High-resolution structure determination by continuous-rotation data collection in MicroED

Abstract

MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.

Figure 1. Data collection strategies in MicroED

(a-b) The initial data collection strategy termed “Still Diffraction” consists of rotating the stage in discrete steps between exposures. This provides data in the form of 2D slices through the 3D reciprocal space (a), and while this is sufficient for structure determination, the data are inherently incomplete because most reflections are only partially recorded (b). (c-d) The improved “Continuous Rotation” method for MicroED samples the reciprocal space continuously as the crystal is rotated (c), which yields much more complete and accurate measurements of reflection intensities (d). In the examples shown here originating from two different crystals, the reflections on the left side of the still diffraction are missing due to crystal orientation while they are present in the continuous rotation data because reciprocal space is being more completely sampled (c versus d, respectively).

Figure 2. Final refined structure of lysozyme at 2.5 Å from Continuous Rotation MicroED data

(a-d) A representative region of the final refined structure of lysozyme originating from the 2-crystal data set is shown, with the 2F_obs-F_calc density map (a; contoured at 1.0σ) showing well defined density around the backbone and sidechains. The final 3D structure is also shown in Supplementary Video 3. To test any potential model bias, residues 27 through 36 were removed from the final refined model and the incomplete model was used to phase and refine the original data. The 2F_obs-F_calc map (contoured at 1.0σ) without the deleted residues (b) shows clearly defined density for both backbone and sidechains where the missing residues (shown in yellow) could easily be placed (c). The F_obs-F_calc map (contoured at 3σ) also shows very strong density for the deleted residues (d). The strong density for the missing residues in the 2F_obs-F_calc and F_obs-F_calc maps indicate the final map does not suffer from model bias. (e-f) Final refined structure of lysozyme at 2.5 Å resolution using data originating from a single crystal. The 2F_obs-F_calc density map (e; contoured at 1.0σ) shown around residues 20-35 shows well-defined density around both backbone and sidechains. The F_obs-F_calc (f; contoured at ±3σ) shows no clear differences between the observed data and the model.

Figure 3. Continuous Rotation improves MicroED data quality

(a-c) Views of the (001) plane show that the intensities from still diffraction data (a) exhibit less variation between high and low intensity reflections when compared to X-ray data (b) and the data collected from continuous rotation (c). Data is displayed using VIEWHKL within the CCP4 suite. (d) The lysozyme MicroED data collected as diffraction stills previously was compared with a data set collected by X-ray crystallography (top). While the data is moderately correlated (Pearson correlation coefficient of 0.56 for all data to 2.9 Å, n = 2,466), there is some spreading of the data. When continuous rotation MicroED data is compared with X-ray data (bottom) it is clear that the two are much more correlated (Pearson correlation coefficient of 0.76 for all data to 2.5 Å, n = 3,950) and the spread of the data is narrower relative to the analysis done with diffraction stills. Overall, this analysis shows that the continuous rotation method in MicroED yields data with high quality compared with X-ray data.