Cryogenic-soft landing imaging mass spectrometry: an ultrapure biological sample preparation platform for cryogenic-electron microscopy

Macromolecular assemblies (MMAs), including protein complexes and viruses—are vital to many biological processes like signal transduction and cellular transport. Their higher-order molecular-structure defines their functions, making it crucial to retrieve their molecular-structure with high-resolution and -accuracy for insights into human health and disease. Single-particle cryogenic-electron microscopy (cryo-EM) has emerged as a leading method for determining MMA structures at atomic resolutions. However, several challenges related to sample preparation, data collection and processing, electron beam-induced damage, and instrumentation limitations hinder further advancements in spatial resolution. This proposal focusses on improvements in the sample preparation aspect. A critical limitation in sample preparation is the vitrification (e.g., plunge freezing) step, where particles localize at the air-water interface, preventing random orientations necessary for 3D structural analysis. This issue combined with the difficulty of achieving uniform ice thickness necessitates extensive sample screening. Another factor is sample heterogeneity, which persists despite the use of pre-fractionation techniques, which are often insufficient to produce ultrapure samples for cryo-EM. Recognizing these limitations, we propose using image-guided native mass spectrometry (nMS) as a sample preparatory tool for cryo-EM. nMS preserves non-covalent interactions within MMAs, maintaining native physiological conditions while transitioning to the gas phase. The native MMA particles can then be gently deposited (“soft-landing”) from the mass spectrometer (MS)s ion beam onto a transmission EM (TEM) grid. This approach could mitigate the challenges of plunge freezing and reduce sample heterogeneity by leveraging the ultrahigh-resolution mass separation and selection capabilities of the MS, while maintaining the higher-order molecular-structure of MMAs. Despite the recent advancements in cryo-EM sample preparation utilizing soft-landing MS incorporating cryogenic conditions, water-vapor deposition, spatial resolution remains inferior to traditional cryo-EM sample preparation techniques. We propose developing a soft-landing unit integrated into a custom-built Orbitrap QExactive ultrahigh-mass range/time-of-flight MS equipped with Timepix-based imaging detector and capable of nMS, featuring six key innovations: (1) image-guided soft-landing utilizing Timepix, which calculates particle arrival time, position, and density with single-ion sensitivity; (2) custom-ion optics for controlled ion deposition; (3) selective deposition of different ion populations onto distinct regions of the same TEM grid; (4) in-situ cryo-cooling using a vibration- and liquid nitrogen-free cryo-microcooler; (5) graphene-coated TEM grids to reduce charging effects; and (6) water-vapor deposition system for controlled vitreous ice application. By developing the soft-landing unit with these innovations, this project aims to improve the sample quality and spatial resolution of cryo-EM, and ultimately yielding high-resolution structures of MMAs.