Hello, My structure contains 2 protein molecules in the asymmetric unit, and they are related by 2-fold rotational NCS to form a dimer. The refinement is nearing completion (1.7 A) and the solvent molecules are now being included. A blob of density has been tentatively identified as being occupied by glycerol, which was present as the cryo-protectant. The issue is that this blob of density lies on the 2-fold NCS rotation axis, and so we need to use 2 glycerol molecules within this density, each at 0.5 occupancy, to conform to the NCS symmetry. This blob of density lies in a surface pocket formed by residues from both NCS-related proteins. When we build in the 2 glycerol molecules and then use phenix.refine, the two glycerols are pushed out of density. Presumably this is because phenix.refine is interpreting this situation as two glycerol molecules sitting on top of on another rather than interpreting it as being a model for a disordered ligand. Can phenix.refine handle this situation? Thanks, Robert
Dear Robert, there was a post on it in the phenixbb that this trick wont work in phenix. The nonbonded interactions are still valid and therefore the two glycerols are moved apart. I have a similar problem with a lysine side chain clashing into the symmetry related side chain of the same amino acid. I am still trying to model it correctly. Best wishes Daniel The original post was: Hi Morton,
I have a symmetrical ligand, malonate -OOCCCOO-, that binds right on the crystallographic axis of a C2 structure. Is the correct approach to set the occupancy to 0.5 and then refine or is there a better way to handle is in Phenix?
The 0.5 trick currently doesn't work in phenix.refine since we don't provide a way to turn off nonbonded interactions between symmetry copies. However, we fully support handling of atoms on special positions and bonds involving symmetry operations. For this to work, you have to give only an asymmetric unit worth of atoms, i.e. in your case half of your molecule. Is the C in the middle exactly on the two-fold axis? If so, delete these atoms from the PDB file: -OOCCCOO- ^^^ Then use the custom bond and angle feature (for the angles you'll need the 2007_04_06 CCI Apps) to define the required bonds and angles, including the bonds to the protein. I hope it is straightforward. If not, could you send me the fragments from you pdb file with the malonate and the piece of protein it is linked to? Then I could work out the bond and angle definitions for you. Ralf On 22.04.2008, at 15:42, Bobby Huether wrote:
Hello,
My structure contains 2 protein molecules in the asymmetric unit, and they are related by 2-fold rotational NCS to form a dimer. The refinement is nearing completion (1.7 A) and the solvent molecules are now being included. A blob of density has been tentatively identified as being occupied by glycerol, which was present as the cryo-protectant. The issue is that this blob of density lies on the 2-fold NCS rotation axis, and so we need to use 2 glycerol molecules within this density, each at 0.5 occupancy, to conform to the NCS symmetry. This blob of density lies in a surface pocket formed by residues from both NCS-related proteins.
When we build in the 2 glycerol molecules and then use phenix.refine, the two glycerols are pushed out of density. Presumably this is because phenix.refine is interpreting this situation as two glycerol molecules sitting on top of on another rather than interpreting it as being a model for a disordered ligand.
Can phenix.refine handle this situation?
Thanks, Robert
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Daniel Frey Department of Biochemistry University of Zurich Winterthurerstrasse 190 8057 Zurich Switzerland [email protected] Tel: +41446355558 www.biochem.uzh.ch/gruetter www.structuralbiology.uzh.ch
Not an answer but another question: What reason would you be keeping NCS with a moderately complete model at 1.7A? I would guess that keeping NCS restraints at such a high resolution and completeness would count against you, if not the solvent exposed side chains having different conformations among the molecules in the asu then it would be different solvent configurations around each molecule in the asu. Or maybe you have a super symmetric assembly, but even then in a case where I have three molecules in the ASU, adding NCS seemed to hurt refinement statistics when I had most (90% or better) residues built. Curious. FR On Apr 22, 2008, at 7:42 AM, Bobby Huether wrote:
Hello,
My structure contains 2 protein molecules in the asymmetric unit, and they are related by 2-fold rotational NCS to form a dimer. The refinement is nearing completion (1.7 A) and the solvent molecules are now being included. A blob of density has been tentatively identified as being occupied by glycerol, which was present as the cryo-protectant. The issue is that this blob of density lies on the 2-fold NCS rotation axis, and so we need to use 2 glycerol molecules within this density, each at 0.5 occupancy, to conform to the NCS symmetry. This blob of density lies in a surface pocket formed by residues from both NCS-related proteins.
When we build in the 2 glycerol molecules and then use phenix.refine, the two glycerols are pushed out of density. Presumably this is because phenix.refine is interpreting this situation as two glycerol molecules sitting on top of on another rather than interpreting it as being a model for a disordered ligand.
Can phenix.refine handle this situation?
Thanks, Robert
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--------------------------------------------- Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
I agree. At least I would try both: with and without NCS, carefully inspect the results and follow whatever works better. Pavel. On 4/22/2008 2:05 PM, Francis E Reyes wrote:
Not an answer but another question:
What reason would you be keeping NCS with a moderately complete model at 1.7A?
I would guess that keeping NCS restraints at such a high resolution and completeness would count against you, if not the solvent exposed side chains having different conformations among the molecules in the asu then it would be different solvent configurations around each molecule in the asu.
Or maybe you have a super symmetric assembly, but even then in a case where I have three molecules in the ASU, adding NCS seemed to hurt refinement statistics when I had most (90% or better) residues built.
Curious.
FR
On Apr 22, 2008, at 7:42 AM, Bobby Huether wrote:
Hello,
My structure contains 2 protein molecules in the asymmetric unit, and they are related by 2-fold rotational NCS to form a dimer. The refinement is nearing completion (1.7 A) and the solvent molecules are now being included. A blob of density has been tentatively identified as being occupied by glycerol, which was present as the cryo-protectant. The issue is that this blob of density lies on the 2-fold NCS rotation axis, and so we need to use 2 glycerol molecules within this density, each at 0.5 occupancy, to conform to the NCS symmetry. This blob of density lies in a surface pocket formed by residues from both NCS-related proteins.
When we build in the 2 glycerol molecules and then use phenix.refine, the two glycerols are pushed out of density. Presumably this is because phenix.refine is interpreting this situation as two glycerol molecules sitting on top of on another rather than interpreting it as being a model for a disordered ligand.
Can phenix.refine handle this situation?
Thanks, Robert
_______________________________________________ phenixbb mailing list [email protected] http://www.phenix-online.org/mailman/listinfo/phenixbb
--------------------------------------------- Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder
gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D
8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
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participants (4)
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Bobby Huether -
Daniel Frey -
Francis E Reyes -
Pavel Afonine