Biology subjects

Rauschenbach, S.

Publications and source records attributed to Rauschenbach, S..

4 recordsLinked to original sources

bioRxiv2026

Membrane proteins retain native architecture through native ESI and soft-landing

Native MS offers a clear picture of membrane protein stoichiometry and interactions, but it lacks direct structural insights at high resolution. Here, we examine the extent to which solution-phase structure and architecture can be retained after native, soft-landing electrospray ion beam deposition (ESIBD) by interrogating several membrane-protein complexes of diverse folds and oligomeric states by cryoEM. The overall protein architectures with secondary structure motifs can be observed after gas-phase transfer, soft landing, and embedding in amorphous ice. Notably, we determined the structure of the ammonium transporter AmtB at sub-3 [A] resolution. It is nearly identical to the structure of the plunge-frozen control and even shows an extended C-terminal segment of AmtB, a dynamic region absent in the solution-phase structure. Our analysis shows that detergent adducts preserve membrane protein structure in vacuum by minimising destabilization of solvent-exposed regions and stabilization through additional polar contacts in vacuo. Molecular dynamics (MD) simulations support these results, suggesting that a monolayer shell of surfactant adducts avoids destabilization driven by unshielded polar residues and disruption of hydrogen bond networks. Overall, our findings provide a structural framework for integrating native MS with cryo-EM showing that gas-phase transfer and surfactant stabilisation preserves key architectural features and high-resolution structure of membrane proteins.

biophysics↗

bioRxiv2025

Cryo-EM Sample Preparation with Soft-Landing and Laser Flash Melting

The preparation of cryo-EM samples by soft-landing mass spectrometry promises to significantly simplify sample optimization, which has remained an important bottleneck in single-particle cryo-electron microscopy (cryo-EM). However, only compacted configurations are observed after soft-landing since the proteins are dehydrated in the process. Here, we demonstrate that proteins can be returned to their native state by depositing a layer of amorphous ice and briefly flash melting the sample to rehydrate the soft-landed proteins. Melting with a 30 {micro}s laser pulse creates a broad ensemble of partially rehydrated conformations. However, a subset of particles recovers their native configuration. This allows us to propose strategies to fully rehydrate the entire ensemble and brings routine sample preparation by soft-landing mass spectrometry within reach. Finally, the experiments also expand the toolbox of microsecond time-resolved cryo-EM for initiating a broader range of protein dynamics.

biophysics↗

bioRxiv2023

Cryo-EM of soft-landed β-galactosidase: Gas-phase and native structures are remarkably similar

Native mass spectrometry (native MS) is a powerful technique that provides information on stoichiometry, interactions, homogeneity and shape of protein complexes. However, the extent of deviation between protein structures in the mass spectrometer and in solution remains a matter of debate. Here, we uncover the gas-phase structure of {beta}-galactosidase using single particle electron cryomicroscopy (cryo-EM) down to 2.6 [A] resolution, enabled by soft-landing of mass-selected protein complexes onto cold TEM grids and in-situ ice coating. We find that large parts of the secondary and tertiary structure are retained from solution, with dehydration-driven subunit reorientation leading to consistent compaction in the gas phase. Our work enables visualizing the structure of gas-phase protein com-plexes from numerous experimental scenarios at side-chain resolution and demonstrates the possibility of more controlled cryo-EM sample preparation. One Sentence SummaryElectrospray ion-beam deposition on cold grids and in-vacuum ice growth enable cryo-EM of mass-selected proteins at 2.6 [A].

biochemistry↗

bioRxiv2021

Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition

Electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA) have revolutionized structure determination of homogeneous proteins. However, obtaining high-resolution structures from heterogeneous samples remains a major challenge, as the various protein states embedded in thin films of vitreous ice may be classified incorrectly, resulting in detrimental averaging of features. Here we present native electrospray ion-beam deposition (native ES-IBD) for the preparation of extremely high-purity cryo-EM samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, mass-filtered, and gently deposited on cryo-EM grids, and subsequently frozen in liquid nitrogen. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected proteins and protein assemblies. SPA reveals that they remain structurally intact, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM structure determination.

molecular biology↗