Re: [ccp4bb] Rfactors stuck very high
Dear Garib, I have run the jellybody refinement and whilst it has not reduced the Rfactors I am going to see whether I can see anything usable in the new maps. many thanks james Dr James Garnett Centre for Structural Biology Division of Molecular Biosciences Level 5 Sir Ernst Chain Building South Kensington Campus Imperial College London London SW7 2AZ Tel: +44 (0) 207 594 5464 Fax: +44 (0) 207 594 3057 From: Garib N Murshudov [ga...@mrc-lmb.cam.ac.uk] Sent: 09 July 2012 12:07 To: Garnett, James A Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rfactors stuck very high Dear James It seems that at this stage you may not be able to distinguish between these space groups. As I see they are subgroup of each other (more precisely P1 C2 I222 with the cell parameters you have) and it would not be easy to find out the exact space group at this stage. Highest space group may be more likely than others in the absence of other evidences. It seems that the problem is that the model is sufficiently far and refinement programs struggle to converge. We had some good results for this type of cases when we used jelly body refinement in refmac with 40-100 cycles and sigma set to 0.01. Phenix may have similar tricks. After that you can try either shelxe or arpwarp or one after another. Although at 2.3A they may not produce results as spectacular as in presentations, they may give you improved maps to work with. regards Garib On 8 Jul 2012, at 22:11, James Garnett wrote: Dear all, I have some troublesome data to ~2.3A and I hope someone can help. My data indexes and scales equally well in I222, C2 and P1 I222 a=46.3 b=76.3 c=81.1 C2 a=111.3 b=46.3 c=76.3 beta=133.2 (reset in I2 a=76.3 b=46.3 c=81.1 beta=90.1) C2 a=94.5 b=76.3 c=46.3 beta=119.6 (reset in I2 a=46.3 b=76.3 c=81.1 beta=90.1) P1 a=46.2 b=60.2 c=60.2 alpha=78.5 beta=67.4 gamma=67.4 I have found a molecular replacement solution in I212121 using an NMR structure of the same protein and MR-ROSETTA/PHENIX (PHASER LLG=128 TFZ=12.3), although I can not refine this below R ~45% and Rfree ~50%. The maps look OK in parts but in other regions the connectivity is much reduced. In case of model bias I have used density modification and also used simulated annealing etc in case it is stuck in a local minima - these did not help. This protein is an Ig-like fold (potential for pseudo-internal symmetry) and so I have also played around with rotations of the structure but this has not helped. Although twinning analysis in all spacegroups suggest there is no twinning I have tried refinement in PHENIX and REFMAC using twin laws but this does not help. I do not detect any off origin peaks in a native Patterson map but I do see peaks suggesting NCS in self rotations in I222 (see below for peaks above 50% origin) - there can only be 1 mol/au though. Does this suggest rotational lattice defects? Eulerian anglesPolar angles Alpha Beta Gamma Peak OmegaPhi Kappa Direction cosines Symmetry: 1 2 Peak1 Origin peak 100.0 0.00.00.0 Peak2 1 10.00.0 180.0 100.0 0.00.03.1 0. 0. 1. Origin peak 100.0 0.00.0 180.0 Peak3 1 10.00.0 180.0 100.0 0.00.03.1 0. 0. 1. Origin peak 100.0 180.00.0 180.0 Peak4 1 10.0 180.0 180.0 100.090.00.03.1 1. 0. 0. 1 20.0 180.00.0 100.090.0 90.03.1 0. 1. 0. Origin peak 100.090.00.0 180.0 Peak5 1 10.0 180.0 180.0 100.090.00.03.1 1. 0. 0. 1 20.0 180.00.0 100.090.0 90.03.1 0. 1. 0. Origin peak 100.090.0 180.0 180.0 Peak6 1 10.0 180.00.0 100.090.0 90.03.1 0. 1. 0. 1 20.0 180.0 180.0 100.090.00.03.1 1. 0. 0. 1 30.00.0 180.0 100.0 0.00.03.1 0. 0. 1. Origin peak 100.090.0 90.0 180.0 Peak7 1 1 270.0 89.9 90.0 51.990.00.0 89.9 1. 0. 0. 1 2 270.0 89.9 270.0 51.945.0 270.0 180.0 0. -0.7067 0.7075 1 3 90.0 90.1 270.0 51.990.0 180.0 90.1
Re: [ccp4bb] immobilized DNA resin
Hi Alex, as you have a DNA binding protein does it have a high pI? If so, good old ion exchange with an S-sepharose column should do it. Cheers James Dr James Garnett Division of Molecular Biosciences, Imperial College London, Level 5, Biochemistry Building, South Kensington, LONDON SW7 2AZ, UK. Tel: +44 (0) 207 594 5464 Fax: +44 (0) 207 594 3057 - Reply message - From: Raji Edayathumangalam r...@brandeis.edu To: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] immobilized DNA resin Date: Sun, Apr 10, 2011 03:38 Hi Alex, Most DNA-binding proteins has decent affinity to heparin columns. Have you tried purifying your protein over heparin columns? Cheers, Raji On Sat, Apr 9, 2011 at 8:44 PM, Alexandra Deaconescu deac...@brandeis.edumailto:deac...@brandeis.edu wrote: Hello ccp4 enthusiasts: I am afraid this is a non-ccp4 related question. Can anyone recommend an immobilized dsDNA chromatographic resin for purification of DNA-binding proteins? GE seems to have something - I was wondering if people have other recommendations? In the age of GST and His tags etc., these are not very much used, but I do not have a tag in this case... Thanks a lot, Alex -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Is my data twinned or not?
Dear all, I have a small three helix bundle domain (~14 kDa) which after data analysis I believed crystallized in either P41212 or P43212 (a=b=121.8 c=118.0) with between 4 and 8 molecules in the ASU and I have data to 3.2A. However after looking at a native Patterson there is a non-origin peak which is also detected in phenix.xtriage and in MolRep. Due to the presence of this pseudo-translation vector I could have any of the P422 space groups. Then to be thorough (and because on some of my images the spots show splitting) I tested for twinning. These tests do not detect obvious twinning (although I^2/I^2 is rather high - see below), however, if I process in P4 assuming merohedral twinning or P222 (a=118.3, b=122.0, c=122.8) assuming pseudo-merohedral twinning, the H-test and Britton-test suggest a twin fraction of ~0.42. All data have similar Rsym values (P422=0.094, P4=0.085, P222=0.082). So my first 2 questions are - 1) Are the twinning tests not revealing twinning due to the presence of the pseudo-translation vector? 2) When I process in P4 or P222 are the twinning fractions from the H-test and Britton-test towards those of a perfect twin because the real point group is actually P422? --- 'P422 - xtriage' Wilson ratio and moments Acentric reflections I^2/I^2:2.466 (untwinned: 2.000; perfect twin 1.500) F^2/F^2:0.745 (untwinned: 0.785; perfect twin 0.885) |E^2 - 1|:0.815 (untwinned: 0.736; perfect twin 0.541) Centric reflections I^2/I^2:3.809 (untwinned: 3.000; perfect twin 2.000) F^2/F^2:0.615 (untwinned: 0.637; perfect twin 0.785) |E^2 - 1|:1.087 (untwinned: 0.968; perfect twin 0.736) NZ test (0=z1) to detect twinning and possible translational NCS --- | Z | Nac_obs | Nac_theo | Nc_obs | Nc_theo | --- | 0.0 | 0.000 |0.000 | 0.000 | 0.000 | | 0.1 | 0.107 |0.095 | 0.217 | 0.248 | | 0.2 | 0.211 |0.181 | 0.340 | 0.345 | | 0.3 | 0.304 |0.259 | 0.432 | 0.419 | | 0.4 | 0.386 |0.330 | 0.488 | 0.474 | | 0.5 | 0.451 |0.394 | 0.538 | 0.520 | | 0.6 | 0.508 |0.451 | 0.578 | 0.561 | | 0.7 | 0.558 |0.503 | 0.616 | 0.597 | | 0.8 | 0.602 |0.551 | 0.652 | 0.629 | | 0.9 | 0.642 |0.593 | 0.677 | 0.657 | | 1.0 | 0.679 |0.632 | 0.705 | 0.683 | --- | Maximum deviation acentric : 0.058| | Maximum deviation centric : 0.031| | | | NZ(obs)-NZ(twinned)_acentric : +0.042| | NZ(obs)-NZ(twinned)_centric : +0.010| --- L test for acentric data using difference vectors (dh,dk,dl) of the form: (2hp,2kp,2lp) where hp, kp, and lp are random signed integers such that 2 = |dh| + |dk| + |dl| = 8 Mean |L| :0.485 (untwinned: 0.500; perfect twin: 0.375) Mean L^2 :0.317 (untwinned: 0.333; perfect twin: 0.200) The distribution of |L| values indicates a twin fraction of 0.01. Note that this estimate is not as reliable as obtained via a Britton plot or H-test if twin laws are available. I also have an NMR structure as a molecular replacement model and a 4 wavelength Ta6Br12 MAD data set and a few derivative IR data sets. Molecular replacement does not work in any of the P422 space groups using MolRep and this may be because there are too many molecules in the ASU or because it is an NMR structure. The highly isomorphous Ta6Br12 data set has been processed to 4A although there is no anomalous signal between 8-4A. The heavy atoms in the 4A isomorphous replacement data seem to have been incorporated although some of the cells are no longer isomorphous. My final question is - 3) Although the lack of anomalous signal above within the MAD data may be due to other issues, is there any way twinning can cause this? I apologise for the long email but before I collect more data I would like to get a few things resolved and hopefully limit the possible space groups to make like much easier (as I feel a little like Alice going deeper into the rabbit hole at the moment). Any responses to these questions and any other suggestions will be very much appreciated. Kind regards James Dr James Garnett Division of Molecular Biosciences, Imperial College London Level 5, Biochemistry Building, South Kensington, LONDON, SW7 2AZ, UK. Tel: +44 (0) 207 594 5464 Fax: +44 (0) 207 594 3057
