[ccp4bb]: Summary: strange NCS/refinement problem

[Dima Klenchin <klenchin@facstaff.wisc.edu>]

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I have received total 16 replies to my inquiry. Many thanks to 
everyone who replied!  

In brief, I described a case of apparent inapplicability of NCS
during refinement where NCS-related molecules behave as if they
were very different. I asked what I might be doing wrong and 
how to deal with such situation properly. (Complete original 
text can be found in the end of this message).  

The general consensus in replies was that most likely this is 
a genuine case of a global disorder for one of the molecules 
in the AU. Such cases frequently manifest themselves by a 
packing where "loose" molecule makes less contacts with 
others, so looking at packing arrangement is advised. Doing 
tests to make sure that the second molecule is actually 
present was recommended too (see below). 
 
Almost everyone agreed that whenever a compelling point for
a disorder in NCS-related molecule(s) is made, generally the 
best way to go is to use TLS refinement [with Refmac]. 
Several refs that dealt specifically with such problem and 
used TLS approach were provided: 
	J Biol Chem, 2002, 277(16):14077-84
	Acta Crystallogr D, 2000, 56 (Pt 3):313-21
	J Biol Chem, 2001, 276(29):27555-61

Several people pointed out that high Vm (6.3 in my case) is 
not by itself a good indication that there is more than one
molecule per AU. In some cases, Vm as high as 7.5 has 
been observed (Structure, 1998, 6(9):1105-16). Several 
options to find out of there is more than one molecule
per AU were mentioned: 
	- Refine "good" molecule as good as possible, then
leave the second one out and see of the density for it 
comes back. 
	- Run ARP/wARP or Resolve in "prime and switch" mode
presenting them with only "good" molecule. See if they are
able to pick a second molecule and see if the NCS operators 
are the same as ones in MR solution. (I have done this 
previously - both programs picked and correctly positioned
couple most conserved parts of the protein with ARP 
generally doing much better job at it). 
	- Do a self-rotation function search and see if 
there are indeed additional significant peaks appearing. 
	- With a "good" molecule fixed, repeat MR 
looking for a second one; in addition to Molrep, try using 
other programs like EPMR, Como, AmoRe, see if the results
are consistent with each other.  

Finally, a possibility of misindexing was mentioned. Trying 
to scale as P1 and seeing how things refine then was an option
in my case. 

Once again, thanks. 

Dima

--------------------------------------------------------------
Original message:
>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 AU. 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 6.3 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? 
>