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This looks perfectly reasonable in terms of initial numbers, so my questions would be: what did you do to the model before you started refining ? Did you delete all the obviously different bits in with the existing sequence ? Strip to ALA and keep only the identical residues ? Refine B-factors after energy minimization ?
At 24% identity at 2.4 Angstrom, I'm not surprised that your R-free goes up during refinement unless you've made particularly shrewd changes to your model but you'd still hope that the (R-free - R) difference would be less than 8% and the geometry would be tight (0.010 or tigher RMS bond length deviations). At 44% or 49% R-free your maps aren't going to look that good. I'd suggest running one cycle of energy minimization and fairly heavily restrained B-factor refinement, calculate maps, then chop out any loops that aren't in reasonable density. I'd look at the hydrophobic core(s), the areas with lowest regional average B-factor, and start making changes then the work outwards. Change at most 10% of the structure, refine again, recalculate maps, lather rinse repeat. It can be tedious but the risk at this stage is that if you make too many changes in a marginal map you can bury yourself in a wrong interpretation.
At this stage I'm not sure whether to advocate chopping non-identical residues back to ALA or not. Not to do so risks burying yourself in a fairly deep pit of phase bias. To do so might actually make the phases worse - I've had two recent fairly tough-ish cases where that seemed to be the case and they were around this sequence identity range.
Good luck, Phil Jeffrey Crystallography Facility Manager Dept Molecular Biology Princeton University
