Energy filtering enables macromolecular MicroED data at sub-atomic resolution - PubMed (original) (raw)

Energy filtering enables macromolecular MicroED data at sub-atomic resolution

Max T B Clabbers et al. Nat Commun. 2025.

Erratum in

Abstract

High-resolution information is important for accurate structure modeling but is challenging to attain in macromolecular crystallography due to the rapid fading of diffracted intensities at increasing resolution. While direct electron detection essentially eliminates the read-out noise during MicroED data collection, other sources of noise remain and limit the measurement of faint high-resolution reflections. Inelastic scattering significantly contributes to noise, raising background levels and broadening diffraction peaks. We demonstrate a substantial improvement in signal-to-noise ratio by using energy filtering to remove inelastically scattered electrons. This strategy results in sub-atomic resolution MicroED data from proteinase K crystals, enabling the visualization of detailed structural features. Interestingly, reducing the noise further reveals diffuse scattering that may hold additional structural information. Our findings suggest that combining energy filtering and direct detection provides more accurate measurements at higher resolution, facilitating precise model refinement and improved insights into protein structure and function.

© 2025. The Author(s).

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1

Fig. 1. Sub-atomic resolution energy-filtered MicroED data.

Energy-filtered diffraction pattern of a stationary proteinase K lamella was recorded over a 420 s exposure at a total fluence of 0.84 e−/Å2. Highlighted areas are magnified in the right panels, and the corresponding peak profiles are plotted.

Fig. 2

Fig. 2. Energy-filtered MicroED data show improved statistics and high-quality maps.

A Comparison of intensity statistics for filtered and unfiltered MicroED data, featuring from left to right: I/σI, CC1/2, and R_pim. B Slice through the structural model showing the electrostatic potential map, the location of the slice in the structure is indicated in the inset. C Maps and hydrogen omit maps are shown for Tyr128. D Maps and hydrogen omit maps for active site residues and waters. Electrostatic potential 2mFo–DFc maps are shown in blue and contoured at 4_σ (B), and 2.2_σ_ (C, D). Difference mFo–DFc maps are shown in green and red for positive and negative density contoured at 3_σ_ (C, D). Hydrogen omit maps are shown in green and contoured at 2.3_σ_ (C, D). Difference peaks marked with an asterisk indicate potential hydrogen atoms that have not been included in riding positions prior to map calculations.

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References

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