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Refinement: Difference between revisions
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Sometimes crystal symmetry combines with non-crystallographic symmetry (NCS) and produces a diffraction pattern resembling higher symmetry space group than what you really have. NCS in this case closely resembles crystallographic symmetry. If resolution is not high enough, the difference in spot positions may be too small to give any detectable problems with indexing, integration and scaling. Even phasing (e.g. molecular replacement) may be successful. But if your R-factor hangs fairly high and you have problems building parts of your structure, it is worth trying to check other space groups. The most straightforward approach is to try processing data in P1, because if that does not bring R-factor down significantly, other space group choices will not solve the problem either. | Sometimes crystal symmetry combines with non-crystallographic symmetry (NCS) and produces a diffraction pattern resembling higher symmetry space group than what you really have. NCS in this case closely resembles crystallographic symmetry. If resolution is not high enough, the difference in spot positions may be too small to give any detectable problems with indexing, integration and scaling. Even phasing (e.g. molecular replacement) may be successful. But if your R-factor hangs fairly high and you have problems building parts of your structure, it is worth trying to check other space groups. The most straightforward approach is to try processing data in P1, because if that does not bring R-factor down significantly, other space group choices will not solve the problem either. | ||
This occurs most often at moderate resolution. However, [ | This occurs most often at moderate resolution. However, [https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0040099 the structure of the ketosteroid isomerase] had to be refined in P1 at atomic resolution, although it refines well in C2221 at lower resolution such as 1.5A. | ||
=== Refining low resolution structures === | === Refining low resolution structures === | ||
Maintaining the secondary structure of your model when refining against weak data can be really challenging. | Maintaining the secondary structure of your model when refining against weak data can be really challenging. When building manually, you may end up with a fairly large number of [[Ramachandran plot]] outliers. | ||
Try [[PHENIX|phenix.refine]] with the keyword "discard_psi_phi=False". Then the psi and phi dihedral angles should be restrained according to the CCP4 monomer library definitions. There was a [http://www.phenix-online.org/pipermail/phenixbb/2007-July/000357.html discussion of it in the phenixbb in July 2007]. Also see the [http://www.dl.ac.uk/list-archive-public/ccp4bb/msg19554.html discussion in the ccp4bb from December 2006]. | Try [[PHENIX|phenix.refine]] with the keyword "discard_psi_phi=False". Then the psi and phi dihedral angles should be restrained according to the CCP4 monomer library definitions. There was a [http://www.phenix-online.org/pipermail/phenixbb/2007-July/000357.html discussion of it in the phenixbb in July 2007]. Also see the [http://www.dl.ac.uk/list-archive-public/ccp4bb/msg19554.html discussion in the ccp4bb from December 2006]. | ||
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Remember that [[Ramachandran plot|phi-psi angles]] are excellent for [[validation]] purposes but only when they are unrestrained. If you restrain them, you lose this option! | Remember that [[Ramachandran plot|phi-psi angles]] are excellent for [[validation]] purposes but only when they are unrestrained. If you restrain them, you lose this option! | ||
You can also try restraining alpha-helices hydrogen bonding, and beta-sheet cross-strand hydrogen bonds. This can be done in [[REFMAC]] | You can also try restraining alpha-helices hydrogen bonding, and beta-sheet cross-strand hydrogen bonds. This can be done in [[REFMAC]] (using ProSMART) and [[PHENIX|phenix.refine]] (using a reference model). | ||
If you are really desperate, another option could be to use harmonic restraints in [[CNS]] to keep your backbone fairly fixed in parts of the map where you believe the secondary structure is correct (most likely alpha-helices). You could also fix main-chain elements completely (in any refinement program), but it is definitely preferable to leave some room for change in the xyz positions, and harmonic restraints are a nice way of doing exactly that. | If you are really desperate, another option could be to use harmonic restraints in [[CNS]] to keep your backbone fairly fixed in parts of the map where you believe the secondary structure is correct (most likely alpha-helices). You could also fix main-chain elements completely (in any refinement program), but it is definitely preferable to leave some room for change in the xyz positions, and harmonic restraints are a nice way of doing exactly that. | ||
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The following advice is specific for [[ccp4dev:Refinement_with_Refmac5|Refmac]]: Changing from simple scaling to Babinet scaling is an important check to exclude mask bulk solvent artifacts, but there, you have to uncheck the "calculate contribution from the solvent region", because this is done by the Babinet scaling, already. | The following advice is specific for [[ccp4dev:Refinement_with_Refmac5|Refmac]]: Changing from simple scaling to Babinet scaling is an important check to exclude mask bulk solvent artifacts, but there, you have to uncheck the "calculate contribution from the solvent region", because this is done by the Babinet scaling, already. | ||
Alternatively, you can optimise the solvent mask parameters by running Refmac with the keyword "solvent optimise". This will write out R and R-free for different combinations of VDW probe, ion probe, and shrinkage sizes. For subsequent Refmac runs you can use the keywords "solvent vdwprobe $VDWPROBE ionprobe $IONPROBE rshrink $RSHRINK" replacing | |||
$VDWPROBE, $IONPROBE, and $RSHRINK with the optimal values from the previous optimisation or you can set these values in the GUI. | |||
If the peaks remain, try gradually reducing the size of the VDW probe. | |||
For [[Phenix|phenix.refine]], the bulk solvent mask may be | For [[Phenix|phenix.refine]], the bulk solvent mask may be optimized using "phenix.refine data.hkl model.pdb optimize_mask=true" - see [http://www.phenix-online.org/documentation/refinement.htm]. | ||
In the case of ''negative'' difference density in a big hydrophobic cavity, one possible reason for a negative difference density are underestimated magnitudes of |Fobs| at very low resolution, either because they are weakened by the beam-stop (half-)shadow, or because they are overloads that have been poorly extrapolated. A simple check for wrongly determined low-resolution |Fobs| is to cut your low resolution data during refinement at a somewhat higher resolution, say 20 A instead of 80 A, and see whether the negative difference density disappears. If, yes, you should check your data processing again. | In the case of ''negative'' difference density in a big hydrophobic cavity, one possible reason for a negative difference density are underestimated magnitudes of |Fobs| at very low resolution, either because they are weakened by the beam-stop (half-)shadow, or because they are overloads that have been poorly extrapolated. A simple check for wrongly determined low-resolution |Fobs| is to cut your low resolution data during refinement at a somewhat higher resolution, say 20 A instead of 80 A, and see whether the negative difference density disappears. If, yes, you should check your data processing again. | ||
The other possibility of course is that the data is good, that this is an accurate experimental result and there really is a void, or at least a cavity where the mean bulk density is lower than in bulk water. One way to test the void theory would be to fill the cavity with O atoms of zero (or very small, say 0.01) occupancy. Hopefully (!) that will prevent Refmac filling the cavity with bulk solvent. One could then try giving these O atoms large B factors, say 200, to smear them out, and then increase the occupancies to titrate the actual bulk density. | The other possibility of course is that the data is good, that this is an accurate experimental result and there really is a void, or at least a cavity where the mean bulk density is lower than in bulk water. One way to test the void theory would be to fill the cavity with O atoms of zero (or very small, say 0.01) occupancy. Hopefully (!) that will prevent Refmac filling the cavity with bulk solvent. One could then try giving these O atoms large B factors, say 200, to smear them out, and then increase the occupancies to titrate the actual bulk density. | ||
Since 2016, so-called [https://www.phenix-online.org/documentation/reference/polder.html Polder maps] in Phenix allow to calculate omit density without filling in water which may obscure a ligand. | |||
== Model correctly placed, but difference density remains after refinement == | == Model correctly placed, but difference density remains after refinement == | ||