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Room 24, Chemistry & Biochemistry Building
Bozeman, MT 59717
United Stateshttps://www.montana.edu/cryo-electron-microscopy/cite this facility
Martin Lawrence
(406) 994-5382
The Cryo-EM core facility at MSU is a regional facility that brings state of the art cryo-EM capabilities, including single particle analysis and cryo-electron tomography, to the Northern Rocky Mountains. Instrumentation includes a 200 kV Talos Arctica with a Gatan K3 camera, 120 kV Tecnai Spirit, and a Vitrobot Mark IV. Collectively, these instruments allow imaging of biological structures at single nanometer to sub-nanometer resolutions, and thus enable 3D modeling of cells, viruses, proteins, nucleic acid, and other soft matter at the atomic level.
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1828765pi:
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08/09/2018title:
A Multi-User Cryo-Electron Microscope for the Cellular and Molecular Life Sciences Community in the Northern Rocky Mountain Regionagency:
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http://www.nsf.gov/div/index.jsp?div=DBIpoName:
Jennifer Wellerawardee:
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EXP PROG TO STIM COMP RESpdPIName:
Charles M LawrenceperfCity:
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10/01/2018transType:
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An award is made to Montana State University and collaborating institutions to support the acquisition of a new 200kV cryo-electron microscopy system. The last decade has seen a revolution in cryo-electron microscopy (cryo-EM). With increasing frequency, single particle analysis (SPA) is providing cryo-EM maps at resolutions on par with X-ray crystallography, resulting in atomic models for a growing number of macromolecular assemblies - ribosomes, transcription complexes, proteasomes, viruses, integral membrane proteins and CRISPR/Cas targeting complexes - all directly from cryo-EM data. Cryoelectron tomography (CET) can now provide 3D images of cellular structures at 2-5 nm resolution, and in favorable cases to sub-nanometer resolution. The new multi-user 200 kV cryo-electron microscope will serve the regional cellular and molecular life sciences community in Montana and the surrounding states. The new system will immediately benefit investigators in at least 5 different departments on two different campuses within the Montana University System (UM and MSU), as well as investigators at regional institutions in a five-state area. In addition, the system will have a significant impact on undergraduate, graduate and post-doctoral education, allowing emerging scientists in the Northern Rockies to develop expertise with modern cryo-EM techniques. Finally, it will greatly enhance K-12 teacher education and outreach programs by MSUs Thermal Biology Institute, the Center for Biofilm Engineering, the Chemistry and Biochemistry REU program, and outreach to Montana's Tribal Colleges through MSU's MAP and Bridges to Baccalaureate Programs. Recent technical advances in cryo-EM allow the structures of macromolecular complexes to be modeled at the atomic level by SPA, and observation of these structures within the native cellular environment with CET. Combined, SPA and CET provide profound biological insight into the biochemical activities and mechanisms of cellular machinery. Toward this end, investigators across the Northern Rockies have initiated cryo-EM studies of CRISPR/Cas targeting complexes, G-protein coupled receptor signaling pathways, archaeal viruses in the "3rd domain" of life, new world hemorrhagic viruses, and iron transport in mammals and pathogenic bacteria. The new 200 kV cryo-EM in the Northern Rockies region will enable groundbreaking investigations of macromolecular and cellular structures at ultrahigh resolution. Data and analyses from these studies will be published in peer-reviewed scientific journals, presented at scientific meetings, and used in both educational and public outreach activities. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.awardeePhone:
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The last decade has seen a revolution in cryo-electron microscopy (cryo-EM), with modern transmission electron microscopes using techniques such as "single particle analysis" or "micro-electron diffraction" now able to image proteins, protein-RNA complexes, protein-DNA complexes and viruses at super high resolution (2 to 4 Angstroms). This allows these macromolecules to be modeled in 3D at the atomic level. Depending on the question at hand, these 3D models can provide critical insight into the function of specific proteins or RNA molecules, mechanistic insight into how they function, or aid in the design of new inhibitors and drugs.
In a third technique, cryo-electron tomography can be used to image cells, or thin sections through cells, to provide 3D structural information on cellular organelles, membranes, filaments, ribosomes, etc.. While the resolution is lower, single nanometer rather than Angstrom resolution, the cell can then be segmented to provide cellular models. Further, repeating structures can be averaged to provide higher resolution macromolecular envelopes, allowing some aspects of cellular structure to be modeled at the pseudo-atomic level.
This NSF MRI award (Award #1828765) was used to bring these capabilities to scientists in the Northern Rocky Mountains. Specifically, MRI funds were used to establish a cryogenic transmission electron microscope core facility at Montana State University - Bozeman, to serve the regional cellular and molecular life sciences community. The major pieces of equipment acquired and installed were a 200 kV Talos Arctica cryogenic transmission electron microscope (cryo-TEM) manufactured by ThermoFisher and a Gatan K3 Direct Electron Detector camera (Figure 1). These were complemented with supporting infrastructure necessary for sample preparation, and computational resources required for processing and analysis of the resulting micrographs. Following space renovation to remediate acoustic, vibrational, thermal and electromagnetic interference, the Talos Arctica/K3 camera was installed during the first six months of 2021. This was followed by commissioning and training of core personnel, and initial test projects in the last half of 2021. 2022 has seen general users coming to the resource for training, data collection and analysis.
The results from the microscope have exceeded our initial expectations. Single particle analysis of the 225 kDa DPSL protein from P. furiosus, which functions to limit iron catalyzed formation of free radicals, yielded a structure at 2 Angstroms resolution (Figure 2). More than 20 structures of ribosomes from Pseudomonas aerugionosa in various assembly and conformational states are also under refinement at resoluitons between 2.4 and 3.2 Angstroms. These structures are expected to inform design of new antibiotics to potentially treat Pseudomonas lung infections in patients with cystic fibrosis. Investigators are also completing models for more than a half dozen structures of CRISPR-Cas integration (Figure 3) and surveillance complexes. In addition, single particle analysis is also providing 2.5 Angstrom structures of Cow Pea Chlorotic Motel Virus (CCMV, Figure 4).
Furhter, the microscope is performing well in cryo-electron tomography experiments, where it has resolved single virion structures of SARS-CoV-2, allowing the number, location and orientation of spike proteins on a single virion to be modeled at nm resolution. Notably, the inner and outer leaflets of the membrane are also very clear. Tomography experiments on lemon shaped viruses from Yellowstone hot springs (Figure 5), and dormant Pseudomonas aeruginosa cells have also been informative. Finally, the microscope has also provided initial results with micro-electron diffraction (micro-ED) using submicron sized crystals of inorganic and small organic molecules. For example, a structure of Faujasite was determined at better than 1.0 Angstrom resolution (Figure 6).
Importantly, the cryo-EM core facility is already showing significant regional impact. In the first year it has served 16 Principal Investigators from five different institutions in three states (Washington State University, University of Idaho, University of Montana, Montana Tech and Montana State University), as well as two industrial users. And in addition to the structural results, it is providing state of the art educational experiences at the undergraduate, graduate and post-doctoral level.
Last Modified: 03/08/2023
Modified by: Martin Lawrence
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