Unexpected Occupancy refiniement for ligands...
Hey, So I am finishing up a model at 1.5 A and I tried to refine my ligands (3) and waters (468) in addition to altloc residues. The residues refine as expected e.g OccA + OccB = 1.0 and all atoms in A have OccA and all the atoms in B have OccB. My ligands on the other are being refined as the following: HETATM 6867 C1 MLA L 3 32.357 -28.612 7.978 0.93 23.77 C C HETATM 6868 O1A MLA L 3 31.209 -28.949 7.502 0.85 24.79 C O HETATM 6869 O1B MLA L 3 32.454 -28.160 9.141 0.70 22.70 C O HETATM 6870 C2 MLA L 3 33.553 -28.727 7.095 1.00 25.46 C C HETATM 6871 C3 MLA L 3 34.774 -28.070 7.627 0.92 28.45 C C HETATM 6872 O3A MLA L 3 34.830 -27.664 8.808 1.00 30.15 C O HETATM 6873 O3B MLA L 3 35.775 -27.948 6.840 1.00 30.23 C O HETATM 6874 HC21 MLA L 3 33.321 -28.281 6.139 1.00 30.56 C H HETATM 6875 HC22 MLA L 3 33.760 -29.776 6.943 1.00 30.56 C H Each atom is getting an individual occupancy assigned, which physically is impossible :) Any light? Could it be my selection syntax? Corrigendum: I think I sent a bug email last night about an error when writing mtz files in refinement. It was an accident. My naming convention was bad, thats why it crashed. Sorry -- Yuri Pompeu
Yuri wrote:
HETATM 6875 HC22 MLA L 3 33.760 -29.776 6.943 1.00 30.56 C H
Each atom is getting an individual occupancy assigned, which physically is impossible :) Any light? Could it be my selection syntax?
Could be- What was your selection syntax? For waters its probably not a good idea to refine occupancy - let the B factor take care of it unless you have really high resolution. For larger ligands I think it makes sense to refine occupancy (for the whole ligand as a single occupancy group) and individual isotropic ADP. The ADP's can only spread the electrons around, cannot correct if the integrated electron density for the ligand is less than expected.
Dear Ed and Pavel, Some background, 1.5A, 0.14/0.16, P 63 2 2. 1- I figured out the occupancy problems. I had to define the ligands as group occupancy rather than individual atoms...(duh!!...) It looks better now. 2- Is there any case/resolution when one should try anisotropic refinement for a ligand? I would think if its covalently attached maybe... 3- About the waters occupancies/B-factors I definetely see some density peaks that correspond to waters (good distance and angles for H-bond interactions with protein backbone on the surface) Obviously they are weaker than peaks for waters that are ordered near the active site. I modelled those in and I got some negative peaks in the mFo-DFc map after isoptropic B refinement. That is what led me to think I should try occupancy refinement. 4-Still on the same topic B-factors, I am also encountering the same problem with surface side chains especially (D, E, Q). I am confident they should be where they are. backbone density looks great. But no matter where I put their side chains, I get negative peaks in the diff. map. Their B-factors usually refine to ~55 vs ~25 for ordered side chains. Yuri On Sat, 13 Aug 2011 12:45:32 -0400, Edward A. Berry wrote:
Yuri wrote:
HETATM 6875 HC22 MLA L 3 33.760 -29.776 6.943 1.00 30.56 C H
Each atom is getting an individual occupancy assigned, which physically is impossible :) Any light? Could it be my selection syntax?
Could be- What was your selection syntax?
For waters its probably not a good idea to refine occupancy - let the B factor take care of it unless you have really high resolution.
For larger ligands I think it makes sense to refine occupancy (for the whole ligand as a single occupancy group) and individual isotropic ADP. The ADP's can only spread the electrons around, cannot correct if the integrated electron density for the ligand is less than expected.
-- Yuri Pompeu
Hi Yuri,
1- I figured out the occupancy problems. I had to define the ligands as group occupancy rather than individual atoms...(duh!!...) It looks better now.
yes.
2- Is there any case/resolution when one should try anisotropic refinement for a ligand?
High resolution, more or less fully occupied. If resolution better than ~1.3-1.2A then probably refine it rather with aniso ADP than iso. There are no general rules, it varies case by case.
3- About the waters occupancies/B-factors I definetely see some density peaks that correspond to waters (good distance and angles for H-bond interactions with protein backbone on the surface) Obviously they are weaker than peaks for waters that are ordered near the active site. I modelled those in and I got some negative peaks in the mFo-DFc map after isoptropic B refinement. That is what led me to think I should try occupancy refinement.
4-Still on the same topic B-factors, I am also encountering the same problem with surface side chains especially (D, E, Q). I am confident they should be where they are. backbone density looks great. But no matter where I put their side chains, I get negative peaks in the diff. map. Their B-factors usually refine to ~55 vs ~25 for ordered side chains.
These side chains are likely to have several conformations and your map shows you only one or a few (2-3?). If this is the case, you should try refining group occupancy for these residues (one occupancy per side chain), so it refines to something less than 1. If you have (you model) more than one conformer, then the sum of group occupancies must be <=1. Pavel.
Hi Yuri, yes, Ed is perfectly right - you don't want to refine occupancies of water at this resolution (1.5A). Regarding the ligand: just make sure the occupancies of ligand atoms are all equal to each other and less than 1 (say 0.9) in your input PDB file. The rest will be taken care of automatically. Pavel. On 8/13/11 8:30 AM, Yuri wrote:
Hey, So I am finishing up a model at 1.5 A and I tried to refine my ligands (3) and waters (468) in addition to altloc residues. The residues refine as expected e.g OccA + OccB = 1.0 and all atoms in A have OccA and all the atoms in B have OccB. My ligands on the other are being refined as the following:
HETATM 6867 C1 MLA L 3 32.357 -28.612 7.978 0.93 23.77 C C
HETATM 6868 O1A MLA L 3 31.209 -28.949 7.502 0.85 24.79 C O
HETATM 6869 O1B MLA L 3 32.454 -28.160 9.141 0.70 22.70 C O
HETATM 6870 C2 MLA L 3 33.553 -28.727 7.095 1.00 25.46 C C
HETATM 6871 C3 MLA L 3 34.774 -28.070 7.627 0.92 28.45 C C
HETATM 6872 O3A MLA L 3 34.830 -27.664 8.808 1.00 30.15 C O
HETATM 6873 O3B MLA L 3 35.775 -27.948 6.840 1.00 30.23 C O
HETATM 6874 HC21 MLA L 3 33.321 -28.281 6.139 1.00 30.56 C H
HETATM 6875 HC22 MLA L 3 33.760 -29.776 6.943 1.00 30.56 C H
Each atom is getting an individual occupancy assigned, which physically is impossible :) Any light? Could it be my selection syntax?
Corrigendum: I think I sent a bug email last night about an error when writing mtz files in refinement. It was an accident. My naming convention was bad, thats why it crashed. Sorry
participants (3)
-
Edward A. Berry -
Pavel Afonine -
Yuri