Scientific MUE: Acquisition of integrated transmission and dual beam (focused ion and scanning) systems for high-resolution biological cryo-electron microscopy by analysis of isolated particles, in situ tomography, and diffraction of microcrystals.

Abstract

We propose establishing a frontier facility, unprecedented in Brazil, focused on selecting and characterizing biological samples for high-resolution structural studies by cryogenic electron microscopy (cryo-EM), revolutionizing structural biology in the last decade. The current implementations of cryo-EM are single particle analysis (SPA), tomography (cryo-ET), and microcrystal diffraction (micro-ED), providing a complete picture of biological processes from isolated proteins and their complexes to preserved ultrastructure in cells and tissue. To this end, our targeted equipment are: the Tundra cryo-TEM (Thermo Scientific), which is a fully automated low-voltage transmission electron microscope (TEM) with rapid sample loading and unloading, decision making aided by artificial intelligence, and simplified data collection for SPA; the Aquilos 2 cryo-FIB SEM (Thermo Scientific), which is a focused ion beam(FIB)/scanning electron microscope (SEM) dedicated to preparing electron-transparent lamellas for cryo-ET - with an integrated fluorescence light microscope (iFLM) for correlative light-electron imaging (CLEM) for the identification of subcellular regions of interest - and nanometer-sized crystals for micro-ED. In addition to expanding and speeding up our research capabilities to unparalleled levels, the Tundra and the Aquilos 2 will integrate a regional multiuser hub facility for iterative cryo-EM specimen preparation at the IFSC/USP, which pioneered the field of macromolecular X-ray crystallography in Latin America thirty years ago. Freshly prepared protein, cells, and crystals will be vitrified on grids with a Vitrobot Mark IV system (Thermo Scientific), granted recently by FAPESP (EMU 2022/04089-5) currently in order. Then, the Tundra will allow for a complete evaluation, optimization, and preliminary data collection for SPA and qualitative visualization of thin lamellae and crystal diffraction (both from the Aquilos 2), but without tilting. Importantly, exchanging samples in and out of the microscopes in minutes, with nearly instantaneous feedback, allows for optimizing sample conditions quickly and efficiently. The best sample grids produced in our Facility by internal and external users will be submitted for SPA, cryo-ET and micro-ED data acquisition worldwide at higher-end platforms hosting 300 kV TEM, such as the Titan Krios, at the Brazilian Nanotechnology National Laboratory (LNNano/CNPEM). The impact of the cryo-EM platform at the IFSC/USP is several folds. Among others, it will (1) constitute a unique hub facility in the state of São Paulo for internal and external users, (2) stimulate the routine application of SPA, which will soon become the primary method for atomic structure determination, (3) help overcome challenges in grid preparation of intrinsically unstable protein samples, which are prone to degradation or aggregation, (4) allow the regional scientific community to study frontier structural biology in situ by cryo-FIB SEM and CLEM, (5) offer a modern and promising alternative to protein crystallography by micro-ED, (6) promote widespread capacitation of students and researchers in frontier techniques through easy access, training and continuous 24/7 hands-on operation, and (7) boost the output of the Titan Krios at the LNNano. (AU)