If you have a high off origin peak at 0 0 0.5 you must have absences
along the 00l axis also consistent with
P63 22.
So you need to test both P6322 and P6 22
And is the tetramer generated by crystal symmetry or is the whole thing
in the asymmetric unit?
Eleanor
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
