An integrated workflow for structural virology with a 100 keV electron microscope.

Area of Science:

• Structural Biology
• Biophysics
• Biochemistry

Background:

• Cryo-electron microscopy (cryo-EM) is crucial for studying flexible and heterogeneous biological samples.
• High-end 300 keV cryo-EM microscopes offer high-resolution data but are expensive and have limited accessibility.
• Limited access to advanced cryo-EM facilities hinders research progress and training.

Purpose of the Study:

• To present the successful user-managed operation of a 100 keV cryo-EM within a structural biology laboratory.
• To detail the practical aspects of installing, maintaining, and operating a cost-effective cryo-EM system.
• To demonstrate the feasibility of independent, high-quality cryo-EM data collection and processing outside core facilities.

Main Methods:

• Installation and daily maintenance of a 100 keV electron microscope.
• Integrated workflow encompassing grid screening, data collection, and data processing.
• Utilized Ceta CMOS camera for low-resolution and Falcon C direct detector for high-resolution reconstructions.

Main Results:

• Demonstrated routine high-quality low-resolution reconstructions and high-resolution reconstructions suitable for atomic model building.
• Showcased a complete workflow using virus surface glycoproteins as case studies.
• Achieved proficiency for beginners in independent microscope operation within one month of regular use and training.

Conclusions:

• User-managed 100 keV cryo-EM enables integrated structural biology research, bridging protein production to structural determination.
• This model provides an accessible training platform, fostering broader expertise in cryo-EM techniques.
• Represents the first successful demonstration of a modern research group independently managing both cryo-EM operations and leveraging its capabilities for structural biology.