Hi Jorge,

I imagine your 222 tetramer makes a sort of 'pancake' which fits into a
cell of 229x229x36 when you apply the 6-fold symmetry. If that was the
case in the crystal, then these would be the cell dimensions that you
would get.

But I suspect you have a situation where the cell repeat has a pancake
that is either slightly shifted in the plane normal to the 6-fold, or
rotated out of that plane by a small number of degrees. So the crystal
would have a doubled unit-cell, with the weak inter-layers. If the two
pancakes had been exactly parallel, and exactly 36A apart, the weak
layers would have disappeared completely, and the situation would reduce
to the smaller cell.

Because of the slight translation/rotation between two adjacent smaller
cells, you get the weaker layers. The perturbation must be really small
that it is much less noticeable at low res, which is where you see the
weak reflections in the l=2n+1 layers. As the res goes up, the ability
to discern the differences goes up, giving the more intense spots in the
outer part of the diffraction pattern. This situation would still occur
in the presence of systematic absences due to an unidentified screw
axis, as suggested by another contributor.

You can try for better ordered crystals, as suggested by someone else.
But to rescue this data set, I would look for one good MR solution, then
use it as a fixed solution and use the same rotation solution (or one
very close to it) to find a second translation solution. This should be
within a small fraction of 0,0,0.5. After rigid body refinement, you
might see the rotation of the 'pancake' clearly. But to get acceptable
R-factors, you must try all the screw axis combinations, 6, 6(1), 6(2),
6(3), 6(4) and 6(5). With a bit of luck, one of these will be much
better than the others.

One final remark: You seem to have cut off the res at 2.8A despite the
significant I/sig(I) statistic in the outer shell, combined with a
benign R-merge. This is understandable if it is due to geometry, but
really, you must go for higher res, and maybe you will get an even
clearer answer. If your data collection system is limiting for the above
cell dimension and res combination, you should try a different facility,
with a larger detector or shorter wavelength, or both. Synchrotrons are
usually good for this sort of thing (of course I am advertising!).

Good Luck.

Pierre
************************************************************************
*******
Pierre Rizkallah, Daresbury Laboratory, Warrington, Cheshire WA4 4AD,
U.K.
Phone:  (+)44 1925 603808      Fax:  (+)44 1925 603124
e-mail: [log in to unmask] html: http://www.srs.ac.uk/px/pjr/

-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
Jorge Iulek
Sent: 27 August 2007 12:48
To: [log in to unmask]
Subject: [ccp4bb] alternating strong/weak intensities in reciprocal
planes - P622

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