Cryo-Electron Microscopy at Environmental Molecular Sciences Laboratory: New Advances, User Science and How to Access
Authors:
James E. Evans* ([email protected], PI), Trevor Moser, Irina Novikova, Amar Parvate, Samantha Powell
Institutions:
Pacific Northwest National Laboratory
Abstract
This project is focused on the operation of a Krios cryogenic transmission electron microscope (Krios G3i) at the Environmental Molecular Sciences Laboratory (EMSL) to advance DOE BER user research in protein and small molecule structural biology and whole cell ultrastructure. The operation of the Krios G3i instrument is a joint funding venture between EMSL and BER. The microscope is available to the general EMSL user community and BER researchers in a 50/50 split allocation.
EMSL users can access this instrument free of charge via the normal EMSL user proposal calls, which permit combining cryo-electron microscopy (cryo-EM) with other EMSL capabilities such as mass spectrometry or super-resolution fluorescence microscopy. Access is offered free of charge for BER users, with Pacific Northwest National Laboratory staff time funded by this current project. The BER access mechanism allows for an expedited submission and review process for “cryo-EM only” projects.
The Krios G3i is fully operational and has been applied to multiple EMSL and BER User projects. The microscope has complete screening, data collection, and image processing workflows for (1) microelectron diffraction of small molecule or protein crystals, (2) single-particle analysis of soluble and membrane protein complexes, and (3) electron tomography of whole cells or isolated organelles. It is equipped with a K3 direct electron detector, Ceta-D camera, phase plate, and BioQuantum energy filter. In addition to semiautomated data collection, the facility has installed automated image processing workflows for real-time monitoring feedback of session quality and full 3D reconstruction of all workflows. To date, the facility has demonstrated sub-Å resolution microelectron diffraction, sub-2 Å resolution from 3D single-particle protein structure determination, and subnanometer resolution for whole-cell tomography. While the facility provides rapid access for samples that arrive frozen on clipped and prescreened grids, users can also begin with samples that arrive in buffer and require all steps of the cryo-EM workflow. In a subset of cases, users can start from a provided gene of interest and employ the cell-free expression system to produce enough protein for structural characterization. The team will highlight several recent user results as well as an example of going from cell-free expression through cryo-EM structure determination in less than 24 hours. The team will also present an overview of EMSL’s 1,000 Fungal Proteins project, which will use the Krios as a core capability and is accepting user proposals for structural and functional characterization of conserved fungal proteins.
Funding Information
Pacific Northwest National Laboratory is operated by Battelle for DOE under contract DE-AC05-76RL01830. This program is supported by the DOE Office of Science, through the Genomic Science program of BER program, under FWPs 74194 and 74195. The work was performed at Environmental Molecular Sciences Laboratory (grid.436923.9), a DOE Office of Science user facility sponsored by the BER program.