problem refining RNA structure at 4Å resolution
Dear phenix users, I solved using MR a low resolution RNA structure taking data up to 4.3Å and a model of a structure with slight differences. However, when I try to refine, Rwork decreases quickly (start 47, final 32 but Rfree remains high (start 48, final 44). The data are pretty good up to 4.0 with I/sigma above 1. The map doesn't point clearly to wrong regions of the model. What can I try to improve the maps and build the correct model? Thanks in advance. Benoit
You are likely suffering from model bias towards your search model and low data:parameter ratio in your refinement.
How many molecules per asu?
How different is the search model vs the target? At this resolution keep in mind that you won't be able to tell much, such as the effect of a single nucleotide substitution or indel unless it results in large changes in the structure.
Cheers
F
On May 29, 2013, at 5:53 AM, Benoît Masquida
Dear phenix users,
I solved using MR a low resolution RNA structure taking data up to 4.3Å and a model of a structure with slight differences. However, when I try to refine, Rwork decreases quickly (start 47, final 32 but Rfree remains high (start 48, final 44). The data are pretty good up to 4.0 with I/sigma above 1. The map doesn't point clearly to wrong regions of the model. What can I try to improve the maps and build the correct model?
Thanks in advance.
Benoit _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb
On Wed, May 29, 2013 at 5:53 AM, Benoît Masquida
I solved using MR a low resolution RNA structure taking data up to 4.3Å and a model of a structure with slight differences. However, when I try to refine, Rwork decreases quickly (start 47, final 32 but Rfree remains high (start 48, final 44). The data are pretty good up to 4.0 with I/sigma above 1. The map doesn't point clearly to wrong regions of the model. What can I try to improve the maps and build the correct model?
Standard advice at this resolution: 1. Use reference model restraints. 2. Use NCS restraints if you have NCS (at this resolution it might be worth trying both the torsion and cartesian parameterizations). 3. Optimize the X-ray/restraint weights. 4. Make lots of omit maps to ensure that you're not being deceived - model bias at this resolution really is scary. Also be careful if the outer resolution shells are incomplete, because Phenix (like Refmac) will by default fill in missing reflections with F-calc to avoid map artifacts, but this can make the model bias worse. 5. Use map sharpening - I would recommend doing this interactively in Coot rather than checking the box in Phenix. You might also want to experiment with more conservative B-factor parameterizations (grouped or TLS), although I have yet to figure out how to do this reliably (and get a better result than tightly restraint individual isotropic refinement). -Nat
participants (3)
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Benoît Masquida -
Francis Reyes -
Nathaniel Echols