Improved applicability and robustness of fast cryo-electron tomography data acquisition

Area of Science:

• Structural Biology
• Microscopy

Background:

• Cryo-electron tomography (cryoET) enables macromolecule characterization within cellular environments.
• Traditional cryoET structure determination is slower than single particle analysis.

Purpose of the Study:

• To improve and evaluate the efficiency and performance of the fast-incremental single-exposure (FISE) tilt series scheme for cryoET data acquisition.
• To enable widespread adoption of cryoET for high-throughput and high-resolution studies.

Main Methods:

• Implemented and tested an improved fast-incremental single-exposure (FISE) tilt series scheme.
• Utilized the latest generation of direct electron detectors and previous generation (pre-2017) double-tilt axis Titan Krios holders.
• Developed methods for compensating x, y, and z-specimen shifts.

Main Results:

• FISE significantly accelerates cryoET data collection when combined with advanced detectors.
• FISE is compatible with older Titan Krios holders, broadening accessibility.
• FISE tilt series data can achieve sub-nanometer resolution averages.
• Specimen shifts in x, y, and z can be effectively compensated.

Conclusions:

• The improved FISE method substantially speeds up cryoET data acquisition.
• This advancement facilitates high-throughput in situ studies and high-resolution structure determination using cryoET.
• The method's compatibility with various hardware broadens its applicability across biological research disciplines.