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FW: [ccp4bb]: pseudo symmetry?

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Dear All,

We are still struggling with this one, which is why we haven't posted a
summary yet. We are now working in the big R3 cell with 4 molecules in the
AU. AMORE didn't work with a single molecule as the search model, but we
were successful when we used the dimer from the small R3 cell - confused?

Anyway, the maps look quite nice, but the refinement is a bit disappointing
: Rfac=25%, Rfree=28% (they were 19 and 22 in the small R3 cell). Also the
overall FOM in REFMAC is 0.51 (it was 0.84). Could this be because the
alternate layers of strong and weak reflections make it difficult to scale
Fobs to Fcalc in REFMAC? The data have been processed with DENZO/SCALEPACK,
maybe the situation could be improved using MOSFLM/SCALA. Unfortunately we
can't get MOSFLM to correctly autoindex in the large cell as the spot
picking routine struggles to find the weaker spots - it just gives us the
small cell. Is there a way to convert a DENZO autoindexing soultion into
MOSFLM format so that we can proceed with this approach??

Thanks once again,


-----Original Message-----
From: david lawson [mailto:david.lawson@bbsrc.ac.uk] 
Sent: Tuesday, March 21, 2000 12:51 PM
To: 'ccp4bb@dl.ac.uk'
Subject: [ccp4bb]: pseudo symmetry?

Dear All,

I'm afraid this is a bit of a long one.........

I wonder if I could get some help on a problem that is driving us mad here.
We have a 1.65 A resolution data set collected from a rhombohedral crystal
(there was also a low resolution pass at 2.8 A). All data were processed and
merged with DENZO/SCALEPACK. If we don't try too hard in the autoindexing we
get the following cell:

rhombohedral setting:	56.646   56.646   56.646   92.678   92.678   92.678
hexagonal setting:	81.960   81.960   93.417   90.000   90.000  120.000 

The data set then processes nicely as R32 with a completeness of 100% and an
overall Rmerge of 4.4% (11.0% for outer shell). If we do it as R3 it gives
virtually identical statistics. In R32 we get one molecule per AU with
Amore. This refines reasonably well (Rfree ~ 24%) but a few bond distances
persitently misbehave. If we do it as R3 with 2 molecules per AU, we can get
the Rfree down to ~ 22%. However the operation relating one molecule to the
other appears to be a near perfect crystallographic one. When the 2
independently refined molecules are superposed the differences are minimal
and I would say not significant. I think the improvement in Rfree is simply
due to allowing the refinement more freedom.

At this point we went back to the raw data and took a closer look. It turns
out that there are alternate layers of strong and very weak reflections. If
we drop the peak picking threshold in DENZO until it finds a significant
number of these weaker reflections we then get the following cell:

rhombohedral setting:	78.313   78.313   78.313   63.129   63.129   63.129
hexagonal setting:	81.987   81.987  187.165   90.000   90.000  120.000

ie. the c axis is twice as long in the hex setting. This also processes
nicely as R32 with a completeness of 100% and an overall Rmerge of 5.0%
(17.3% for outer shell). When we tried to run AMore on this it gave some
very strange results - some problem with defining the Cheshire cell for non
primitive space groups (although it does say in the manual that you can get
problems with rhombohedral cells). We assumed the weak reflections mean that
we have a repeating unit consisting of two of the "small" cells with the
molecules in virtually the same orientation. So we generated a second copy
of the molecule from our small R32 cell by applying half a unit cell
translation along c. This model was then put into rigid body refinement in
REFMAC, but Rfree was very high ~ 60% even though the packing looked fine.

If we look at the outputs from truncate (attached) they are a bit strange,
in particular for the big cell. Look at cumulative intensity distributions
and moment2 values for acentrics. There is also a bit of a bump in the
Wilson plot at about 2 Ang resolution.

As an added complication there is a very strong non-crystallographic 2-fold
axis relating one half of the molecule to the other, so that it looks
virtually identical if you invert it. This means you have to be very careful
which way up your MR solution is. 

We have tried submitting the data to the "Crystal twinning server"
(http://www.doe-mbi.ucla.edu/Services/Twinning/) but the twin fraction is
less than 2%.

So does anyone have any suggestions as to how we proceed? Obviously the easy
way out is to ignore the weak data and go with the small cell - after all an
Rfree of below 25% is definitely publishable!!

Many thanks in advance

Dave Lawson