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Re: [ccp4bb]: ligands in a 2-fold axis



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Daan Virtual wrote:
 > Isn't there another possibility, i.e. that the ligand binds with 50%
 > occupancy in the two possible orientations around a crystallographic
 > two-fold? Thus, looking at each monomer, the ligand interacts with the
 > protein in two different orientations with very similar contacts with
 > exactly 50% occupancy each. In order to refine this one would have to
 > switch off the interactions between the two
 > "alternate conformations" of the ligand - that is possible in CNS. As
 > long as there isn't any ligand atom lying exactly on the two-fold this
 > should work fine.

That is exactly what we decided to be the case in Rb. capsulatus
bacterioferritin (1JGC). There is a heme with its pseudo-2-fold
axis on a crystallographic 2-fold axis. The density is of course
perfectly symmetrical.

Heme is not quite symmetrical, but the binding site is: the axial
ligands are the same methionine in two symm-related molecules. Thus
it binds with equal occupancy in two orientations, and when averaged
over the crystal the site is occupied by a perfectly symmetrical
50:50 mixture which satisfies the symmetry of the higher space group.

The ligand was definitely involved in crystal packing, but through
a symmetrical chain: protein-methionine-iron-methionine-protein.
The asymmetry is only in the vinyl/methyl substituents.

For refinement in CNS we placed one heme at 50% occupancy, allowing
crystallographic symmetry to generate another 50% on top of it.
Initially we fixed all the heme atoms and refined only protein. Then
for the final refinement we used a very modest fiddling with the
CNS script which is described in the CNS FAQ (It's the last faq under
"refinement" at http://cns.csb.yale.edu/v1.1/faq/text.html or
in the documentation distributed with CNS) to turn off
interactions between hemes.)

The structure contained the same symmetrically ligated heme at
two other places; on NCS rather than crystallographic 2-folds.
By dropping to a lower spacegroup (as Rongsheng intimated),
we could turn the crystallograhic case also to noncrystallographic.
However this does not eliminate the problem, as the density indicates
a symmetrical ligand, it must be composed of a 50:50 mixture of the
ligand in two orientations, and to model that it is necessary to reduce
occupancy to 0.5 and include the ncs-mate, turning off interactions
between them. For good measure, we also enforced NCS between the two
superimposed hemes. The refinement program took advantage of the split
molecule to refine two conformations for the propionates, which are
intrinsically symmetrical.

Ed


> On Tue, 3 Dec 2002, Eleanor J. Dodson wrote:
> 
>>Rongsheng Jin wrote:
>>>
>>>Hi there,
>>>
>>>My protein crystallized in the P21212 space group. It forms a homo-dimer
>>>around the crystallographic 2-fold axis. There is one small molecule
>>>ligand binds to each protomer. The interesting thing is that the 2-fold
>>>axis goes through the middle of the ligand molecules, although the
>>>ligand itself is not perfectly 2-fold symmetric. It ends up with two
>>>ligands overlap with each other while the 2-fold axis goes in the middle.
>>>Does anybody know how to do the refinement (CNS?) without reducing to the
>>>p21 space group?
>>>
>>>Thanks,
>>>
>>>Rongsheng
>>
>>
>> First - if this is happened your spacegroup is not P21212 - it is P21
>>with an NCS axis relating the two protein molecules..
>>
>> You must first process the data with P2/m symmetry -
>>once you have merged h k l and -h -k l you have lost the information
>>about the ligand orientation. (lets hope you collected enough redundant
>>data to cover the larger asym unit!!)
>>
>> Then impose NCS restraints on the protein and look for the ligand in
>>the diff map..
>>
>>Eleanor
>>