Opportunities Linking Omics and Structural Biology at PNNL: Excelling at Cryo-EM

Authors:

Trevor Moser, Irina Novikova, Amar Parvate, Samantha Powell, and James E. Evans*

Institutions:

Pacific Northwest National Laboratory (PNNL)

Goals

This project is focused on the operation of a new state-of-the-art cryogenic Transmission Electron Microscope (Krios G3i) at the Environmental Molecular Sciences Laboratory (EMSL) to advance DOE BER user research in protein/small-molecule structural biology and whole-cell ultrastructure. The operation of EMSL’s new Krios G3i instrument is a joint funding venture between EMSL and DOE BER and the microscope is available to the general EMSL user community and DOE/BER researchers in a 50/50 split allocation.

Abstract

This project was designed to rejuvenate cryo-electron microscopy (cryo-EM) research at EMSL with a new microscope and new semi-automated or automated workflows. EMSL users can access this new instrument free of charge via the normal EMSL user proposal calls which permit combining cryo-EM with other capabilities at EMSL such as mass spectrometry or super-resolution fluorescence microscopy. Access is offered free of charge for DOE BER users with PNNL staff time funded by this current project. The DOE BER access mechanism allows for an expedited submission and review process for cryo-EM only projects.

The new KriosG3i microscope is fully operational and has been applied to multiple EMSL and DOE BER user projects. The microscope has complete screening, data collection, and image processing workflows for: (1) micro-electron 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 semi-automated data collection, researchers have installed automated image processing workflows for real-time monitoring feedback of session quality and full 3D reconstruction of all workflows. To date, the team has demonstrated sub-angstrom resolution micro-electron diffraction, sub-2 angstrom resolution from 3D single-particle protein structure determination, and sub-nanometer resolution for whole-cell tomography. While researchers can provide rapid access for samples that arrive pre-frozen on clipped and pre-screened grids, they can also begin with samples that arrive in buffer and require all steps of the cryo-EM workflow. In a subset of cases, researchers can also start from a provided gene of interest and employ or cell-free expression system to produce enough protein for structural characterization. This poster presentation will highlight several of the recent user results as well as demonstrate an example of going from receiving a gene clone through cell-free expression and cryo-EM structure determination in less than 9 days. The team will also showcase the new user-friendly AutoMicroED software, which permits quick and direct determination of small molecule structure even from heterogenous datasets to accelerate science discovery with micro-electron diffraction.

Image

Figure 1. Benchmarking examples of each of the cryo-electron microscopy sample workflows available to users on the new Krios G3i microscope at the Environmental Molecular Sciences Laboratory. Top row shows raw data while bottom row shows reconstructed volumes from samples ranging from small molecules through whole cells.

Funding Information

Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy under Contract DE-AC05-76RL01830. This program is supported by the U. S. Department of Energy, Office of Science, through the Genomic Science program, Biological and Environmental Research (BER) Program, under FWPs 74194 and 74195. The work was performed at EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the BER Program.