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.
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Barends, T.R. et al. Nature 505, 244–247 (2014).
Chapman, H.N. et al. Nature 470, 73–77 (2011).
Boutet, S. et al. Science 337, 362–364 (2012).
Shi, D., Nannenga, B.L., Iadanza, M.G. & Gonen, T. eLife 2, e01345 (2013).
Iadanza, M.G. & Gonen, T. J. Appl. Crystallogr. 47, 1140–1145 (2014).
Leslie, A.G.W. & Powell, H.R. in Evolving Methods for Macromolecular Crystallography (eds. Read, R.J. & Sussman, J.L.) 41–51 (Springer, 2007).
Battye, T.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).
Dauter, Z. Acta Crystallogr. D Biol. Crystallogr. 55, 1703–1717 (1999).
Evans, P.R. Acta Crystallogr. D Biol. Crystallogr. 67, 282–292 (2011).
Evans, P.R. & Murshudov, G.N. Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).
Winn, M.D. et al. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
McCoy, A.J. et al. J. Appl. Crystallogr. 40, 658–674 (2007).
Sauter, C. et al. Acta Crystallogr. D Biol. Crystallogr. 57, 1119–1126 (2001).
Adams, P.D. et al. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Gjønnes, J. et al. Acta Crystallogr. A 54, 306–319 (1998).
Oleynikov, P., Hovmöller, S. & Zou, X.D. Ultramicroscopy 107, 523–533 (2007).
Grigorieff, N., Ceska, T.A., Downing, K.H., Baldwin, J.M. & Henderson, R. J. Mol. Biol. 259, 393–421 (1996).
Vincent, R. & Midgley, P.A. Ultramicroscopy 53, 271–282 (1994).
Yonekura, K., Maki-Yonekura, S. & Namba, K. Biophys. J. 82, 2784–2797 (2002).
Gonen, T. Methods Mol. Biol. 955, 153–169 (2013).
The authors thank J. Hattne, F.E. Reyes, D. Olbris, H. Tietz and M. Stumpf for helpful discussions. Work in the Gonen lab is supported by the Howard Hughes Medical Institute. A.G.W.L. is supported by the Medical Research Council (U105184325) and Biotechnology and Biological Sciences Research Council (BBSRC; BB/F020384/1) and Collaborative Computational Project Number 4 (CCP4).
The authors declare no competing financial interests.
Integrated supplementary information
Lysozyme microcrystals were visualized by cryo-EM prior to data collection and a representative crystal is shown (inset, scale bar represents 1 μm). Well diffracting crystals were typically on the order of 2 μm x 2μm x 0.5 μm in size. The crystal shown is a wedge that measured as 0.5 μm thick at the center and 0.15 μm thick at the edges. A representative diffraction pattern from a lysozyme microcrystal collected under continuous rotation. This individual frame was collected over 4 s at a dose of ∼0.01 e-/Å2/s and a rotation rate of 0.09° s-1. The diffraction pattern shows visible reflections extending to ∼ 2 Å resolution and beyond. The contrast of this image was enhanced in Fiji25 to enable spot visualization for this figure.
(a) A single crystal was exposed continuously for 120 frames for a final accumulated dose of 12.0 e-/A2 (0.1 e-/A2 per frame). A representative region from diffraction patterns originating from the same crystal in the same orientation is shown as a function of accumulated dose. It can be seen qualitatively that the intensities of the reflections decrease as dose is accumulated, especially at higher resolutions (see arrows in top left panel), and this effect becomes more severe for all reflections at the higher dosages (8.0 e-/A2 and 12.0 e-/A2). (b) In order to more quantitatively investigate the negative effects of radiation damage, intensities in the resolution bins of 2 - 3 Å (2 randomly chosen reflections) as well as 3 - 6 Å (7 randomly chosen reflections) were selected and their normalized intensity as a function of accumulated dose was analyzed. It was found that the higher resolution reflections suffer relatively early (∼2 e-/Å2 of total accumulated dose, blue), whereas the intensities between 3 Å and 6 Å do not decrease drastically until approximately 5 to 6 e-/Å2 of total accumulated dose (black). Error bars represent standard error of the mean.
The raw data collected on the TEM is first converted to a file compatible with MOSFLM by an in-house script. MOSFLM then uses automated spot finding to determine the position of the reflections on the images. Following spot finding MOSFLM uses the relative positions of each reflection to determine crystal lattice parameters and the crystals orientation. This then allows the location of each reflection within the data set to be predicted, properly indexed, and integrated. After merging and scaling, process of converting the many raw diffraction patterns into one reflection file is complete.
(a-b) Diffraction patterns near the (001) major planes of lysozyme were used to assess the level of dynamic scattering by looking at the systematic absences along the a* axis for both still diffraction data (a) and continuously rotated diffraction data (b). The data from (a) and (b) come from two different crystals. (c-d) The intensity of reflections along a* was quantified and plotted for the still diffraction and continuously rotated diffraction patterns (the portion of the diffraction patterns indicated by the box inset). The plot from still diffraction (c) clearly show small but significant levels of intensity coming from dynamic scattering for reflections (2n+1,0,0), which are forbidden in P43212 symmetry. The intensities of these forbidden reflections are greatly reduced when continuous rotation is used (d).
Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Results (PDF 7000 kb)
Representative intensities from a single crystal continuously bombarded by the electron beam were plotted and the decay was tracked as a function of total accumulated dose. The higher resolution data begins to decay first with the lower resolution data withstanding the effects of the radiation for most of the dosage test. The area from the diffraction patterns shown is between approximately 2.5 Å to 7 Å. (MP4 392 kb)
A representative complete 3D electron diffraction data set from a single lysozyme microcrystal collected by continuous rotation.
Diffraction patterns were collected as the stage was continuously rotating at 0.09° s–1. Each pattern represents a 0.36° wedge of data and the entire data set covers about 44° and its comprised of 121 individual frames. (MP4 5323 kb)
2Fobs-Fcalc density around the complete lysozyme model at 2.5 Å resolution (contoured at 1.0σ). The structure originates from the 2-crystal data set. (MP4 2656 kb)
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Nannenga, B., Shi, D., Leslie, A. et al. High-resolution structure determination by continuous-rotation data collection in MicroED. Nat Methods 11, 927–930 (2014). https://doi.org/10.1038/nmeth.3043
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