The 34-34-34 cell does not predict all the spots, does it?
from the diffraction pattern it seems only the 34-34-170 or 34-34-340
cell can predict all spots, so the structure should be solved in the
one that predicts all spots.
The procedure I would use is to take a 180º dataset, sacrificing some
resolution if necessary, then integrate in P1 and solve the structure
by MR or if possible by just placing the double helices in the cell.
If you then still have disorder of the bases this means that the dsRNA
can fit in the density in several ways, either up and down, or, less
likely, if the ends of the 10 bp duplexes are not clear, even
displaced by one of more bases along the long axis. In principle there
is no reason why the differences in sequence ("inside" structure)
always have to lead to significant differences in the "outside"
structure (the phosphates) and thus affect the crystal packing. They
usually do, but not always (some dimeric transcription factors
crystallised with asymmetric dsDNA duplexes have disordered bases, as
long as the bases with which the protein interacts are the same the
protein doesn't necessarily care that much about the rest...).
Once you have your structure clear in P1, you can introduce the
possible symmetry axes one by one; and once you have your spacegroup
clear, collect as high as possible resolution data with the minimal
wedge necessary and as high a dose as possible without inducing
radation damage.
If your duplexes fit in two ways, "up" and "down", refinement should
be possible with both at partial occupancy.
Mark J. van Raaij
Dpto de Bioquímica, Facultad de Farmacia
Universidad de Santiago
15782 Santiago de Compostela
Spain
http://web.usc.es/~vanraaij/
On 29 Jan 2009, at 10:45, Margriet Ovaere wrote:
Dear all,
There were some comments about detector issues, but these can be
ruled out, to my opinion, since the lines appeared on different
beamlines.
Default settings of mosflm (spot picking) finds the cell 34 34 34 90
90 90 (pointless indicating P41212)
Structure was solved by SAD phasing on the phosphates in this space
group. Double helices stack in continuous helices, the backbone is
well defined in the (refined) density maps but the individual bases
are messy (purines and pyrimidines seemed to overlap) + obviously
not all spots were covered and the duplex does not fit in the A.U.
For this reason the integration was repeated in the higher cell 34
34 170
Space group most probably P212121, but solutions can be found in
P41212 as well (still disordered bases)
There are also indications that the 41 screw axis is rather a pseudo
axis than a pure crystallographic one, also in the small cell
Reindexing the cell to 34 34 340 also gives a solution, which
supports the theory of Holton
Rmerg is around 5% for the small cell, about 8% for the 170Å cell
(both in P41212)
Which refinement procedure would be best to follow?
kind regards
Margriet
Margriet Ovaere
Chemistry Department K.U.Leuven
Biomolecular Architecture
Celestijnenlaan 200 F
B-3001 Heverlee (Leuven)
Tel: +32(0)16327477
Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm for
more information.
