[phenixbb] TaBr SAD Autosol and Phaser

Randy Read rjr27 at cam.ac.uk
Thu Apr 5 01:52:49 PDT 2018

Dear Ian,

If you could share the data (off-list, of course), I would be interested in looking to see why the cluster SAD phasing is giving results that are so much poorer than for individual Ta atoms.  It’s possible that something isn’t being done optimally — this feature in Phaser hasn’t been exercised as much as other features.

If SIR hasn’t worked, that might well imply that the native and TaBr crystals aren’t completely isomorphous.  In that case, cross-crystal averaging could be very useful.  If you can approximately define the volume covering the protein in the TaBr-phased map, you can cut that density out and use it to phase the native crystal by molecular replacement.  (Actually, just rigid-body refinement usually works and gives you operators near the identity, which makes life easier.)  Depending on how different the crystal forms are, cross-crystal averaging of the two maps might yield enough improvement to either start to see the individual atoms in the clusters or to place helices more accurately.  Once you have a map from averaging or a model that has higher resolution features, log-likelihood-gradient completion in Phaser is more likely to distinguish the individual atoms, which should help to push the resolution for which you get good phases.

I’m not sure why Phaser isn’t reproducing the FOM values you got in AutoSol.  If you have only provided the SAD data to AutoSol, then it should be using Phaser internally.  Have you specified different resolution limits in the two approaches?  

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: rjr27 at cam.ac.uk
Cambridge CB2 0XY, U.K.                               www-structmed.cimr.cam.ac.uk

> On 5 Apr 2018, at 00:22, Ian S <iscrystallo at gmail.com> wrote:
> Hi Phenixbb, 
> I trying to phase a structure soaked in TaBr and am running into some technical problems. My anomalous signal goes out to about 4.5 and I have a 1.9A native data set. SIR hasn't worked so far.
> Autosol SAD has given me several convincing solutions when searching for Tantalum (not TX), finding 11 sites (not clustered) at 6 angstroms: FOM =0.478, map=0.1.3, and corr of 0.82 and a final score of 37+-15.71. By only playing around with the sites I can get an overall score of 73 +- 4.58, map skew to 0.5, and a RMS density to 0.79, thought the R-factor increases to .43 and the FOM . 
> Perhaps not suprisingly at this resolution, the map is uninterpretable (maybe some helices) so I'm trying to improve the phases by model building and by using Phaser and giving it TX sites. 
> If I put the Ta sites in Phaser with the same dataset as Autosol, I get a FOM of .342. If I use the TX label it drops to 0.143. What is happening here? I know that typically TaBr has one or two major sites (my protein isnt massive, only about 1200 aa predicted in the ASU) , so I use only the top two major sites and have the same problem. Does this indicate that the Ta sites Autosol found are not in fact the sites I'm looking for? Or maybe that there really are 10 TaBr sites all with low occupancy? Any suggestions would be appreciated. 
> Thank you. 
> Best,
> Ian
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