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

Abstract

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of β-galactosidase using single-particle cryo–electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM.

Instrumentation at the Leading Edge of Proteomics

“…Improvements in mass analyzer m/z ranges and resolution, transfer optics for large ions, IMS devices, implementations of MS/MS methods, and back-end software have all made native MS a rapidly growing and accessible platform in academic and industrial settings. Native MS is also being leveraged to improve other structural biology techniques, such as soft landing of mass-selected protein complexes for further structural characterization (e.g., cryo-electron microscopy). − ,− Hydrogen–deuterium exchange and protein footprinting have also seen substantive gains due to steady improvements in instrumentation − , , At the peptide level, methods for cross-linking MS (XL-MS) have enabled large-scale structural proteomics studies owing in part to the advanced instrumentation discussed herein. ,, Proximity-dependent labeling has also emerged to help map interactions and corroborate structural studies. − As some of the more demanding studies within the field, structural proteomics at both the peptide (XL-MS) and the protein (native MS) level will likely continue to progress at rapid rates that mirror the exciting instrument developments sure to manifest in coming years.…”

Molecular Dynamics Simulations of Soft and Reactive Landing of Proteins Desorbed by Argon Cluster Bombardment

“…Molecule orientation can be guided using optical or hexapole fields . In addition, conformation and reaction stability of proteins can be improved by cryo methods, that reduce thermal diffusion and structure fluctuations. , …”

Probing the Stability of a β-Hairpin Scaffold after Desolvation

“…To correlate gas-phase data of biomolecules to data obtained in the native environment, conditions for nMS are chosen to conserve properties and native structural features after ionization and desolvation. However, the assumption that structures and function of gas-phase biomolecular ions produced by soft ionization techniques such as electrospray ionization (ESI) mirror those in the condensed phase remains a subject of debate, largely due to ambiguous structural insights afforded by solitary MS-based investigations. , Structural information can be obtained when combining nMS with ion mobility-mass spectrometry (IM-MS), surface labeling techniques like hydrogen–deuterium exchange (H/DX) − or chemical cross-linking, fragmentation techniques, , and gas-phase spectroscopic methods. − While coarse structural features of native folds were found to be preserved, especially for large proteins or protein complexes, − highly charged species may undergo substantial or partial unfolding due to increased Coulomb repulsion, leading to the loss of local structural features. Hitherto, most nMS studies aimed at obtaining more detailed gas-phase structural information examined smaller-scale secondary structural elements, particularly α-helical polypeptides. ,, …”