Capturing Hidden Regulatory Mechanism of Human Membrane Proteins via Soft- landing Mass Spectrometry-Guided Cryo-EM (360G-Wellcome-342948_Z_25_Z)

Membrane proteins function as dynamic conformational ensembles shaped by lipid composition, ligand occupancy, and intrinsically disordered cytoplasmic regions (IDRs). Conventional single-particle cryo-electron microscopy (cryo- EM) primarily resolves dominant conformations from solution-averaged populations, leaving low-abundance regulatory states structurally inaccessible. This limits mechanistic understanding of membrane protein regulation and disease-associated conformational variation. This programme establishes a state-resolved structural framework integrating native mass spectrometry (native MS) with cryo-EM using electrospray ion beam deposition (ESIBD)-based soft-landing. By mass-selecting defined ligand- and lipid-bound charge states prior to vitrification and exploiting reduced dielectric screening in the gas phase, the approach shifts conformational equilibria to stabilise regulatory regions under-represented in solution. Mass-defined populations are then structurally characterised by cryo-EM to test whether electrostatic and compositional modulation enables visualisation of otherwise obscured conformations. The research progresses from bacterial membrane proteins to regulatory IDRs in human transporters, culminating in analysis of ligand- and lipid-associated states in the disease-relevant solute carrier SLC45A4. This work moves structural biology from passive ensemble observation toward controlled state enrichment. The programme will define how lipid environment, ligand binding, and electrostatic interactions reshape conformational equilibria in membrane proteins, establishing a platform for resolving regulatory heterogeneity across transporters and ion channels.