High-resolution cryo-EM using beam-image shift at 200 keV

Abstract

Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200 keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200 keV using beam-image shift and analyzed. This analysis shows that the instrument beam tilt and particle motion initially limited the resolution to 4.9 Å. After particle polishing and iterative rounds of aberration correction in RELION, a 2.8 Å resolution structure could be obtained. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV.

Keywords: RELION; aldolase; single-particle cryo-EM.

Figure 1

Data-collection strategy for micrographs collected with beam-image shift. (a) Representative image at intermediate magnification. Red cross, focus area; white squares, exposures. The scale bar is 5 µm. Each exposure was collected with image-shift beam tilt. (b) Overview of image-shift values from Leginon for the beam-tilt data set. The units shown are µm.

Figure 2

Single-particle analysis of aldolase without beam-tilt correction. (a) Representative micrographs with minimal (left) and obvious (left) beam-tilt-induced objective astigmatism. Inset: cropped power spectrum. The scale bar is 100 nm. (b) Histogram of CTF resolution limits across the data set using CTFFIND4. (c) Representative 2D class averages calculated using RELION. The scale bar is 200 Å. (d) 3D classification results for selected particles after 2D classification. Dashed boxes indicate classes with particles used for subsequent 3D refinement. (e) Sharpened reconstruction after 3D refinement using RELION filtered to 4.9 Å resolution. (f) FSC curves for final reconstruction.

Figure 3

Improved resolution and map quality using beam-tilt refinement. (a) Strategy for grouping micrographs. Micrographs were grouped into 25 groups (5 × 5), 100 groups (10 × 10) and 400 groups (20 × 20). (b) The effect of group size on beam-tilt refinement and subsequent resolution estimation for refined 3D structures. (c) Sharpened 3D reconstruction for particles placed into 400 micrograph groups filtered to 3.8 Å resolution. (d) FSC curves for 3D reconstruction in (c). (e) Beam-tilt measurements for each group displayed with respect to microscope beam-image shift for X coordinates (black) and Y coordinates (gray). Dashed lines show least-squares fit where R ² = 0.96 (beam tilt X) and R ² = 0.64 (beam tilt Y).

Figure 4

Iterative CTF refinement with particle polishing improves the overall resolution to 2.8 Å. (a) Initial 3D structure at 4.9 Å resolution. Following the first CTF refinement and 3D refinement to obtain a structure at 3.8 Å resolution (b), continued CTF refinements alongside Bayesian particle polishing allowed resolution and B-factor improvements (c, d, e), ultimately allowing the determination of a 2.8 Å resolution structure (e). (f) FSC curves for 3D reconstructions from (a) to (e).

Figure 5

Final aldolase reconstruction at 2.8 Å resolution. (a) Sharpened aldolase reconstruction at 2.8 Å resolution. (b) Example densities and models for aldolase at 2.8 and 4.9 Å resolution. (c) FSC curve for the final reconstruction.