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* These authors contributed equally
Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.
Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-Å-10 Å. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.
Biomedical research improves our understanding of human health and disease through the development of new technologies. High-resolution imaging is transforming our view of the nanoworld - permitting us to study cells and molecules in exquisite detail1,2,3,4,5. Static information of dynamic components such as soft polymers, protein assemblies, or human viruses reveals only a limited snapshot of their complex narrative. To better understand how molecular entities operate, their structure and function must be ....
1. Loading the specimen holder for liquid-EM
A liquid-TEM operating at 200 kV was used for all liquid-EM imaging experiments and a cryo-TEM operating at 300 kV was used for all cryo-EM data collection. Representative images and structures of multiple viruses are presented to demonstrate the utility of the methods across various test subjects. These include recombinant adeno-associated virus subtype 3 (AAV), SARS-CoV-2 sub-viral assemblies derived from the patient serum, and simian rotavirus double-layered particles (DLPs), SA11 strain. First, comparisons are demons.......
New opportunities are presented to streamline current liquid-EM workflows by using new automated tools and technologies adapted from the cryo-EM field. Applications involving the new microchip sandwich technique are significant with respect to other methods because they enable high-resolution imaging analysis in liquid or vitreous ice. One of the most critical steps in the protocol is producing specimens with the ideal liquid thickness to visualize exquisite details at the nanoscale level. Ideal regions of interest are i.......
The authors acknowledge Dr. Luk H. Vandenberghe (Harvard Medical School, Department of Ophthalmology) for providing purified AAV-3. This work was supported by the National Institutes of Health and the National Cancer Institute (R01CA193578, R01CA227261, R01CA219700 to D.F.K.).
....Name | Company | Catalog Number | Comments |
Acetone | Fisher Scientific | A11-1 | 1 Liter |
Autoloader clipping tool | ThermoFisher Scientific | N/A | Also SubAngstrom supplier |
Autoloader grid clips | ThermoFisher Scientific | N/A | top and bottom clips |
Carbon-coated gold EM grids | Electron Microcopy Sciences | CF400-AU-50 | 400-mesh, 5-nm thickness |
COVID-19 patient serum | RayBiotech | CoV-Pos-S-500 | 500 microliters of PCR+ serum |
Methanol | Fisher Scientific | A412-1 | 1 Liter |
Microwell-integrad microchips | Protochips, Inc. | EPB-42A1-10 | 10x10-mm window arrays |
TEMWindows microchips | Simpore Inc. | SN100-A10Q33B | 9 large windows, 10-nn thick |
TEMWindows microchips | Simpore, Inc. | SN100-A05Q33A | 9 small windows, 5-nm thick |
Top microchips | Protochips, Inc. | EPT-50W | 500 mm x 100 mm window |
Whatman #1 filter paper | Whatman | 1001 090 | 100 pieces, 90 mm |
Equipment | |||
DirectView direct electron detector | Direct Electron | 6-micron pixel spacing | |
Falcon 3 EC direct electron detector | ThermoFisher Scientific | 14-micron pixel spacing | |
Gatan 655 Dry pump station | Gatan, Inc. | Pump holder tip to 10-6 range | |
Mark IV Vitrobot | ThermoFisher Scientific | state-of-the-art specimen preparation unit | |
PELCO easiGlow, glow discharge unit | Ted Pella, Inc. | Negative polarity mode | |
Poseidon Select specimen holder | Protochips, Inc. | FEI compatible;specimen holder | |
Talos F200C TEM | ThermoFisher Scientific | 200 kV; Liquid-TEM | |
Titan Krios G3 | ThermoFisher Scientific | 300 kV; Cryo-TEM | |
Freely available software | Website link | Comments (optional) | |
cryoSPARC | https://cryosparc.com/ | other image processing software | |
CTFFIND4 | https://grigoriefflab.umassmed.edu/ctffind4 | CTF finding program | |
MotionCorr2 | https://emcore.ucsf.edu/ucsf-software | ||
RELION | https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page | ||
SerialEM | https://bio3d.colorado.edu/SerialEM/ | ||
UCSF Chimera | https://www.cgl.ucsf.edu/chimera/ | molecular structure analysis software package |
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