in a variety of places vs not knowing the centroid of all these locations. Remember, you're averaging over trillions of unit cells. If you collect a different dataset from a similar crystal and re-refine the structure the final x-y-z coordinate assigned to the atom will not change all that much. The full-width at half-maximum (FWHM) of this kernel_B distribution is: fwhm = 0.1325*sqrt(B) and the probability of finding the nucleus within this radius is actually only about 29%. The radius that contains the nucleus half the time is about 1.3 times wider, or: r_half = 0.1731*sqrt(B) That is, for B=25, the atomic nucleus is within 0.87 A of its average position 50% of the time (a volume of 2.7 A^3). Whereas for B=50, it is within 1.22 A 50% of the time (7.7 A^3). Note that although B=50 is twice as big as B=25, the half-occupancy radius 0.87 A is not half as big as 1.22 A, nor are the volumes 2.7 and 7.7 A^3 related by a factor of two. Why is this important for comparing two structures? Since the B factor is non-linear with disorder, it is important to have a common reference point when comparing them. If the low-B structure has two atoms with B=10 and B=15 with average overall B=12, that might seem to be significant (almost a factor of two in the half-occupancy volume) but if the other structure has an average B factor of 80, then suddenly 78 vs 83 doesn't seem all that different (only a 10% change). Basically, a difference that would be significant in a high-resolution structure is washed out by the overall crystallographic B factor of the low-resolution structure in this case. Whether or not a 10% difference is significant depends on how accurate you think your B factors are. If you kick your coordinates (aka using noise in PDBSET) and re-refine, how much do the final B factors change? -James Holton MAD Scientist On 2/25/2013 12:08 PM, Yarrow Madrona wrote: Hello, Does anyone know a good method to compare B-factors between structures? I would like to compare mutants to a wild-type structure. For example, structure2 has a higher B-factor for residue X but how can I show
[ccp4bb] How to compare B-factors between structures?
Hello, Does anyone know a good method to compare B-factors between structures? I would like to compare mutants to a wild-type structure. For example, structure2 has a higher B-factor for residue X but how can I show that this is significant if the average B-factor is also higher? Thank you for your help. -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] How to compare B-factors between structures?
Hello, Does anyone know a good method to compare B-factors between structures? I would like to compare mutants to a wild-type structure. For example, structure2 has a higher B-factor for residue X but how can I show that this is significant if the average B-factor is also higher? Thank you for your help. -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697 -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] How to compare B-factors between structures?
Thanks Nat, I was planning on plotting B-factors vs. residue anyway, so maybe this will save me time. I will take a look. -Yarrow Not a CCP4 solution, but the structure comparison program in the Phenix GUI will plot B-factors for different structures of the same protein. (I am happy to make additions or modifications to this, but so far I haven't received much feedback.) -Nat On Mon, Feb 25, 2013 at 12:08 PM, Yarrow Madrona amadr...@uci.edu wrote: Hello, Does anyone know a good method to compare B-factors between structures? I would like to compare mutants to a wild-type structure. For example, structure2 has a higher B-factor for residue X but how can I show that this is significant if the average B-factor is also higher? Thank you for your help. -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697 -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] How to compare B-factors between structures?
Thank you Manfred, To be honest I have tried to understand the Pearson linear CC since reading the nature crystallography methods paper by Karplus and diederichs, but I am having trouble. I do not clearly understand what it represents. Do you know any good resources that could help me out? -Yarrow If you have two sets of numbers which correspond ideally, you should calculate a correlation coefficient, to be precise a Pearson linear CC. This is independent of scaling. If you want to compare mutant vs native, you probably have to calculate residue average B-factors, because you won't have the exact same number of atoms. Best, Manfred On 25.02.2013 21:08, Yarrow Madrona wrote: Hello, Does anyone know a good method to compare B-factors between structures? I would like to compare mutants to a wild-type structure. For example, structure2 has a higher B-factor for residue X but how can I show that this is significant if the average B-factor is also higher? Thank you for your help. -- Dr. Manfred. S. Weiss Helmholtz-Zentrum Berlin für Materialien und Energie Macromolecular Crystallography (HZB-MX) Albert-Einstein-Str. 15 D-12489 Berlin GERMANY Fon: +49-30-806213149 Fax: +49-30-806214975 Web: http://www.helmholtz-berlin.de/bessy-mx Email: mswe...@helmholtz-berlin.de Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V. Aufsichtsrat: Vorsitzender Prof. Dr. Dr. h.c. mult. Joachim Treusch, stv. Vorsitzende Dr. Beatrix Vierkorn-Rudolph Geschäftsführung: Prof. Dr. Anke Rita Kaysser-Pyzalla, Thomas Frederking Sitz Berlin, AG Charlottenburg, 89 HRB 5583 Postadresse: Hahn-Meitner-Platz 1 D-14109 Berlin http://www.helmholtz-berlin.de -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
[ccp4bb] Does Scala merge anomalous/non-anomalous?
Hello CCP4 users, My column labels from scala include: SIGF_New(+) and F_New as well as F_New(-) and SIGF_new(-) But also contains: SIGF_New, F_New DANO_New and SIGDANOW_NEW When I refine (phenix) using SIGF_NEW(+) and SIGF_New(-) my completeness does not match what comes out of the scala log file (97%). Instead it is only 90% But when I refine using Intensities and let phenix.refine run truncate I get the expected completeness of 97% in my log file. Is there something special you have to do in Scala to tell it to combine anomalous and non-anomalous data for refinement using structure factors? I don't need the anomalous data so I don't need to keep it separate. Thanks. -Yarrow -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
[ccp4bb] calculating dialectric properties of enzyme active site
Hello CCP4 list readers, Does anyone know how to calculate the dielectric properties of an enzyme active site? I would like to compare the polarity/hydrophobicity of similar proteins and different mutants. Thank you. -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] calculating dialectric properties of enzyme active site
Thanks Boaz, CCP4mg looks similar to Pymol. Is there any advantage to using it over pymol? I have the used APBS plugin for pymol but I am really looking for just the selection around the active site and found this hard to do using APBS (although I am not very good at using APBS). -Yarrow ccp4mg is another good program for this. Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yarrow Madrona [amadr...@uci.edu] Sent: Saturday, October 06, 2012 4:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] calculating dialectric properties of enzyme active site Hello CCP4 list readers, Does anyone know how to calculate the dielectric properties of an enzyme active site? I would like to compare the polarity/hydrophobicity of similar proteins and different mutants. Thank you. -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697 -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
[ccp4bb] Calculating water accessible volume in active site
I have a buried active site and would like to determine if there is room for a water molecule in various mutants. Does anyone know of a good program to calculate this? I have heard of GRID and VOID but have never used them. Thanks. -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
[ccp4bb] offtopic: packing gel filtration columns
From: Yarrow Madrona amadr...@uci.edu Date: July 12, 2012 7:39:57 PM PDT To: Peter Hsu hsuu...@u.washington.edu Subject: Re: [ccp4bb] offtopic: packing gel filtration columns It is also likely that the clogging due to NAOH is due to crapped out protein. Try cleaning with guanadine. On Jul 12, 2012, at 6:51 PM, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, Sorry for the slew of offtopic posts, but does anyone here have any experience repacking the large 120mL Superdex75/200 columns? Any advice/tips on doing it? I've got an older column that's gotten clogged while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not sure the bossman would be thrilled w/buying another column. Thanks for any ideas. Peter
[ccp4bb] Fwd: [ccp4bb] offtopic: packing gel filtration columns
Subject: Re: [ccp4bb] offtopic: packing gel filtration columns We do it pretty routinely in our lab with great results. To do it right you have to invest in a reservoir sold by GE. It screws onto the end of the column. It allows you to pour the entire slury (resin and water) into the resin in one shot. The reservoir is capped with an adapter and the pump turned onto 10ml/min or so (less than the hardware pressure limit of 0.5 mPascal). When the resin is packed you take the water and reservoir off. Then stick in the plunger and continue to pack. when pushing down the plunger you have to loosen the fitting that connects the akta tubing to the plunger tubing, so that the excess liquid has somewhere to go. When you tighten the plunger to hold it in place you will need to screw the fitting in ASAP. It is much easier to see than explain. Be careful of over tightening the plunger as this will actually unscrew a fitting inside the plunger and the column will leak. GE had a tutorial video on their website at one time. I don't know if it is still up. It's one of those things that has to be done or seen rather then explained. Good luck. On Jul 12, 2012, at 6:51 PM, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, Sorry for the slew of offtopic posts, but does anyone here have any experience repacking the large 120mL Superdex75/200 columns? Any advice/tips on doing it? I've got an older column that's gotten clogged while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not sure the bossman would be thrilled w/buying another column. Thanks for any ideas. Peter
[ccp4bb] scalepackvirus rejections and Rrin
Hello, I am using scalepackvirus and I noticed that the rejection list grows but does not disappear in later rounds of scaling from the log file as in scalepack. I am assuming that the rejections are treated the same way as in scalepack but for some reason are not removed from the log file. Does anyone know if this is correct? Also I wondered if there is any way to get a redundancy dependent R value from scalepack. Thank you. -Yarrow -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] scalepackvirus rejections and Rrin
Thank you for your help, I had the write rejection file set to 0.5 and probability at 0.001. It seems that I had so many rejections that it was hard to get rid of all of them. I have obtained a script to keep running scalepack until all of the rejections are gone from the log file. It takes a very long time to run but it is better than hitting enter over and over. Although I have read the manual I am still having some difficulty understanding the difference between rejection file and the probability. If rejection file is set to 0.5 I'm guessing that if a reflection has a 50% probability of being an outlier it will be written to the log file. But I don't understand how the rejection probability interplays here. Thanks for any help you can provide. -Yarrow Yarrow, as far as I know, scalepack, scalepackvirus and the other variants only differ by their respective array sizes (Someone from Wladek's or ZO's lab may be more suitable to comment on this). The rejections in the log file are controlled by the parameters 'write rejection file xxx' and 'rejection probability'. You may want to read up on those in the manual. I believe that historically the way it was supposed to work is that rejection candidates are written to the log file for examination and then in subsequent rounds are written to the 'reject' file. The latter is determined by the 'write rejection file' parameter. No, scalepack does not output the rmeas or rpim values. I have some recollection that Manfred Weiss at EMBL wrote a program which could analyze scalepack data written with 'no merge original index' to yield those parameters, you may want to look into this. The name of the fortran source is 'rmerge.f'. Unfortunately the link to the original source repository is dead, but I have a copy around, which I could share. Another way would be to go the route of scalepack unmerged data - pointless - scala, although I have never tried that myself. Good luck Carsten -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yarrow Madrona Sent: Tuesday, December 06, 2011 11:04 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] scalepackvirus rejections and Rrin Hello, I am using scalepackvirus and I noticed that the rejection list grows but does not disappear in later rounds of scaling from the log file as in scalepack. I am assuming that the rejections are treated the same way as in scalepack but for some reason are not removed from the log file. Does anyone know if this is correct? Also I wondered if there is any way to get a redundancy dependent R value from scalepack. Thank you. -Yarrow -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697 -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
[ccp4bb] New or alternative to 3D Zalman monitor for coot/pymol?
Hello CCP4 mail subscribers, I know there have been previous threads regarding the use of a Zalman monitor for using coot and pymol on these message boards. I am wondering if there is a new alternative since the Zalman monitor is no longer being produced. I am in a crystallography lab and we are looking for a less expensive alternative to the SGI work station that just blew-up. We are thinking of buyng a mac and using something like a Zalman monitor. Alternatively we are thinking of running Fedora with a Zalman monitor. Thank you for your help. -Yarrow -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
Re: [ccp4bb] New or alternative to 3D Zalman monitor for coot/pymol?
Thank you. Would you happen to know off hand what the difference in quality is between the ZM-M215W, M240W and M220W? -Yarrow Hi Yarrow, You might want to try ASI. They apparently ordered 100 of these last year. Just a month ago it appeared that they still had a few in stock (see below for the prices and contact information). Eric Eric Ortlund, Ph.D. Assistant Professor Department of Biochemistry Emory University School of Medicine 1510 Clifton Road, NE, Room G235 Atlanta, GA 30322 Tel 404-727-5014 Fax 404-727-2738 eric.ortl...@emory.edu - SKU 101685 ZAL LCD 21.5W ZM-M215W DVI 3D $435.00 Darin From: Ortlund, Eric [mailto:eort...@emory.edu] Sent: Friday, April 29, 2011 9:39 AM To: Darin Suffridge; Oliver Kao Subject: FW: Zalman Quote Hi Darin, Our Zalman monitor is working well and I am just writing to ask if the prices have come down on these for 2011? Thank you, Eric Eric Ortlund, Ph.D. From: Oliver Kao oliver@asipartner.com Date: Wed, 14 Jul 2010 16:44:11 -0400 To: Eric Ortlund eort...@emory.edu Subject: Zalman Quote Zalman LCD ZM-M215W 21.5in Wide 1680x1050 1:1 5ms DVI 3D Black Retail Steroscopic polarized passive 3D monitor $435 Zalman LCD ZM-M240W 24inch Wide 1920x1080 1:1 5ms DVI VGA 3D Black Retail $568 Oliver Kao Account Executive ASI- Atlanta Phone: (800) 746-6274 x 343 Fax: (678) 502-1392 Email and MSN: oliver@asipartner.com On 5/30/11 12:38 PM, Yarrow Madrona amadr...@uci.edu wrote: Hello CCP4 mail subscribers, I know there have been previous threads regarding the use of a Zalman monitor for using coot and pymol on these message boards. I am wondering if there is a new alternative since the Zalman monitor is no longer being produced. I am in a crystallography lab and we are looking for a less expensive alternative to the SGI work station that just blew-up. We are thinking of buyng a mac and using something like a Zalman monitor. Alternatively we are thinking of running Fedora with a Zalman monitor. Thank you for your help. -Yarrow -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697 This e-mail message (including any attachments) is for the sole use of the intended recipient(s) and may contain confidential and privileged information. If the reader of this message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this message (including any attachments) is strictly prohibited. If you have received this message in error, please contact the sender by reply e-mail message and destroy all copies of the original message (including attachments). -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697
