nativeESIBD_mempro: Combining Cryo-EM and Native ES-IBD for Atomic-level Structure Analysis of Membrane Proteins

The analysis of intricate structures, flexible conformations, and chemical information of membrane proteins remains a highly crucial challenge.

Our aim is to create a novel pathway for thorough investigation of membrane protein structures, conformations, and their various interactions. We propose an interdisciplinary approach, native electrospray ion beam deposition (ESIBD) combined with cryo- EM.

Membrane protein samples will be prepared by transferring proteins gently into the gas phase by native electrospray ionization, leaving their structure and conformation intact.

After chemical filtering in the mass spectrometer, they are gently deposited on TEM grids held at low temperatures in a vacuum.

This method allows to utilize the chemical selectivity of the mass spectrometer to make samples of precisely selected protein complexes which facilitates the discrimination of flexible conformations and further reveals structural details such as ligand interactions.

The project will involve the investigation of well-established membrane protein systems such as aquaporin Z, outer membrane proteins (OmpS), and ammonium transporter (AmtB) to demonstrate the feasibility of the limitations of this new pathway. Then the structure of membrane proteins with or without functional lipids will be studied.

Ultimately, we aim to image the transient interaction of highly complex membrane proteins like G-Protein coupled receptors or the BAM complex.

In conclusion, native ESIBD has the potential to empower structural biology by widening the scope of structural analysis, and showing conformational variety, interactions, and flexibility of the proteoforms of membrane proteins.