Seeing tobacco mosaic virus through direct electron detectors

Area of Science:

• Structural Biology
• Microscopy Techniques
• Biophysics

Background:

• Direct electron detectors (DEDs) have revolutionized electron cryo-microscopy (cryo-EM), enabling atomic-resolution structure determination.
• Comparative characterization of DED performance is essential for optimizing data acquisition and processing protocols.

Purpose of the Study:

• To comparatively characterize the performance of K2 Summit and Falcon II DEDs for cryo-EM.
• To investigate image processing aspects, including motion correction strategies and the impact of dose deposition on high-resolution structure determination.
• To establish optimal recording and processing conditions for achieving high-resolution cryo-EM structures.

Main Methods:

• Utilized tobacco mosaic virus (TMV) as a model system for cryo-EM studies.
• Recorded data on K2 Summit and Falcon II direct electron detectors.
• Applied micrograph-based and segment-based motion correction techniques.
• Analyzed the effects of radiation damage on TMV structure at the atomic level.

Main Results:

• Compared 3D image reconstruction quality from K2 Summit and Falcon II DEDs.
• Demonstrated that radiation damage preferentially affects negative carboxyl chains in a side-chain specific manner.
• Achieved a high-resolution cryo-EM map of TMV at 3.35Å resolution, with potential for 3.2Å resolution.

Conclusions:

• Direct electron detectors significantly enhance cryo-EM capabilities for atomic-resolution structure determination.
• Optimized processing strategies and careful dose management are crucial for mitigating radiation damage and achieving high-resolution maps.
• This study provides a benchmark for DED performance and establishes conditions for high-resolution cryo-EM structure determination.