On Thursday, 26 January 2017 11:03:12 PM Claire Smith wrote:
> Hello,
>
> I would like to ask a question related to a recent thread regarding
> bad/missing density.
>
> I have used SAD to solve the structure of a protein at about 2.6Å
> resolution. Phenix build a good portion of it (about 50%) and the density
> in this region is good. However, we cannot see ther rest 50%. Rfree is
> currently at 32%. No twinning is suggested.
>
> How can we "find" the missing 50%? We have tried MR-SAD with no significant
> improvement. We know the missing mass is there because we ran the crystals
> on a gel, and the protein is intact.
>
> I know with MR sometimes the space group can make a difference, but with
> experimental phasing (SAD), if the space group were incorrectly
> identified, would we have gotten a solution to the Se substructure? This
> seems correct because the visible 50% of the protein make total sense. So,
> since we have a correct substructure, can I conclude with confidence that
> the space group is correctly identified?
>From your description it sounds unlikely that the unit cell is correct but
the space group is wrong. However the symptoms do match a possible
missed supercell, such that you collected and refined data from only
a subset of the true unique data.
I will try to describe this in words but a picture would be so much easier...
I will try ascii art. Please view in a fixed spacing font.
Suppose the true cell looks like this:
-----------------------------------
| AAA | AAA BBBBBBB |
| AAAABBBBB | AAAAB BBB |
| AAA BBB | AAA |
-----------------------------------
I.e. suppose you have pseudo-translational NCS such that domain A
superimposes perfectly on itself but domain B does not.
If you incorrectly index that cell edge as being only 1/2 its true length,
you measure only half of the true data but it refines nicely to
describe a fully-occupied A and a mess in the region where B should be.
(superimposed 1/2 intensity ghosts made noisier by the missing data).
Yes I've hit this in real life, with an even messier case of cell-edge
tripling rather than doubling.
If you want to pursue this possibility, you should go back to the
diffraction images and look really hard for weak spots in between the
indexed spots.
good luck,
Ethan
> Of course, it could be that the missing bit is very flexible and does not
> scatter coherently, but then, wouldn't we expect a lower Rfree?
An Rfree of 0.32 for 2.6A refinement doesn't sound that bad.
> Thanks so much!
>
> Claire
>
--
Ethan A Merritt, Dept of Biochemistry
Biomolecular Structure Center, K-428 Health Sciences Bldg
MS 357742, University of Washington, Seattle 98195-7742
