Hi Yanming,
when you use your two correctly placed molecules have you tried to
calculate a composite omit map using CNS. I assume your crystal lattice
does not look very well packed when you only have two molecules per asu
? That would be a good indication that your third molecule is truly
there if the packing would make sense.
Even if you only have two molecules, you should try to run DM using
twofold NCS that would still increase your signal to noise by a factor
of 1.4. I would for example start a DM run from e.g. 2.5 A and extend to
1.8 A in perhaps 200 cycles for a first quick look without improving
your mask derived from one molecule. Then check with Coot in the
difference density map if there is a hint for your third molecule.
Good luck,
Juergen
Yanming Zhang wrote:
> Hi ,
> Please help me on this structure:
> Data 1.8A: cell 84.892 84.892 172.580 90 90 90 with tetrahedral index
> Space group: P43212 or P43, Final refinement Rfree indicates P43 is
> better so most likely P43(also the systematic absence indicate P43 is it)
> Matthew coeffient indicates: P43 3mol/a.u(2.58,52%) 2mol/a.u(3.87, 68%)
>
> MR: Both Molerep and Phaser find out 2mol/au initially. Refinement
> reaches Rfree 30% , R 27% finally. WE think, comparing with 1.8
> resolution, The R and Rfree are too high. Also considering the 1.8A
> resolution, there should be 3 molecules in total. So we engaged into
> the effort to find out the 3rd molecule.
>
> Phased MR by MOLREP did find out the 3rd, so did Phaser(fix two, find
> out the 3rd). Both solutions gave relatively poorer statistics but
> packing is perfect. The 3rd molecule, solved by phased MR of MOLREP
> and PHaser respectively, can not superpose showing slightly different
> orientations, but position is similar. Refinement indicates PHased
> MOLREP MR gives lower Rfree.
>
> Using totally 3 molecule, When I restrain or constrain NCS during
> refinement(used TLS),R free goes way up (R~30, Rfree > 36%). If I
> refine without NCS,R factors slip right away to 27/30 but this strange
> thing
> happens: two copies of the original solution refines very well - low B
> factors,very good looking map. The 3rd copy, however, B factors are
> high and the map is bad, no good density match with the model.And the
> 3rd molecule did no help to Rfree or R.
>
> Story ends with my questions:
> 1,It seems the 3rd copy non-exist or globally disordered, under this
> circumstance, can I say 'because of the 3rd molecule is globally
> disordered so Rfree and R stay high (2mol/a.u with Rfree 30% R 27%,
> 1.8A data)?
> 2,Anything I should worry about in the process finding out the 3rd?
> 3,Are there suggestions to further lower down Rfree (the reasonable
> Rfree should be around 22%)?
>
> THANKS
> Yanming
>
--
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone: +1-206-616-4510
FAX: +1-206-685-7002
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