CryoEM at 100 keV: a demonstration and prospects

Area of Science:

• Structural Biology
• Biophysics
• Microscopy

Background:

• Current single-particle cryo-electron microscopy (cryoEM) typically uses higher electron energies (200-300 keV).
• Lower electron energies (e.g., 100 keV) are predicted to offer an improved information-to-damage ratio for biological samples.
• Existing cryoEM instruments in the 100-120 keV range are not optimized for cryoEM's specific requirements, such as high-defocus imaging and specialized detectors.

Purpose of the Study:

• To investigate the feasibility and potential of operating single-particle cryo-electron microscopy at 100 kV.
• To assess the resolution achievable with 100 kV electron energy for various biological macromolecules.
• To identify current technological limitations and practical advantages of 100 kV cryoEM.

Main Methods:

• A standard commercial 200 kV field-emission gun (FEG) microscope was operated at 100 kV.
• A side-entry cryoholder was utilized for sample preparation and imaging.
• A commercial hybrid pixel camera, typically used for X-ray detection, was adapted for low-dose 100 keV electron imaging.

Main Results:

• Five diverse single-particle specimens (hepatitis B virus capsid, bacterial ribosome, catalase, HSP, hemoglobin) were successfully imaged.
• Reconstructions of 3D structures were achieved with resolutions ranging from 8.4 Å to 3.4 Å.
• The study demonstrated the capability of obtaining high-resolution data at 100 kV despite using non-standard detector configurations.

Conclusions:

• Single-particle cryoEM at 100 kV is a viable approach, offering potential benefits in cost and imaging quality.
• Current technological limitations, particularly in detector technology and illumination coherence, need to be addressed for widespread adoption.
• This work supports the development of future cryoEM instruments optimized for lower voltages, aiming for more accessible structure determination.