Do you have continuous chains all the way through? Any possibility of domain swapping?
leo - Leonard Chavas - Synchrotron SOLEIL Proxima-I L'Orme des Merisiers Saint-Aubin - BP 48 91192 Gif-sur-Yvette Cedex France - Phone: +33 169 359 746 Mobile: +33 644 321 614 E-mail: leonard.cha...@synchrotron-soleil.fr - > On 05 May 2017, at 17:12, Roger Shek <roger.s...@stonybrook.edu> wrote: > > Hello Randy, > > I appreciate all your help. Phaser found 12 molecules after some long > computing time. However, it looks like some molecules are clashing. Though, > the electron density looks pretty good. However, where the molecules are not > clashing (the two gray and orange molecules), there is still the original > problem, but the ED looks good when contoured up to ~>4 sigma. The rest of > the molecules found do not have that problem and looks good at 1.5 sigma > although the clashes are still an issue. Any tips on how to proceed? I ran > phaser from scratch with the search model as a monomer and not using the > three molecules found as the search model. > > <1dotfivesigma.PNG><molecules.PNG><not clashing.PNG><contoured down.PNG> > > On Wed, Apr 26, 2017 at 4:52 PM, Randy Read <rj...@cam.ac.uk> wrote: > Dear Roger, > > You left a lot of very relevant detail out of your presentation of the > problem! It seems that your data have been merged as P6222, but then you’re > using Phaser to expand that back out to P1 and search for multiple copies. > Assuming the data merged well in point group 622, then either that’s the > right symmetry or the crystal is twinned. I’m guessing from the 2nd moments > in the Phaser log file that it probably is twinned, but the presence of tNCS > will mask the twinning in those moment statistics. > > I’m guessing you haven’t really looked at the log file in any detail and > tried to understand the warnings it is giving you. You’re searching for 4 > copies, but the suggestion of 12 is more likely. If you had 622 point group > symmetry, there would be 12 symmetry related copies, but then that wouldn’t > explain the presence of tNCS, which requires more than one unique copy per > asymmetric unit. However, it does fit with the idea that the true symmetry > is somewhat lower. So trying P1 instead of all the many other subgroups > (which Phaser would have listed for you before you set the symmetry to P1) is > a good first choice, but you have to look for the right number of copies. > > The next warning is that the default NMOL of 2 (where NMOL is the number of > tNCS-related copies in the same orientation) doesn’t fit with the analysis. > It makes more sense to have 3 copies related by a sort of lattice tripling, > i.e. the original copy, one translated by about 1/3,2/3,0 and a third > translated by 2/3,1/3,0 (remember that 2*2/3=4/3 is equivalent to 1/3, after > taking account of the unit cell translation). So you need to run a job > specifying NMOL 3. I’m not surprised it didn’t work with the wrong NMOL. On > the other hand, when you turn off the tNCS correction, any potential solution > that places copies related by the Patterson vector will look good, because it > explains the biggest feature in the data, the systematic modulation caused by > the tNCS. > > Finally, you didn’t mention that you have hundreds of solutions. When you > don’t get a unique solution, you have to start worrying about what went > wrong. There’s no reason to believe that the first one is correct. > > By the way, why did you set the estimated RMS error to 2A? Did you try a run > first in which you specified the sequence identity? That usually works > better, and any model that is so bad that 2A is a suitable RMS error is very > unlikely to work. If you give too large a number, you’re effectively > throwing away a large amount of information, because Phaser doesn’t expect to > be able to fit data beyond 3.6A resolution and therefore omits those data > from the calculation. > > If you’re lucky, if you run a job setting NMOL to 3, specifying that there > are 12 copies in the a.u. and looking for 12 copies with an appropriate > setting for the model quality, you might get something promising! > > Randy > > ----- > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: +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 26 Apr 2017, at 22:27, Roger Shek <roger.s...@stonybrook.edu> wrote: > > > > actually I turned off corrections for tNCS because it failed to find a MR > > solution. The logfile is attached. > > > > On Wed, Apr 26, 2017 at 4:19 PM, Randy Read <rj...@cam.ac.uk> wrote: > > Could you send me the logfile from the job that found 3 copies? Normally > > when there is tNCS and Phaser uses it, you get the right multiple of > > copies. So I’m concerned that it didn’t actually use the tNCS for some > > reason. > > > > Best wishes, > > > > Randy > > > > ----- > > Randy J. Read > > Department of Haematology, University of Cambridge > > Cambridge Institute for Medical Research Tel: +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 26 Apr 2017, at 22:07, Roger Shek <roger.s...@stonybrook.edu> wrote: > > > > > > I searched for 4 copies, but Phaser only found 3. It's possible there is > > > an additional copy of the molecule in the ASU > > > > > > On Wed, Apr 26, 2017 at 2:40 AM, Randy Read <rj...@cam.ac.uk> wrote: > > > Dear Roger, > > > > > > You didn’t mention this, but did you search for 2 copies (or an even > > > number of copies) and, if so, did Phaser take account of the > > > translational NCS in the calculation? > > > > > > Best wishes, > > > > > > Randy Read > > > > > > ----- > > > Randy J. Read > > > Department of Haematology, University of Cambridge > > > Cambridge Institute for Medical Research Tel: +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 26 Apr 2017, at 02:23, Roger Shek <roger.s...@stonybrook.edu> wrote: > > > > > > > > Hi everyone, > > > > > > > > We have a structure that phaser found a solution for in P1, even though > > > > there is a large off-origin Patterson peak indicating pseudo NCS. > > > > However, the 2mFo-Fc map needs to be contoured up to >3 sigma to be > > > > reasonable, going lower makes it look like what I normally see at <1 > > > > sigma. If you look at the Fo-Fc map, there are large areas of positive > > > > and negative density. The 2mFo-Fc map looks very convincing, you can > > > > see the tyrosines, phenyl rings, etc...Also phaser gave LLG of >400 and > > > > a TFZ of ~14. Rfree is ~0.45. Does anyone know what's going on? > > > > > > > > Thanks, > > > > Roger > > > > > > > > -- > > > > Roger Shek > > > > > > > > Stony Brook University > > > > Graduate Student in Biochemistry and Structural Biology (PhD) > > > > Stony Brook Integrative Structural Biology Organization > > > > Cell: (808) 386-3879 > > > > > > > > > > > > > > > -- > > > Roger Shek > > > > > > Stony Brook University > > > Graduate Student in Biochemistry and Structural Biology (PhD) > > > Stony Brook Integrative Structural Biology Organization > > > Cell: (808) 386-3879 > > > > > > > > > > -- > > Roger Shek > > > > Stony Brook University > > Graduate Student in Biochemistry and Structural Biology (PhD) > > Stony Brook Integrative Structural Biology Organization > > Cell: (808) 386-3879 > > <phaser.log> > > > > > -- > Roger Shek > > Stony Brook University > Graduate Student in Biochemistry and Structural Biology (PhD) > Stony Brook Integrative Structural Biology Organization > Cell: (808) 386-3879
