Evaluating the Volta phase plate for improved tomogram alignment in cryo-electron tomography

Area of Science:

• Cryo-electron microscopy and tomography
• Structural biology
• Cellular imaging

Background:

• The Volta phase plate (VPP) enhances low-frequency phase contrast in cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET).
• Its effect on tomogram alignment, critical for 3D reconstruction, has not been thoroughly investigated.

Purpose of the Study:

• To systematically assess the impact of the VPP on tomogram alignment in cryo-ET.
• To evaluate the VPP's utility for imaging thick and crowded biological specimens.

Main Methods:

• Utilized sub-2 Å single-particle analysis (SPA) and analytical contrast transfer function (CTF) modeling.
• Acquired and compared cryo-ET datasets with and without the VPP.
• Quantitatively analyzed tilt-axis orientation measurements and local tilt-series alignment.

Main Results:

• Despite high-frequency information loss, the VPP enhances low-frequency signal crucial for alignment.
• VPP tomograms show increased contrast, even after denoising, for challenging specimens.
• Improved tilt-axis orientation measurements and a higher success rate for local tilt-series alignment were observed with the VPP.

Conclusions:

• The VPP significantly improves contrast and tilt-series alignment robustness in cryo-ET.
• These benefits are vital for studying cellular ultrastructure and protein organization in crowded cellular environments.
• While VPP may lower resolution for subtomogram averaging, its alignment advantages are critical for moderate-resolution structural studies.