[phenixbb] handling of alternate conformations

[Ulrich Baumann]

Sorry, my mail before got corrupted. Thanks to Pavel for the swift reply  
and help.

My first question is whether NCS constraints are also applied to alternate  
conformations.

My second question concerns the atom count of the conformers, where the  
parts with non-alternating conformations seem to be included as well.


Thanks again,

Ulrich

On Mon, 16 Jul 2007 19:50:03 +0200, Pavel Afonine <PAfonine at lbl.gov> wrote:

> Hi Ulrich,
>
> One thing I'm puzzled about (before reading your email until the end)...
> Normally we use NCS restraints at lower resolutions and availability of
> high resolution data may suggest to turn NCS restrains off completely
> (at least it's a definitely good thing to try). The alternative
> conformations are normally visible at high resolution (exactly where the
> NCS restrains are most likely useless if not harmful). So, it's
> interesting how you arrived at the situation when you have both:  need
> to use NCS restraints and alternative conformations.
>
>> we have a complex with 4 chemically different polypeptide chains in the
>> asymmetric unit, composition A3B3CD. For chains C and D we see  
>> additional
>> density, which appears to belong to three alternating conformations  
>> which
>> can be thought as rigid-body motions (for the whole chains C and D,  
>> let's
>> call these conformers 1,2 and 3, but we label them conventionally in the
>> file with the prefix A,B,and C) with roughly estimated occupancies of
>> about 0.3, 0.3 and 0.4 (we can really see most of them). A and B have no
>> visible alternating conformations (occupancies set to 1.0). From the  
>> atom
>> count it appears that phenix reads in three conformers called A,B and C
>> with A: A3B3C(1)D(1), B: A3B3C(2)D(2) and C: A3B3C(3)D(3).
>>
>
> OK, now I see. Did you try TLS to model this? Not sure if it's best
> thing to do if you actually see the density for alternative
> conformations, but just out of curiosity...
>
>> Phenix.refine says it has these 3 conformers:
>> conformer A A:2700 atoms,
>> conformer B:2700 atoms, common with A 2100,
>> conformer C:2700 atoms, common with A 2100, common with B 2100.
>>
>> Refinement yields an R/Rfree of about 0.210/0.325.
>>
>
> Yes, the gap between R and Rfree looks not good (too big).
>
>> If we treat the three conformers of polypeptide chains C and D as
>> independent chains with occupancy each 1.0 (this is possible 'cos there
>> are no repulsive contacts between them, fortunately), we get R/Rfree of
>> 0.243/0.291 and phenix applies NCS restraints to all chains.
>>
>> The resolution is about 2.9 A. We use phenix.refine from the CCI_APPS,
>> version 2007-03-26-1903, SUSE Linux.
>>
>> My questions is: is the difference due to the lack of NCS between
>> conformers or are the occupancies mistreated somwhow? I am asking  
>> because
>> I would not consider the whole A3B3C(x)D(x) particle as a conformer but
>> only chains C and D ......
>>
>> Is there a possibility to apply NCS restraints to conformers?
>
> 1)
> - If you do:
> % phenix.refine model.pdb data.hkl main.ncs=true
> phenix.refine tries to automatically determine NCS groups and use them
> in NCS restraints. This may be not good in your very specific case.
> - If you do:
> % phenix.refine model.pdb data.hkl main.ncs=true  
> ncs_groups_selelctions.txt
> where ncs_groups_selelctions.txt is the file where you provide
> selections for NCS groups manually, then phenix.refine will use them but
> it will try to optimize them automatically. This is also may be not good
> in this specific case.
> - Most likely you need to specify your own selections for NCS groups but
> disable their automatic correction:
> % phenix.refine model.pdb data.hkl main.ncs=true
> ncs_groups_selelctions.txt  ncs.find_automatically=False
> Look "Atom selection examples" paragraph in phenix.refine Documentation
> to see the syntax for selections involving alternative conformations.
>
> 2) Once you figured out how you deal with NCS selections, add to your
> normal refinement run group occupancy refinement, so on top of
> coordinates and B-factors refinement you also refine one occupancy
> factor per selected chain (that you believe in alternative conformation):
>
> % phenix.refine model.pdb data.hkl main.ncs=true
> ncs_groups_selelctions.txt  ncs.find_automatically=False
> strategy=individual_sites+individual_adp+group_occupancies
> refine.occupancies.group="chain A and resid 1-50"
> refine.occupancies.group="chain B and resid 1-50"
>
> The command above will do three macro-cycles of NCS restrained xyz and
> b-factors refinement, plus group occupancy refinement: one q per chain A
> and one q per chain B (all other non-selected occupancies will be fixed
> to original input values).
>
> Please not that currently we have no specific constraints on occupancies.
>
> I hope Ralf may have more comments on this (he implemented NCS  
> restraints).
>
> Pavel.
>
> PS> I will be away (not available by email) from Tuesday 17 until Monday  
> 23.
>
>
>
>
>
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-- 
Prof Ulrich Baumann
Departement für Chemie und Biochemie
Freiestrasse 3
Ch-3012 Bern, Switzerland
phone + 41 (0)31 631 4320/4343
fax + 41 (0) 31 631 4887