My fault for not changing the thread subject! I'm sorry!
Greetings to ccp4bb readers!
This is a long-delayed reply to requests that I received after I
posted an observation on the ccp4bb almost three years ago. (I think it
was early 2012). The paper had not yet been published (pending
finalization of experiments unrelated to structural
biology) and so I was not free to distribute any of the structure
factors to those who were interested. The paper now has been published,
and the coordinates and structure factors have been released. The
following is the peculiarity that made this of interest.
I refined a structure with 1.55 A data of a homotrimeric protein
bound to an inhibitor. Although the inhibitor added to the enzyme
contained a five-membered ring, the inhibitor we observed in two of the
three active sites had the ring opened. We therefore
discovered that the ring-opened form is also an inhibitor. However, in
the remaining active site, we discovered
both the ring-opened and ring-closed forms of the inhibitor.
(The active sites are identical, but crystal contacts create asymmetry
among the three). The two forms of the inhibitor were positioned
slightly differently in this active site, and so the
electron density at this resolution clearly identified the presence of
both forms, even though there was much overlap in their positions. I
used the CNS occupancy refinement script, and then normalized the
results so that the two occupancies added up to 1,
and so I came up with occupancies of 0.88 (ring-opened) and 0.12
(ring-closed). Then having all inhibitor molecules in the model, I
calculated maps with another program, which substituted Fcalc for
missing reflections, and could no longer see the lower occupancy
form in the electron density. I re-calculated maps in CNS, and again
saw both forms in the electron density. So I calculated new maps in CNS
the same way as before, but turned on the option to substitute missing
reflections with Fcalc: and the lower occupancy
form was missing from the density in the resulting maps.
So, this was the difference - when I used zero for missing
reflections, I was able to see both forms; but when I substituted Fcalc
for missing reflections in maps, I could only see the predominant form.
The data set was 99.6% complete overall, 99.2%
in the highest resolution shell; with a multiplicity of 5.8 (5.3 in the
highest shell). So there didn't seem to be many missing reflections to
be substituted. The test set contained roughly 2% of the total number of
observed unique reflections.
Some had replied to me, saying that they were interested in
using the coordinates & structure factors for running tests;
however, as I said, I was not free to distribute them at the time. Now
they are publicly available: PDB ID 4K9G. I hope this helps
all who are interested. Thank you.
Gregg
