FLEXR-MSA: electron-density map comparisons of sequence-diverse structures.

Proteins with near-identical sequences often share similar static structures. Yet, comparing crystal structures is limited or even biased by what has been included or omitted in the deposited model. Information about unique dynamics is often hidden in electron-density maps. Currently, automatic map comparisons are limited to sequence-identical structures. To overcome this limitation, we developed FLEXR-MSA, which enables unbiased electron-density map comparisons of sequence-diverse structures by coupling multiple sequence alignment (MSA) with electron-density sampling. FLEXR-MSA generates visualizations that pinpoint low-occupancy features on the residue level and chart them across the protein surface to reveal global changes. To exemplify the utility of this tool, we probed electron densities for protein-wide alternative conformations of HSP90 across four human isoforms and other homologs. Our analysis demonstrates that FLEXR-MSA can reveal hidden differences among HSP90 variants bound to clinically important ligands. Integrating this new functionality into the FLEXR suite of tools links the comparison of conformational landscapes hidden in electron-density maps to the building of multi-conformer models that reveal structural/functional differences that might be of interest when designing selective ligands.