Cryo-EM Versus X-Ray Crystallography in Resolving Allosteric Conformational States of Multidomain ABC Transporters: A Structural and Mechanistic Benchmarking Study

Abstract

Multidomain ATP-binding cassette (ABC) transporters adopt distinct allosteric conformational ensembles critical to substrate translocation and drug resistance mechanisms. Despite substantial progress, persistent ambiguities remain regarding which structural methodology—cryo-electron microscopy (cryo-EM) or synchrotron-based X-ray crystallography—more faithfully captures physiologically relevant intermediate states. Here, we systematically benchmarked both approaches against five therapeutically significant ABC transporter homologs, combining molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and cross-validation with small-angle X-ray scattering (SAXS) datasets. Cryo-EM consistently resolved occluded-intermediate conformations at sub-3-Å resolution, whereas X-ray crystallography introduced lattice-packing artifacts that distorted nucleotide-binding domain (NBD) dimerization interfaces. These findings redefine quality metrics for ABC transporter structural determination and have direct implications for structure-based drug design targeting multidrug resistance efflux pumps.