Re: [ccp4bb] Question on calculation of RMSD
I would first calculate least square superposition of the first monomer between 2 structures (one to be fixed and one to be moved) using a program such as lsqkap or any other programs suggested by others. You may need to define a relevant region for the superimposition. Then take the output of the moved structure and calculate another least square superposition of the second monomer in the dimer to the second monemer of the fixed structure. Look at the rotation and translation matrix from the output (or log file). That will give you information of how the second monomer is different in the 2 structures. You can calculate the orientation difference (in degrees) from trace of the rotation matrix. Alternatively, after the first monomers are superimposed, you can use program lsqman (or other programs that calculate rmsd of the second monomers without doing least square superposition) if you prefer an rmsd value instead of a rotational angle. George Wisedchasri On Sun, 14 Nov 2010, E rajakumar wrote: Dear All I have two structures of homo-dimeric protein complex with different DNA. I want to calculate RMS deviation between second monomer from these two complexes by fixing superposed first monomer. This I require to know what is the effect of DNA on relative orientation of two monomers in the dimer. Previously I was using MOLEMAN2 to do this calculation. Please can you suggest me any other program to do this calculation. Thanking you Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile)
Re: [ccp4bb] FOM: Phaser vs SigmaA
Thank you very much. I am in total agreement with you that HL coefficients are better description of the phase probability and in fact I have been using the HL coeffs all these times for phase combination and density modification. My earlier post was just to make it easy for a comparison between weights calculated by sigmaA and Phaser. If I take HL coeffs from Phaser and calculate FOM, I will also get the same unusually low FOM. (I am not familiar with how to compare HL coeffs directly but based on the HL equation, the higher the coeff values for the same phase set, the higher the phase probability. The HL coeffs from Phaser are much smaller (A=0.3, B=0.28, C,D=0) than HL coeffs converted from SigmaA (A=0.95, B=0.88). The values are mean abs. from the mtz files.) The reason I am curious about the low FOM (or HL coeffs as you prefer) from Phaser is because I am trying to compare phase combination results between the partial structure and low resolution experimental phases. I use poly alanine alpha-helices as a partial structure and apparently when using HL coeffs calculated from sigmaA or Refmac results, the model phases contribute quite a large model bias after phase combination which also get carried through density modification. The model bias seems less (obviously because of lower weight from the model) when I use result from Phaser and and the map after density modification somehow seems more satisfying. BTW, I am testing a method to solve previously determined strutures starting with experimental low resolution phases and that is why I was curious to know how Phaser gives so much different weight and HL coeffs from sigmaA. I know that you can change weight by using a scale factor during phase combination (also available in CCP4 clipper utility) or bluring the phase probability. The easiest would be to arbitarily scale it down by half. But if anybody knows a good criteria to properly down weight phase probability, please let me know. Many thanks, George Wisedchaisri On Thu, 21 Oct 2010, Clemens Vonrhein wrote: Hi George, On Wed, Oct 20, 2010 at 04:58:34PM -0700, 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). What do you need FOM values for - apart from just looking at them? You don't need them for calculating maps since both SIGMAA and Phaser (I assume) output map-coefficients directly (an amplitude and weight). And you don't need them in refinement since both programs probably output Hendrickson-Lattmann coefficients - which are a much better description of phase probability. Density-modification: same thing. 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. Are you using the FOM columns in density modification? Why? Most modern programs will allow you input of Hendrickson-Lattmann coefficients (and also output those). And the initial map can be calculated with the map coefficients anyway. It also saves me from having to blur the phase probability distribution in order to down weight FOM when FOM is too high. FOM columns in a MTZ file are maybe useful to calculate statistics versus resolution ... but nearly everything you would do with FOM (attach it to an amplitude and phase) can be done much better with Hendrickson-Lattmann coefficients. E.g. running DM after a MR solution, I would use LABIN FP=F SIGFP=SIGF PHIO=PHWT FOMO=FOM - HLA=HLA HLB=HLB HLC=HLC HLD=HLD - FDM=FWT PHIDM=PHWT It still reads the FOM column - but only to analyse it against resolution to determine a good phase-extension scheme. Internally, it will always use the HLA-D values ... afaik. Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
[ccp4bb] FOM: Phaser vs SigmaA
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] difficult P1 crystal
We had a P1 case with 8 molecules/asu and the crystal diffracted to 2A resolution. Initial indexing showed that the data could be indexed in P212121 space group but the data could not be merged in this space group (Rsym 60%). P1 was the only space group that we could merge the data (Rsym 10%). After solving the structure, the major differences we saw in each monomer are at the N and C termini. They adopt different conformations that prohibit the molecules to follow the symmetry that you would otherwise have in P212121. The reference is given below. Structures of Mycobacterium tuberculosis DosR and DosR-DNA complex involved in gene activation during adaptation to hypoxic latency. Wisedchaisri G, Wu M, Rice AE, Roberts DM, Sherman DR, Hol WG. J Mol Biol. 2005 Dec 2;354(3):630-41. PDB 1ZLJ George Wisedchaisri On Thu, 30 Sep 2010, Mark J van Raaij wrote: we had a P1 case with 12 mols/au, about 30% sequence id, 2.4A resolution, which was solved after a lot of trials with phaser. Other problems of these crystals were that they took months to grow and invariable presented multiple lattices (we did not try microbeam). See: Crystallization of the avian reovirus double-stranded RNA-binding and core protein sigmaA. Hermo-Parrado XL, Guardado-Calvo P, Llamas-Saiz AL, Fox GC, Vazquez-Iglesias L, Martínez-Costas J, Benavente J, van Raaij MJ. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 May 1;63(Pt 5):426-9. Epub 2007 Apr 20. PMID: 17565188 Crystal structure of the avian reovirus inner capsid protein sigmaA. Guardado-Calvo P, Vazquez-Iglesias L, Martinez-Costas J, Llamas-Saiz AL, Schoehn G, Fox GC, Hermo-Parrado XL, Benavente J, van Raaij MJ. J Virol. 2008 Nov;82(22):11208-16. Epub 2008 Sep 17. PMID: 18799570 Quoting Mario Milani mario.mil...@mi.infm.it: Thank you for the suggestions. The data resolution is 1.9 so i can try different heavy atoms techniques ... anyway i am really puzzled by the peculiar assembly in the crystal and on the possible causes... does anyone know about similar cases? mario Mario, beside what you were mentioning, I would definitely try a quick soak (10-30 seconds) of the crystals in cryo conditions supplemented with halides such as NaBr, or NaI, at pretty high concentrations (say 0.5 M), then directly freezing without backsoak. If the crystals survive the treatment, with that amount of mols per unit cells and surely some good NCS, you should be able to phase pretty easily. Well, of course SeMet would be the other option... ciao, s On Sep 30, 2010, at 1:54 PM, Mario Milani wrote: Dear all, i have a 30 kDa protein that crystallize so far in three different conditions but with the same space group. It initially looks like tetragonal (I4, a=141, b=141, c=208) and then results triclinic (P1, a=141, b=141 c=144, alpha=119, beta=119, gamma=90), hosting about 24 mol. in the unit cell. Other data: self rotation shows the presence of 4 peaks with chi=180; molecular replacement shows the presence of a pseudo-translation peak; DLS made at protein concentration close to crystal growth conditions shows a Rh compatible with something like a tetramer with low polydispersity (about 15%). Do you have any experience with similar asymmetric associations? Do you have any suggestions, beside the addition of ligands to the crystal growth conditions, in order to get a simpler crystallographic assembly? I have some models (with sequence identity less than 25%) in order to try MR but all trials so far did not solve the structure (using balbes, molrep, phaser and epmr). Any suggestion is welcome. Thank you, Mario Milani -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
