Dear Stefano,
At this resolution, re-refining your model may not yield a good absolute
scale, as you have noticed. If you have a homologous X-ray structure for
the virus, then you could try rigid-body fitting that into the map at
different scales and see which one fits best (so without re-refinement).
Best would be to have an independent measurement of the pixel size on
the same scope, for example by solving a known structure. Perhaps some
of your colleagues at the institute have determined the pixel size at
this magnification already?
HTH,
Sjors
On 09/17/2018 11:08 AM, Stefano Trapani wrote:
>
> Dear all
>
> We are refining the atomic model of an icosahedral capsid based on a
> cryoEM map obtained from samples of intact viruses. The nominal pixel
> size of the map is 1.21 A and the resolution (according to Relion) is
> 3.9 A.
>
> We have tried to finely calibrate the map pixel size by running
> several refinements of the atomic model at different map magnification
> levels (phenix.auto_sharpen + phenix.real_space_refine with 2ry
> structure restraints and no ADP refinement).
>
> Results are quite confusing (see attached graphs, the nominal pixel
> size is represented by the black thick vertical line):
>
> * the best map-to-model fit is found at a rather larger pixel size
> (~1.35A) than the nominal one (1.21 A);
> * however the resulting icosahedral assembly is quite loose around
> 1.35 A pixel size (according to PISA predictions the 60-meric
> would not be stable);
> * also, quite a few Ramachandran conformations initially in favoured
> regions shift towards less favoured (though allowed) regions as
> the map-to-model fit increases.
>
> Do you have any suggestion on how to interpret these results or a
> better procedure (if any) to better evaluate the map magnification ?
>
> Cordially
>
> --
> Stefano Trapani
>
> Maître de Conférences
> http://www.cbs.cnrs.fr/index.php/fr/personnel?PERS=Stefano%20Trapani
> -------------------------------------
> Centre de Biochimie Structurale (CBS)
> 29 rue de Navacelles
> 34090 MONTPELLIER Cedex, France
>
> Tel : +33 (0)4 67 41 77 29
> Fax : +33 (0)4 67 41 79 13
> -------------------------------------
> Université de Montpellier
> CNRS UMR 5048
> INSERM UMR 1054
> -------------------------------------
>
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--
Sjors Scheres
MRC Laboratory of Molecular Biology
Francis Crick Avenue, Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44 (0)1223 267061
http://www2.mrc-lmb.cam.ac.uk/groups/scheres
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