I’ll relay some ideas that seem ancient these days. But you are where I was over a decade ago. Dubious molecular replacement solution, stuck refinement in the low resolution. Some tips: [1] Complete, well measured (not over exposed), low resolution reflection bins. There is a tendency to attempt to collect high resolution reflections to attempt to extend the resolution, but molecular replacement (simple things like pattersons) are low resolution issues. [2] If you have a large complex, or a large unit cell, think about symmetry inside your ASU. Native pattersons and self rotation function evaluation. [3] Experimental phasing. Use large clusters (TaBr, for example) and grab a well measured experimental dataset. IsoDiff or AnomDiff maps can help guide the molecular replacement solution. But the derivatives also provide an unbiased, external source of phase information if your MR solution is subtly incorrect, or incomplete. [4] Keep track of your derivatives, they will be quasi isomorphous and can be helpful for multi crystal DM. In my opinion Rfree at this resolution on an absolute scale is insufficient to say you’ve ’solved’ the structure or can distinguish among competing hypothesis (is the MR solution correct? Is conformation with Rfree of X better than one of Y?) Reach out if you want to discuss more, Francis Reyes
On Oct 15, 2024, at 3:22 AM, [email protected] wrote:
Dear colleagues, I am currently engaged in an endeavor to unravel the crystal structure through molecular replacement techniques. Regrettably, the resolution attained stands at a rather low mark, approximately 4.3 Angstroms, which poses significant challenges. Despite exhaustive efforts directed towards optimizing the crystal conditions, there has been no discernible improvement in the resolution. The structure in question is confined to a specific domain, and we have at our disposal two distinct conformational models sourced from the PDB database: one derived from X-ray data and the other from Cryo-EM. Our prime objective is to meticulously discern which of these two conformations aligns most accurately with our experimental context. To this end, we have employed both structures (X-ray and Cryo-EM) as templates for molecular replacement, yet the refinement process has hit a roadblock, hindering our progress.
We are seeking a collaborator with experience in dealing with low-resolution data to assist in solving the structure. We are open to sharing first or corresponding authorships in the near future for this paper.
Warm regards
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