On possibility for #5, the B factors all dropping to the lower limit
during refinement. If you are including all of your low resolution data
(which you should) but have not used a model for the bulk solvent scattering
of X-rays (which would be bad) then you will observe this result. The
refinement program will attempt to overestimate the amplitudes of the
high resolution Fc's to match the overestimated low resolution Fc's.
Check you log files to ensure you bulk solvent correction is operating
correctly.
Dale Tronrud
Jorge Iulek wrote:
Dear all,
Please, maybe you could give some suggestions to the problem below.
1) Images show smeared spots, but xds did a good job integrating them.
The cell is 229, 229, 72, trigonal, and we see alternating strong and
weak rows of spots in the images (spots near each other, but rows more
separated, must be by c*). They were scaled with xscale, P622 (no
systematic abscences), R_symm = 5.3 (15.1), I/sigI = 34 (14) and
redundancy = 7.3 (6.8), resolution 2.8 A. Reciprocal space show strong
spots at h, k, l=2n and weak spots at h, k, l=2n+1 (I mean, l=2n
intensities are practically all higher than l=2n+1 intensities, as
expected from visual inspection of the images). Within planes h, k,
l=2n+1, the average intensity is clearly and "much" *higher at high
resolution than at low resolution*. Also, within planes h, k, l=2n, a
subjective observation is that average intensity apparently does not
decay much from low to high resolution. The data were trucated with
truncate, which calculated Wilson B factor to be 35 A**2.
2) Xtriage points a high (66 % of the origin) off-origin Patterson peak.
Also, ML estimate of overall B value of F,SIGF = 25.26 A**2.
3) I suspect to have a 2-fold NCS parallel to a (or b), halfway the c
parameter, which is "almost" crystallographic.
4) I submitted the data to the Balbes server which using
pseudo-translational symmetry suggested some solutions, one with a good
contrast to others, with a 222 tetramer, built from a structure with 40
% identity and 58% positives, of a well conserved fold.
5) I cannot refine below 49 % with either refmac5, phenix.refine or CNS.
Maps are messy, except for rather few residues and short stretches near
the active site, almost impossible for rebuilding from thereby. Strange,
to me, is that all programs "freeze" all B-factors, taking them the
program minimum (CNS lowers to almost its minimum). Might this be due to
by what I observed in the reciprocal space as related in "1" ? If so,
might my (intensity) scaling procedure have messed the intensities due
to their intrinsic "property" to be stronger in alternating planes ? How
to overcome this ?
6) I tried some different scaling strategies *in the refinement step*,
no success at all.
7) A Patterson of the solution from Balbes also shows an off-origin
Patteron at the same position of the native data, although a little lower.
8) Processed in P6, P312 and P321, all of course suggest twinning.
I would thank suggestions, point to similar cases, etc... In fact,
currently I wondered why refinement programs take B-factor to such low
values
Many thanks,
Jorge
