Re: [ccp4bb] How to apply correction factors for translational NCS in PHASER?
Hi, If there is more than one strong unique Patterson peak, then Phaser currently doesn't know how to interpret the tNCS. If all the peaks can be interpreted as multiples of one one vector (taking symmetry into account) then you can use the NMOL argument of the tNCS command. Otherwise, you can get Phaser to only take account of the top peak by setting the minimum peak height. If you post or email the list of peaks, I might be able to give some specific advice (although I'll be on my way to the Cold Spring Harbor school most of the day). Best wishes, Randy Read Randy J. Read On 22 Oct 2013, at 23:35, Niu Tou niutou2...@gmail.com wrote: Dear All, I have a MR case which may contain two different molecules, ideally 1:1 ratio. Solvent content analysis indicates most likely to be 4~6 hetero molecules. The data shows at least 5 strong translational NCS vectors. When I tried to run PHASER, it always said correction factors NOT applied, but I have chosen to count it in the interface. Is there anyway I could use these NCS information, either by checking some options in the GUI of modifying the script? Many thanks! Best, Niu
Re: [ccp4bb] expanding reflections from C2221 to P21
I suspect the problem is that sftools hasn't been modernised to alter all the cell dimension records used in current MTZ files. I can't remember right now but maybe the DCELL command in cad might fix it. Randy Read Randy J. Read On 2 Oct 2013, at 17:13, wtempel wtem...@gmail.com wrote: Hello, I would like to expand a reflection data set in mtz format from C2221 to P21. The purpose is to obtain consistent R-free flags based on a structure already refined in C2221 for a related data set that I suspect is pseudo-C2221 but real P21. Primitive cell dimensions are: 37.6 126.1 40.61 89.99 117.6 90.01, C-centered: 37.6 71.99 126.1 90 89.99 90.01 pointless provides the following matrix: pointless Reindex operator from input cell to lattice cell: [h,h+2l,-k] h' = ( h k l ) ( 1 1 0 ) ( 0 0 -1 ) ( 0 2 0 ) /pointless In sftools, I loaded the C2221 data set and did sftools$ reindex matrix 1 0 0 0 0 -1 -.5 .5 0 with the transposed (to account for the presumably inverted order of factors in the program?) inverse matrix of the one listed above with the aim of restoring the primitive asymmetric unit. I was encouraged seeing sftools report new cell dimensions matching the expected primitive cell. Then I did sftools$ expand 4 I expected now to have a workable P21 version of my C2221 data set, but molecular replacement (MOLREP) with my C2221 model failed to place even a single copy of the model. Thus, I must have misused sftools by issuing commands that were either wrong or in the wrong order or my application of linear algebra was mistaken. Any ideas out there? Thanking you in advance, Wolfram Tempel
Re: [ccp4bb] Help! weird thing
If you've used the older version of Phaser distributed with CCP4 then the TFZ for the second copy is not meaningful in the presence of tNCS. Just putting something in the same orientation with the right relative translation will explain the modulation of the data so it will score well. However if you get a large TFZ with the latest version of Phaser then this means something, because it's already accounting for the modulation. Best wishes, Randy Read Randy J. Read On 16 Mar 2012, at 18:33, xiaoyazi2008 xiaoyazi2...@hotmail.com wrote: Thanks all! In addition to the pseudo translation problem, the model I used for MR is really a partial model with low quality. It could make it more complicated. I guess the MR solution is right since phaser gave very high Z-score (up to 60, maybe it is not real, too high to be true). It is possible that P1 is the hope. Are there any cases that structures were determined only by experimental phasing with pseudo translation? Nice weekend! zhihong On Mar 15, 2012, at 1:54 AM, herman.schreu...@sanofi.com wrote: Dear Zhihong, Refinement stuck at 55% Rfree (which is essentially random), means that you do not have found the correct MR solution. For me the prime suspect is the space group. In my experience pseudo translation or any almost crystallographic NCS will easily confuse automatic space group determination programs like pointless and it is often trickey to find out which symmetry is crystallographic and which is non-crystallographic. Since P21 is fairly low symmetry, I would reprocess your data in P1 and just naively run all your favorite molecular replacement programs (phaser, molrep, epmr, phenix, etc.) in P1. I would try them all since there are subtle difference between these programs and one may succeed where the others fail. Once you have a solution which refines (Free Rfactor drops below say 40% in P1) you can try to figure out what the true, higher symmetry space group may be. Your true space group may even be P1, with pseudo P21 symmetry! Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of xiaoyazi2008 Sent: Thursday, March 15, 2012 6:12 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help! weird thing Dear all, Thank you very much for all the great suggestions on my case. Yes, I run the latest version of Phaser in Phenix. The analysis showed that there is one non-origin distinct peak more than 15 angstroms from the origin. 44.1% origin:FRAC 0.000 0.042 0.500 (ORTH -15.72.8 103.5) I managed to find four copies with the latest Phaser. After 50 cycles of rigid body refinement and 50 cycles of jelly body refinement, Rfree/R goes around 55/52. It is really hard for me to do model building at this point, because there is pretty much no new density. Compare to model building and refinement with normal dataset (no pseudo translation NCS), are there any special tricks or tips for structure determination from dataset with pseudo translation? Thanks again! Have a nice evening or morning or afternoon! Zhihong On Mar 12, 2012, at 10:16 AM, Randy Read wrote: Airlie points out that what I said about the ccp4i interface wasn't correct! In order to keep the ccp4i interface in synch with the version of Phaser, we've started distributing the ccp4i files with the source code. The ones on our website are for an older version of Phaser, but the latest ones will come with the Phenix download that gives you the latest executable. Apologies to anyone who was quick enough to download and install the wrong ccp4i files already! Best wishes, Randy Read On 12 Mar 2012, at 16:47, Randy Read wrote: Yes, the current version of Phaser will do the same test that xtriage carries out, and if it finds a sufficiently high non-origin Patterson peak, it will automatically characterise the translational NCS and use this for molecular replacement. This is working pretty well in our tests. In the near future you will be able to get the current version of Phaser as part of the upcoming CCP4 release, but at the moment the easiest way to get it is to download a recent version of Phenix. You should be able to run that through ccp4i by downloading and installing the updated GUI files from our website (and getting ccp4i to interpret the command phaser as phenix.phaser). Best wishes, Randy Read On 12 Mar 2012, at 16:06, Anastassis Perrakis wrote: Hi - I agree with Garib that its likely a pseudo-translation issue. I also agree with that the advice he gives is correct, but ... ... since I am evidently less smart to follow all these steps, I like to use phenix.xtriage that will tell me if there is pseudo-translation or not, and will give a p-value for that being significant. Its at the end of the text output. I am not sure if Phaser
Re: [ccp4bb] twin refinement in refmac
I'm worried when you say that you use the initial job's output MTZ. Refmac replaces F with a detwinned F in the output file so you wouldn't be refining against your measured data in the subsequent round. Best wishes Randy Read Randy J. Read On 2 Mar 2012, at 02:00, wtempel wtem...@gmail.com wrote: Dear CCp4ers, A good morning to everyone. Today, I have a structure that I initially refined in space group P6522, 1mol/asu. Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; I/sigma 3 2.61-2.55A: Rsym=39.6%, I/sigma 10 50.00-6.13: Rsym=6.4% Some mild anisotropy in the resolution limits is apparent on the diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in the other. Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A resolution, with some difference density for loops that cannot be interpreted with reasonable geometry. Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage does not suggest merohedral twinning. Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my model to a homotetramer and ran Refmac with amplitude based twinning. (Would this be a reasonable input to twin refinement?) From the output coordinates: REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS :4 REMARK 3 TWIN DOMAIN :1 REMARK 3 TWIN OPERATOR : H, K, L REMARK 3 TWIN FRACTION : 0.269 REMARK 3 TWIN DOMAIN :2 REMARK 3 TWIN OPERATOR : -K, -H, -L REMARK 3 TWIN FRACTION : 0.171 REMARK 3 TWIN DOMAIN :3 REMARK 3 TWIN OPERATOR : K, H, -L REMARK 3 TWIN FRACTION : 0.258 REMARK 3 TWIN DOMAIN :4 REMARK 3 TWIN OPERATOR : -H, -K, L REMARK 3 TWIN FRACTION : 0.302 Does this establish twinning versus underestimated symmetry? And what do I need to know about my free-R? Did refmac assign a new flag? Whereas the output file's flags are all 1s and 0s, the input file had 0 ... 19. During the first run, Rfree dropped to 28%. But on a subsequent run, Rfree was stuck 30% when I used the initial job's output MTZ. Many thanks in advance for your helpful comments. Wolfram Tempel
Re: [ccp4bb] Anomalous signal for chlorides
Dear Yuri, In addition to the other good options that have been presented, you can use the log-likelihood gradient maps in Phaser to find anomalous scatterers. We find this to be very sensitive, and it has the advantage of being iterative (i.e. when you find some anomalous scatterers, this improves your model and thus the sensitivity for finding additional sites). When you're starting from a refined model, we think it is best to look for purely imaginary scatterers to add to the real scattering in your model. In the ccp4i GUI, choose the SAD with molecular replacement partial structure mode, provide the data (with F+ and F-), the wavelength, and the current model, then turn on LLG-map completion and select the Complete with purely anomalous scatterer option. If you want to see the initial LLG map, set the number of completion cycles to 0 and turn on the option to output log-likelihood-gradient map coefficients. Open these in coot as a difference map, choosing the columns FLLG_AX and PHLLG_AX. If you let Phaser complete the sites, then the final LLG map should be nearly flat and you have to look at the PDB file containing the sites that it found. Best wishes, Randy Read On Nov 26 2011, Yuri Pompeu wrote: Hi Boaz, Yes indeed, the phosphate group of the molecule looks quite beautiful at 1.17A and it has a really big peak 18sigma! Is there a utility for calculating anomalous maps, or is it simply an option in the refinement program? -- Randy J. Read
Re: [ccp4bb] Phaser_EP
I thought it would be good to follow up with the solution to this problem, in case someone else has been suffering with it in silence. It turns out that, at some point, the Windows version of CCP4 release 6.1.13 included a newer version of Phaser (2.2.1), but the ccp4i interface for that release is designed for Phaser version 2.1.4. Because there were some small syntax changes between those versions, some jobs will fail, particularly those for SAD phasing. The easiest fix is to replace the Phaser executable with the 2.1.4 version, which you can obtain by contacting us. So if anyone else has been having issues with this, please let us know! In the not too distant future, version 6.2 of CCP4 should come out, and that will contain Phaser version 2.3 as well as compatible ccp4i GUIs. Regards, Randy Read On May 28 2011, Ylva Lindqvist wrote: After updating CCP4 includin Phaser_EP I can not run it - it can not read my SIGFs - what is wrong? The error message is SYNTAX ERROR: Use SIGFPOS SIGF(+) or SIG+ but my mtz-labels are LABIN F+ = F_Fe1(+) SIGF+ = SIGF_Fe1(+) F- = F_Fe1(-) SIGF- = SIGF_Fe1(-). I have changed them with SFTOOLS but that did not help - always the same error message. Ylva Lindqvist Dept. of Medical Biochemistry Biophysics Div. of Molecular Structural Biology KAROLINSKA INSTITUTET Tomtebodavägen 6 S-171 77 Stockholm, Sweden http://www.msb.mbb.ki.se/
Re: [ccp4bb] Phaser_EP
Dear Ylva, If you're running from the ccp4i interface, it looks like a mismatch between Phaser version and ccp4i version. Some of the syntax changed between the version of Phaser released with CCP4 6.1 and the version that will be coming out with the imminent 6.2 release. So, for instance, you can specify the LABIN as SIG+, SIGF(+) or SIGFPOS but not any more as SIGF+. Could you tell me (perhaps off-list) whether you have a pre-release of CCP4 6.2 and maybe check whether you've told the interface to use a different binary for Phaser (e.g. phenix.phaser)? Thanks! Randy Read On May 28 2011, Ylva Lindqvist wrote: After updating CCP4 includin Phaser_EP I can not run it - it can not read my SIGFs - what is wrong? The error message is SYNTAX ERROR: Use SIGFPOS SIGF(+) or SIG+ but my mtz-labels are LABIN F+ = F_Fe1(+) SIGF+ = SIGF_Fe1(+) F- = F_Fe1(-) SIGF- = SIGF_Fe1(-). I have changed them with SFTOOLS but that did not help - always the same error message. Ylva Lindqvist Dept. of Medical Biochemistry Biophysics Div. of Molecular Structural Biology KAROLINSKA INSTITUTET Tomtebodavägen 6 S-171 77 Stockholm, Sweden http://www.msb.mbb.ki.se/
Re: [ccp4bb] The meaning of B-factor, was Re: [ccp4bb] what to do with disordered side chains
In this case, I'm more on ZO's side. Let's say that the refinement program can't get an atom to the right position (for instance, to pick a reasonably realistic example, because you've put a leucine side chain in backwards). In that case, the B-factor for the atom nearest to where there should be one in the structure will get larger to smear out its density and put some in the right place. To a good approximation, the optimal increase in the B-factor will be the one you'd expect for a Gaussian probability distribution, i.e. 8Pi^2/3 times the positional error squared. So a refined B-factor does include a measure of the uncertainty or error in the atom's position. Best wishes, Randy Read On Apr 1 2011, James Holton wrote: I'm not sure I entirely agree with ZO's assessment that a B factor is a measure of uncertainty. Pedantically, all it really is is an instruction to the refinement program to build some electron density with a certain width and height at a certain location. The result is then compared to the data, parameters are adjusted, etc. I don't think the B factor is somehow converted into an error bar on the calculated electron density, is it? For example, a B-factor of 500 on a carbon atom just means that the peak to build is ~0.02 electron/A^3 tall, and ~3 A wide (full width at half maximum). By comparison, a carbon with B=20 is 1.6 electrons/A^3 tall and ~0.7 A wide (FWHM). One of the bugs that Dale referred to is the fact that most refinement programs do not plot electron density more than 3 A away from each atomic center, so a substantial fraction of the 6 electrons represented by a carbon with B=500 will be sharply cut off, and missing from the FC calculation. Then again, all 6 electrons will be missing if the atoms are simply not modeled, or if the occupancy is zero. The point I am trying to make here is that there is no B factor that will make an atom go away, because the way B factors are implemented is to always conserve the total number of electrons in the atom, but just spread them out over more space. Now, a peak height of 0.02 electrons/A^3 may sound like it might as well be zero, especially when sitting next to a B=20 atom, but what if all the atoms have high B factors? For example, if the average (Wilson) B factor is 80 (like it typically is for a ~4A structure), then the average peak height of a carbon atom is 0.3 electrons/A^3, and then 0.02 electrons/A^3 starts to become more significant. If we consider a ~11 A structure, then the average atomic B factor will be around 500. This B vs resolution relationship is something I derived empirically from the PDB (Holton JSR 2009). Specifically, the average B factor for PDB files at a given resolution d is: B = 4*d^2+12. Admittedly, this is on average, but the trend does make physical sense: atoms with high B factors don't contribute very much to high-angle spots. More formally, the problem with using a high B-factor as a flag is that it is not resolution-general. Dale has already pointed this out. Personally, I prefer to think of B factors as a atom-by-atom resolution rather than an error bar, and this is how I tell students to interpret them (using the B = 4*d^2+12 formula). The problem I have with the error bar interpretation is that heterogeneity and uncertainty are not the same thing. That is, just because the atom is jumping around does not mean you don't know where the centroid of the distribution is. The u_x in B=8*pi^2*u_x^2 does reflect the standard error of atomic position in a GIVEN unit cell, but since we are averaging over trillions of cells, the error bar on the AVERAGE atomic position is actually a great deal smaller than u. I think this distinction is important because what we are building is a model of the AVERAGE electron density, not a single molecule. Just my 0.02 electrons -James Holton MAD Scientist On Fri, Apr 1, 2011 at 10:57 AM, Zbyszek Otwinowski zbys...@work.swmed.edu wrote: The meaning of B-factor is the (scaled) sum of all positional uncertainties, and not just its one contributor, the Atomic Displacement Parameter that describes the relative displacement of an atom in the crystal lattice by a Gaussian function. That meaning (the sum of all contributions) comes from the procedure that calculates the B-factor in all PDB X-ray deposits, and not from an arbitrary decision by a committee. All programs that refine B-factors calculate an estimate of positional uncertainty, where contributors can be both Gaussian and non-Gaussian. For a non-Gaussian contributor, e.g. multiple occupancy, the exact numerical contribution is rather a complex function, but conceptually it is still an uncertainty estimate. Given the resolution of the typical data, we do not have a procedure to decouple Gaussian and non-Gaussian contributors, so we have to live with the B-factor being defined by the refinement procedure. However, we should still improve the estimates of the B-factor,
Re: [ccp4bb] FOM: Phaser vs SigmaA
Sorry for the late reply -- I've been travelling. You're correct, the current version of Phaser produces figures of merit (and HL coefficients) based entirely on the initial guess about the quality of model. The version currently in Phenix (and coming soon to the upcoming CCP4 release) can refine the RMS values, which will give results similar to those from SIGMAA or Refmac. This will be turned on by default when we're happy that it's almost always best to carry out the refinement. (The worry is that it might be unstable when the model is very incomplete, which might be the case with your helices.) Regards, Randy Read On Oct 21 2010, Goragot Wisedchaisri wrote: Hi, I have helices that I did rigid body refinement with Phaser (after phased rotation and phased translation in Molrep). I compare FOM output by Phaser to the FOM computed by sigmaA using the Phaser refined coordinates and found that FOM from Phaser is only about half (~0.25) of FOM from SigmaA (~0.5). I'm running Phaser using ccp4 version 6.1.13. I remember a while back that Phaser used to calculate a priori sigmaA estimation based on assumed model rmsd error. I am not sure if this a priori SigmaA weight is also output in the FOM column. If this is not the case, could anyone point me to a documentation of how Phaser calculates FOM. The Phaser wiki and J App Crys paper does not seem to have detail information on this. I could just use SigmaA or do refinement in Refmac but I have to say that I like the low FOM from Phaser because model bias seem to be much less after density modification. It also saves me from having to blur the phase probability distribution in order to down weight FOM when FOM is too high. But I still would like to know how Phaser currently calculates the unusually low output FOM. Many thanks, George Wisedchaisri
Re: [ccp4bb] Parallelization of Phaser and other programs part of CCP4
There's OpenMP parallelization of some of the most CPU-intensive parts of molecular replacement in Phaser, in the versions released recently in Phenix and hopefully in the next CCP4 release. However, OpenMP is not turned on by default in the compilation of the binaries released with Phenix, so the feature is more or less hidden at the moment. We're working on a mechanism to distribute binaries with OpenMP turned on. Regards, Randy Read On Oct 27 2009, Christian Rausch wrote: Dear colleagues, is there a parallelized version of Phaser? How about other programs that are part of the current CCP4 distribution? Thank you, Christian Rausch
Re: [ccp4bb] Two Equally Good MR Solutions Found by Phaser
Hi, If you search with a dimer, then the version of Phaser in CCP4 doesn't know that there is internal symmetry in your model, and it will find two solutions that are equivalent by the internal symmetry, but would not be equivalent if the model were not symmetric. The latest version of Phaser (which is available in Phenix, and should be in the next CCP4 release) identifies internal symmetry and would recognise the two solutions as being equivalent. You can check this by reading the two solutions into coot, using ssm superpose to superimpose the first monomer of the first solution on the second monomer of the second solution, and seeing whether the transformation is some combination of crystallographic symmetry and an allowed origin shift. Best wishes, Randy Read On Oct 21 2009, X Xiong, Cellular Molecular Medicine wrote: Hi All, Thanks for all the replies, I would like to add more information, after reindex it to P21212, the cell parameter is a=88.71, b=116.26, c=55.12, the molecule is a long rod like head to head dimer with a length of 110Å (55Å long for each monomer) and we used the dimer to search the orthorhombic data, two solutions were found as shown in the original thread which can be both refined to almost the same very good R/Rfree, coot was used to generated the symmetry related molecules from both solutions and the two solutions had the same packing but translated along the b axis by 31.9Å, thus clashed into each other and made a mess in the overlap region, so I think they are not crystallographically the same solutions, unless the origin of the cell in P21212 can be placed on b-axis arbitrarily, and I think Phaser will also prune the crystallographically same solutions. Interestingly, the most prominent pseudo-translational peak (1/3 of the origin peak), has a fractional vector 0.5000 0.2741 0.5000, and the fraction on b axis = 0.2741*116.26 = 31.8755Å, and that is exactly how long the two solutions translated along the b-axis, I don't know what that means, does these information verify this is the second case Eleanor suggested? if so should I keep the messy overlapped region and set the rest 0 occupancy to check the density? Thanks for all the suggestions so far! Xiaoli From the Pattersn peak it seems very likely that you have two molecules in the asymmetric unit seperated by the very vector that seperates your two MR solutions, and both MR solutions are correct? Or is that not possible? Is there no room for 2 molecules in the asymmetric unit, and the Patterson peak isa function of the highly repetitive dimer Eleanor If that is so you need to set the occupancy of any differences in the solution to 0.00 and check from the maps after refinement if you can see which copy of the molecule fits the difference density best - it would nice if you had a TRP/ALA pair of residues or something very distinctive.. Eleanor X Xiong, Cellular Molecular Medicine wrote: Dear Crystallographers, We got a highly repetitive dimeric protein solved by SeMet-SAD in P21 crystal form, and I am now trying to solve a dataset collected from a non-reproducible orthorhombic crystal of the same protein using the structure refined from P21 data. From the Scala statistics, the orthorhombic crystal diffracted to 2.2Å with an I/sigma of 3.1 at outmost shell; 98% complete overall, 89% complete 43.4-7.0Å, 99% complete 2.32-2.2Å, no twinning was detected. Due to the incompleteness at low resolution, it was hard to determine which orthorhombic space group it is in so data was scaled in P222. Very strong pseudo-translational symmetry has been detected by self-Patterson, as shown for reindexed data P21212 (space group later found by Phaser): Order No. Site Height/RmsGrid Fractional coordinates Orthogonal coordinates 111 128.24 0 0 0 0. 0. 0. 0.00 0.00 0.00 2 13 13 57.5160 44 38 0.5000 0.2741 0.5000 44.37 31.88 27.55 322 33.75 0 7 0 0. 0.0414 0. 0.00 4.82 0.00 4 14 14 16.0960 50 38 0.5000 0.3150 0.5000 44.37 36.63 27.55 5 12 12 15.7560 37 38 0.5000 0.2324 0.5000 44.37 27.03 27.55 633 12.28 0 13 0 0. 0.0836 0. 0.00 9.72 0.00 7 1507.0660 57 38 0.5000 0.3574 0.5000 44.37 41.56 27.55 8446.18 0 72 0 0. 0.4503 0. 0.00 52.36 0.00 9995.68 5 0 5 0.0410 0. 0.0683 3.64 0.00 3.76 10555.36 2 20 2 0.0142 0.1254 0.0206 1.26 14.59 1.14 11 11 115.3358 31 38 0.4852 0.1909 0.5000 43.06 22.20 27.55 12663.98 5 0 2 0.0435 0. 0.0286 3.86 0.00 1.58 13773.82 2 27 3 0.0168 0.1659 0.0334 1.49 19.30 1.84 14883.68 0 0 5 0. 0. 0.0722 0.00 0.00 3.98 15 10 103.4160
Re: [ccp4bb] cad gives a fortran runtime error in line 869 : comparing phase sets
For the first part of your question, I usually use sftools from the command line to combine things from different MTZ files, so you could give that a try if cad is giving you trouble. For the second part of the question, if you're staying within CCP4, then cphasematch is probably the program you want, as it checks for origin shifts for you. Similar things can be done in Resolve or in the phenix tool get_cc_mtz_mtz (which uses Resolve). Regards, Randy Read On Apr 11 2009, hari jayaram wrote: Hi I have multiple phase sets from separate sharp ( experimental phasing) and phaser molecular replacement runs. I have the values as hendrickson latman coeffs as output by phaser and sharp. I am imagining i can use phistats to compare these phase sets which are probably on different origins . The first problem I have is that i need to combine the multiple HLA files from a couple of mtz files into one mtz file for input to phistats . I am trying this with cad and I get the error in ccp4-6.0.99e At line 869 of file /home/hari/ccp4-6.0.99e/src/cad.f Fortran runtime error: End of record I am hoping that cad can blindly take mtz columns from these files and put them into the same file . My questions are : How can I put these multiple sources of phases into one file . My second question is what is a good way to compare these phase sets . The detailed error from cad is reproduced below Thanks for your help hari *** * Information from CCP4Interface script *** The program run with command: /home/hari/ccp4-6.0.99e/bin/cad HKLIN1 /home/hari/newtry/gio_12.1.mtz HKLIN2 /home/hari/AdiC/eden_flat_75.0pc.mtz HKLOUT /home/hari/newtry/phasereden.mtz has failed with error message At line 869 of file /home/hari/ccp4-6.0.99e/src/cad.f Fortran runtime error: End of record ***
Re: [ccp4bb] brute force molecular replacement
Hi, If you were desperate for BRUTE, I could probably dig out the source code. My somewhat newer program BEAST also does a brute-force search, and you could get that by downloading an older version of CCP4 or, again, directly from me. Our new program Phaser also has brute-force rotation and translation search options, in addition to the faster FFT-based searches. If you wanted to do a full 6D brute-force search for a molecule, you'd have to use some tricks, but it would be possible. However, the reason we suggested to CCP4 that they should deprecate BEAST is that we haven't found a case where doing a brute-force search solves a structure that can't be solved faster with Phaser using the fast search methods. (If anyone has such a case, we'd be delighted to learn what it has to teach us!) Is there a particular reason you'd like to try a brute-force search? Regards, Randy Read On Jan 24 2009, Xie Jiabao wrote: Dear all, From where can I download the molecular replacement program BRUTE? What are the other brute force programs for molecular replacement out there? Thanks in advance, Xie
Re: [ccp4bb] Phaser job on CCP4 suite
Hi, I've just double-checked this on my installation (6.0.99e still, but shouldn't make a difference), and this command works. I think that you've ended up with an older version of Phaser coming first in your path. The syntax of the PACK command has changed (to allow it to behave in a way that more users wanted), and an older version will give that error message. If you're on a Unix machine, typing which phaser will tell you which phaser executable comes first in your path. It could be an earlier CCP4 installation, or a very old Phenix installation. (More recent Phenix installations avoid this issue by calling the executable phenix.phaser, to make the name unique.) Another way to tell is from the version number in the program banner. This should be 2.1.4 for the version distributed with CCP4 6.1. If that doesn't solve your problem, you can get in touch directly. Regards, Randy Read On Dec 3 2008, wangsa tirta ismaya wrote: Dear all, We are trying to run phaser (for molecular replacement) with the automated search option. The spacegroup is P21 and there are 4 molecules in the ASU. We fed phaser with 1 molecule and let the program generates the symmetry related molecules and packs them into the ASU. As the model grows larger, we observe there is clash between the molecule. SInce we knew that the molecule was funnily oriented (when we run phaser using the initial model, which is much smaller), we expect this problem may appear. Therefore we tried to lower the clashes distance (default 3.0) or increase the maximum allowed number of clashes (default 10). Unfortunately, the program is terminated with this error: - MODE MR_AUTO HKLIN /wangsa/building/native.run31.mtz TITLE rerun phaser COMPOSITION PROTEIN SEQ /home/wangsa/cmtbr.pir NUMBER 4 ENSEMBLE ensemble1 PDBFILE /wangsa/building/phaser5.P21.modelP21212.A.pdb IDENT 100.0 SEARCH ENSEMBLE ensemble1 NUMBER 4 PACK BEST SYNTAX ERROR: Number not present or not valid - The same problem appeared even when we put the values back to default (max 10 clashes and distance 3.0 A) IF we left the run option setting window open during this faulty run (and just changed the value and clicked run). It went back to normal if we first close the run option setting window (and click rerun to open a new run option setting window). Has anyone experience the same problem or could this be a bug? we are using the CCP4 program suite 6.1.0 (CCP4interface 2.0.3). Many thanks in advance, Wangsa
[ccp4bb] Post-doctoral position available with Phaser team
We are looking to expand the team developing our program Phaser, for use within both the CCP4 and Phenix packages. Our goals include expanding the functionality to new phasing experiments (in addition to the options for molecular replacement and SAD that exist in the current version), and enhancing the automation of Phaser runs. The ideal applicant would be someone with experience solving macromolecular crystal structures and programming in both C++ and Python. However, if you only have experience in one of those areas, but a strong desire to expand your skills, then you might be the right person. If you're interested, please look at the formal advertisement (http://www.admin.cam.ac.uk/offices/hr/jobs/vacancies.cgi?job=4017), and submit an application as described there. I'm also happy to answer any informal enquiries. The position is funded by a grant from the Wellcome Trust, which extends to May 2013. Please note that the closing date for applications is 2 October 2008. Regards, Randy Read
Re: [ccp4bb] Phaser error
This turns out to be a failure in the (rarely exercised) code to read in the substructure with a .ha file. We almost always use PDB files from Hyss or SHELXD to enter the initial substructure, then Phaser .sol files if there's a reason to carry on refinement. The bug has now been fixed for the version of Phaser that will appear in CCP4 6.1 and the next release of Phenix. In the meantime, you can work around it by using PDB files and not HA files for the substructure. Many thanks to Sarah for reporting the bug! Randy Read On Jun 9 2008, Wisecarver, Sarah N wrote: I am attempting to solve a 28kDa protein structure by sulfur anomalous scattering in Phaser for EP (2.1.2). Phaser crashes immediately and I get the following error in the log file. Thanks in advance for your input. Sarah Wisecarver EXIT STATUS: SUCCESS CPU Time: 0 days 0 hrs 0 mins 0.01 secs (0.01 secs) Finished: Fri Jun 6 15:15:02 2008 /pre /html *** * Information from CCP4Interface script *** The program run with command: phaser has failed with error message child killed: segmentation violation *** #CCP4I TERMINATION STATUS 0 child killed: segmentation violation #CCP4I TERMINATION TIME 06 Jun 2008 15:15:02 #CCP4I MESSAGE Task failed
Re: [ccp4bb] DM - NCS averaging after phaser molecular replacement run-confused about coefficients
On Apr 4 2008, hari jayaram wrote: Hi everyone, I have a phaser molecular replacement solution for my membrane protein which crystallized in spacegroup P3. The diffraction data is good to about 3.3 A. The model I used had 39% homology to the given protein. A solvent content analysis suggests that there probably are three dimers in the ASU ( to give about 67% solvent) Phaser sucessfuly found two dimers in the ASU with good density and a third dimer with very weak almost non-existent density. I am now trying to do some NCS-averaging using DM to see If I can improve the desnity for dimer 3 and have a question about the different co-efficients I should be using for the averaging DM run. Question1: The phaser output mtz file has a PHWT and a PHIC . For carrying out DM with flattening, averaging and histogram matching which phases do I use PHIC or PHWT along with observed Fo. Also for the weight do I use the FOM. Question2: The output mtz from DM run either way above , now has a PHIDM and a PHWT along with a FWT and FC. Which coefficients should I be using to get a map after DM for building. FWT and PHWT or Fo and PHIDM or FWT and PHIDM. Thank you for your help Hari In the MTZ file produced by Phaser, the FWT/PHWT pair provide the 2mFo-DFc map coefficients that should reduce model bias. I'd suggest starting DM from these coefficients, which you can do by adding FDM=FWT PHIDM=PHWT to the LABIN command for DM. (If you want to do this from the current ccp4i GUI for DM, the only way is to use the RunView Com File command.) After running DM, the map coefficients to view in coot are FDM/PHIDM. Good luck! Randy Read
Re: [ccp4bb] wich program to test an alternative phaser solution
As suggested earlier, you can use the TOPFILES command to get Phaser to produce the files for more solutions. However, instead of rerunning the whole job, you can produce the PDB files alone by feeding the .sol file into a packing (MR_PAK) job, or both PDB and MTZ files by feeding the .sol file into a rescoring (MR_LLG) job. Randy Read On Jan 15 2008, Vellieux wrote: Dear all, We have a phaser output with 2 alternative solutions. Not differing much by the statistics. Phaser provides the solution with the highest LLG. However I am not convinced by that solution. What program should be used with the input PDB and the Euler angles and translation parameters (found in the .sol file) to generate the alternative solution? Thank you in advance, Fred.
Re: [ccp4bb] tricky molecular replacement
The procedure of cutting out electron density, putting it into a large unit cell, and backtransforming to get structure factors can be tricky (as you've discovered), so we put some instructions on our webpage: http://www-structmed.cimr.cam.ac.uk/phaser/density_as_model.html The last time I tried that it worked, but let me know if you run into any problems with your case. Randy Read On Nov 4 2007, Jan Abendroth wrote: Hi all, thanks a lot for the various responses. When I tried to use a map as the serach model, I ran into various problems: again, the starting point is a weak, yet convincing molecular replacement solution in the hexagonal crystal form (1mol/asu) and no MR solution in P1 (2mol/asu, 2-fold in SRF). a) using phaser and defining the search model though DM map of the MR solution in the hexagonal form: Phaser stops as two space groups were used, p1 for the data set and P6... for the map b) - fft to create map after MR and DM of hexagonal form (map in P6..., asu) - mapmask to cover MR solution (in P6..., asu) - mapcutting using map and mask from prev steps (P6.., asu) - sfall to generate FC, phiC in large P1 cell: fatal disagreement between input info and map header c) same steps as in (b), however, using P6... and full unit cell - mapcutting: maprot dies with ccpmapin - Mask section lsec: recompile d) same steps as in (b), however, using P1 throughout - sfall dies with: Fatal disagreement between input info and map header e) same steps as in (c), however, using P1 and full unit cell - should not be different from case (d) - mapcutting: maprot dies with ccpmapin - Mask section lsec: recompile Any ideas? I btw. use the osx binaries from the ccp4 webpage. Thanks for any input! Cheers Jan On 11/2/07, Edward A. Berry [EMAIL PROTECTED] wrote: One other idea idea: 1. Solvent flattening on the hexagonal crystal 2. use the flattening mask to cut out the density of one molecule, put in a large P1 cell for calculating structure factors 3. Use the structure factors from the density of the hexagonal crystal to solve the triclinic crystal by molecular replacement. 4. If 3 works, multicrystal averaging to improve both crystals til the map is traceable. Jan Abendroth wrote: Hi all, I have a tricky molecular replacement case. One protein in two different crystal forms: hexagonal with 1 mol/asu, triclinic with 2 mol/asu (based on packing and self rotation). No experimental phases are available this far, however, there is a distant homology model. For the hexagonal crystals, phaser gives a solution with really good scores (Z 9, -LLG 50) and a good packing. While the correct solution is way down the list in the RF, the TF can separate it from the bulk of bad solutions. Slight changes in the model give the same solution. Maps are somehow ok, however, not good enough to enable arpwarp to build the model. It does not totally blow up either. For the triclinic crystal form with 2 molecules related by a two-fold which is not parallel to a crystal axis, phaser does not find a solution. Neither does molrep using the locked rotation function with the two-fold extracted by the SRF. As the homology between the data set should be higher than between the model in the data sets and the search model, I tried a cross rotation function between the two data sets. Strong peaks there should give the relation between the orientation of the molecule in the hexagonal crystal (that I believe I can find). With two rotations known and one translation undefined, I'd be left with only one translation that needs to be found. Then averaging within P1 or cross crystal might improve the density... Almn appears to be the only program in ccp4 that can do a cross rotation using Fs only, right?? I used the P1 data as hklin, the hexagonal data as hklin2. Almn comes back with two strong peaks (see below), however, now I am lost: - the first two peaks appear to be the same - are the Euler angles the ones I could use in a peak list for eg. Phaser? - does this procedure make sense at all? - any other ideas? Thanks a lot Jan almn.log: ## Peaks must be greater than 2.00 times RMS density 52.2161 Eulerian angles Polar angles Alpha Beta Gamma Peak OmegaPhi Kappa Direction cosines PkNo Symm: 1 2 Peak 1 1 1 1 323.7 143.4 18.5 540.892.9 62.6 143.80.4594 0.8867 -0.0511 1 1 2 323.7 143.4 78.5 540.883.2 32.6 145.90.8364 0.5351 0.1184 1 1 3 323.7 143.4 138.5 540.875.62.6 157.20.9674 0.0441 0.2495 1 1 4 323.7 143.4 198.5 540.871.9 332.6 174.40.8439 -0.4373 0.3108 1 1 5 323.7 143.4 258.5 540.8 107.2 122.6 167.0 -0.5149
Re: [ccp4bb] opinion : phaser RMSD vis-a-vis LLG, Z-score
On Nov 2 2007, Bryan W. Lepore wrote: neglecting e-density, packing, etc. - what matters most to people when running phaser - LLG or Z-score? in what function (RF, TF)? Depends on the purpose. From the LLG, I want to see that it is positive (negative means that I'm being too optimistic about the quality of the model, i.e. the RMS error is higher or the completeness lower than assumed), and I would like to see it increase as the solution becomes more complete (i.e. RF score increases with TF, which increases for RF on second molecule, and so on). But to assess the significance of a solution, I'd place more weight on the LLG. For various reasons, the RF can be unclear, particularly when there is high symmetry or high NCS, so a correct orientation can have a low Z-score. But usually the Z-score for a correct translation is a better indication. In some cases with multiple molecules in the a.u., the Z-score gets higher for molecules later in the search. further, if you 'refine' the RMSD to find the best RMSD, what is the more robust indicator - LLG or Z-score? LLG is the one that tells you how well your model fits the data, and the assumed RMSD is one of the parameters of the model. At some point, we will refine the assumed RMSD in Phaser (against the LLG), but the signal can be very weak for incomplete models, so we'll have to be careful about how we implement that. lastly - is it better to have a high LLG/Z-score with poor discrimination from other peaks or the other way around? Zscore and discrimination tend to be highly correlated, and we don't really have a good feel for what different absolute values of LLG mean. Anyway, it's the discrimination from other peaks that convinces you that the top solution is the correct one, and not one of the other solutions. If there's poor discrimination, none of the solutions is correct or you need some additional information to sort them out. This can be things like how well they behave in subsequent refinement, or whether the model can be used to find sensible positions for anomalous scatterers. Randy Read
Re: [ccp4bb] opinion : phaser RMSD vis-a-vis LLG, Z-score
On Nov 3 2007, Randy J. Read wrote: Depends on the purpose. From the LLG, I want to see that it is positive (negative means that I'm being too optimistic about the quality of the model, i.e. the RMS error is higher or the completeness lower than assumed), and I would like to see it increase as the solution becomes more complete (i.e. RF score increases with TF, which increases for RF on second molecule, and so on). But to assess the significance of a solution, I'd place more weight on the LLG. Oops! Tassos just pointed out my slip here. I'd meant to say that to assess the significance of a solution, I'd place more weight on the Z-score, not the LLG. Randy Read
Re: [ccp4bb] phaser and percent composition result [ part 2]
On Sep 18 2007, Bryan W. Lepore wrote: i wrote (ensemble) / (sequence) to get a percent composition i forgot to emphasize that i do not mean the Vm or the Z composition, but the composition as one would enter e.g. COMPosition ENSEmble mol1 FRACtional 0.22 or COMPosition SCAttering SCATTERING -bryan In the current version of phaser, if you give the command VERBOSE ON, then you get information about molecular weights and the fraction of the asymmetric unit that each ensemble represents. But it's a good point -- I've frequently wanted to know myself what Phaser thought the fractional composition was. So I've just changed the imminent release so that it will print out a brief summary of the fractional composition to the normal (non-verbose) logfile. Regards, Randy Read
Re: [ccp4bb] phaser + phased-TF
On Aug 31 2007, Bryan W. Lepore wrote: if it doesn't now, will phaser be able to run a phased translation function in the future? -bryan Yes, that's on our long to-do list! Randy Read
Re: [ccp4bb] problems with phaser for MR
Yes, this is an inconsistency between the version of the Phaser executable and the version of ccp4i. To get around the problem that filenames, particularly in Windows, can have spaces in them, the ccp4i GUI now wraps filenames with quotation marks. But this required a corresponding change in the Phaser parser. You're running the version of ccp4i that puts in quotation marks with the older version of Phaser (probably from Phenix) that doesn't know about them. To avoid this confusion in the future, Ralf Grosse-Kunstleve has changed things so that the version of Phaser in Phenix is called phenix.phaser. From the next release of Phenix on, there won't be a confusion between installations. In the meantime, our FAQ page has instructions on how to configure ccp4i so that it finds the executable that you want. Randy Read On Jun 10 2007, Ed Wright wrote: Dear All, I can't seem to read my mtz files with 'PHASER for MR' in version ccp4i 6.0.2 running on either a linux or irix system. I can read the mtz files with virtually every other CCP4 subroutine. Any ideas/suggestions? The error I get in the log file is * *** Phaser Module: READ MR DATA FROM MTZ FILE 1.3.1 *** * FATAL RUNTIME ERROR: Cannot open MTZ file: /home/elias/mergedata/p4/ccp4/sort_p4.mtz EXIT STATUS: FAILURE
Re: [ccp4bb] Phaser TFZ=100.0 ?
On Mar 12 2007, Andy Purkiss wrote: Quoting Andrew Wong [EMAIL PROTECTED]: I was using Phaser for some MR with a data set in P1 (may not actually be P1, but..), and in every run I always got a list of solutions that all have TFZ=100.0, with a very small LLG (around 0 or even in the negative). The RFZ is like 4 or less. The solution(s) are definitely wrong, but I'm just curious why Phaser is doing that? Could the TFZ=100.0 'solutions possibly mask out the real solutions? Is this not because the translation search has no fixed origin (for the first molecule). In other words, all points are the same and placing the molecule fixes the origin allowing search for a second molecule. I will check what my P1 search showed, when I get into work! Yes, that's basically it. Because there's no translation search for the first molecule in P1, there's no TFZ score, so the value of 100 is put in arbitrarily as a placeholder. I can see that would be a bit confusing when there are multiple alternative solutions. Perhaps we should just repeat the RFZ score. On the particular case at hand, if the LLG scores are all around zero, perhaps the model resembles the true structure less than expected for the level of sequence identity (for instance because of unexpected flexibility), or the assumed content of the asymmetric unit is too low? Randy Read
Re: advice
On Jan 22 2007, Eaton Lattman wrote: Will someone knowledgeable tell me what the present state of full 6 dimensional searches in molecular replacement? Presumably you're referring to systematic 6D searches, not stochastic ones like in EPMR or QoS. Do you mean can it be done on current hardware or is it worth doing? If the former, then it's doable, though slow. In Phaser, for instance, you can generate a complete list of rotations (using the fast rotation function with keywords to prevent clustering and to save all solutions), then feed that big list of rotations to the fast translation search. In a typical problem that would probably run on a single processor in significantly less time than the average PhD, and could be made reasonably quick with a cluster. If the latter, our feeling is that it isn't worth it. We've tried the full search option on a couple of monoclinic problems (where it's only a 5D search), and nothing came up with the full list of orientations that didn't come up with the first hundred or so orientations. We conclude that, even in the most recalcitrant cases, the rotation search gives a better than random indication of whether an orientation is correct, so it's not necessary to search through all possible orientations. However, we do feel that it can be worthwhile to try a reasonably large number of orientations in difficult cases. Best regards, Randy Read P.S. When we generate our list of orientations, we use Lattman angles to get reasonably even sampling of rotations.
