[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]

Re: [ccp4bb]: strange NCS/refinement problem (twinning?)



Dear Dima,

i would try two more things,

try some more MR programs (EPMR! and AMORE, and so on) and see whether
or not the second solution is the same in all cases. Maybe the second molecule
is badly oriented.
May be Your second molecule is not fulfilling the NCS. Can be that helices or
domains have different orientation. It is importend to do Rigid-Body-refinement
with increasingly smaller rigid regions.

But before refining anything I would definitely run Resolve. Using
prime-and-switch phasing to reduce model bias. See what is coming back of
the second molecule. You may even want to trace the second molecule again,
whatever comes back. Than run resolve again, and trace, and so on. Between each
of these phasing steps You can calculate FOM's from the new model using SigmaA.
Worked for me in a case of 22% sequence identity with differently oriented helices.

With friendly regards
(:hristopher
 
 
 
 

Dima Klenchin wrote:

***  For details on how to be removed from this list visit the  ***
***          CCP4 home page http://www.ccp4.ac.uk         ***

Hello all,

I am facing a problem I am not sure how to address.
how to approach (short of looking for a different xtal form).

Good data set to 2A, indexes very well by P2 (67.947 76.813 98.441,
beta=101.21) or P1 (alpha and gamma within 0.5 close to 90).
Two molecules per cell. Scaled as P2, systematic absences indicate
P21 very clearly.

Molrep finds what appears to be a good solution readily. Problems
start after that: When I restrain or constrain NCS during refinement,
R free goes way up (R~30, Rfree > 40%). If I refine without NCS,
R factors slip right away to 27/29 but this strange thing
happens: one copy of the protein refines very well - low B factors,
very good looking map, two ligands totalling >100 non-H atoms
show up perfectly well on 1fofc map. A completely different
story with another copy of the molecule: B factors are sky
high and the map looks crappy with most of it being probably
model bias. Matthews coefficient is 6.1 with just one molecule...

At this point I have tried a lot of things hoping to find
an error in previous steps and nothing shows up. Can this be
twinned crystal? Yates server does not appear to think so.
Short of trying to find a new well-diffracting crystal form,
is there a reasonale solution?

Thanks so much for any help,

Dima

-- 
Dr. Christopher Lehmann
 
Center for Advanced             fon:    ++(301)-738-6126
Research in Biotechnology       fax:    ++(301)-738-6255
9600 Gudelsky Drive             e-mail: lehmann@umbi.umd.edu
Rockville, MD 20850, USA                

web: http://www.geocities.com/dr_christopher_lehmann/