Abstract
Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.
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Acknowledgements
The Gonen laboratory is supported by funds from the Howard Hughes Medical Institute. The Eisenberg laboratory is supported by HHMI, NIH grant 1R01-AG029430 (D.E.), and DOE grant DE-FC02-02ER63421 (D.E.). The Calero laboratory is supported by NIH grants RO1GM120292 and RO1DK102495 (G.C.) and BioXFEL-STC1231306 (G.C.). The Hinck laboratory is supported by NIH grants R01-GM58670 and R01-CA172886 (A.P.H.).
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D.S. and T.G. designed the experiment; M.J.d.l.C. and F.E.R. prepared lysozyme, xylanase, thaumatin, trypsin, proteinase K, and thermolysin samples; J.R. prepared tau peptide samples; S.C.W., S.K.K., C.S.H., A.P.H., and C.G. prepared TGF-βm–TßRII samples; M.J.d.l.C., D.S., J.R., and S.C.W. collected data; M.J.d.l.C., J.H., P.S., M.R.S., D.C., S.C.W., and G.C. analyzed data and refined and determined models; J.H., J.R., and M.R.S. prepared figures; M.J.d.l.C., J.H., and T.G. wrote the manuscript with contributions from all authors.
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Integrated supplementary information
Supplementary Figure 1 X-ray and corresponding MicroED diffraction pattern from protein tau.
(Left) When extracted from hanging drops, a cluster of microneedle crystals of the amyloid-forming protein tau diffracts as powder to no better than 4.2 Å using a rotating anode X-ray source. Physically breaking these needle clusters and selecting individual sub-micron thick crystal fragments yields diffraction to atomic resolution by MicroED (right) and a structure solvable by direct methods. The X-ray diffraction pattern was collected over a 6° oscillation range; the MicroED pattern spans a 0.6° wedge. See main text for data collection details.
Supplementary Figure 2 X-ray and corresponding MicroED diffraction pattern from Zn2+-NNQQNY.
The structure has previously been solved by X-ray diffraction, albeit at lower resolution [Nelson et al. (2005) Nature 435, 773–778]. It was readily redetermined by direct methods from the MicroED data [Sawaya et al. (2016) Proc Natl Acad Sci 113, 11232–11236]. For the MicroED pattern the inset shows a close-up of the spot indicated by the blue circle.
Supplementary Figure 3 X-ray and corresponding MicroED diffraction pattern from Cd2+-NNQQNY.
This structure was not previously solved by X-ray diffraction, but was readily determined by direct methods from the MicroED data [Sawaya et al. (2016) Proc Natl Acad Sci 113, 11232–11236]. For the MicroED pattern the inset shows a close-up of the spot indicated by the blue circle.
Supplementary Figure 4 X-ray and corresponding MicroED diffraction pattern from GNNQQNY.
The structure has previously been solved by X-ray diffraction, albeit at lower resolution [Nelson et al. (2005) Nature 435, 773–778]. It was readily redetermined by direct methods from the MicroED data [Sawaya et al. (2016) Proc Natl Acad Sci 113, 11232–11236]. For the MicroED pattern the inset shows a close-up of the spot indicated by the blue circle.
Supplementary Figure 5 X-ray and corresponding MicroED diffraction pattern from lysozyme.
The X-ray diffraction pattern displays multiple lattices. No optimization of crystal growth was done; instead crystals were sonicated and probed by MicroED. The obtained resolution was as good as what was obtained from the parent crystal. For the MicroED pattern the inset shows a close-up of the spot indicated by the blue circle.
Supplementary Figure 6 X-ray and corresponding MicroED diffraction pattern from TGF-βm–TβRII.
The X-ray diffraction pattern was collected at an X-ray free-electron laser, but the crystals diffracted better under MicroED. No optimization of crystal growth was done; instead crystals were vortexed with glass beads and then probed by MicroED. For the MicroED pattern the inset shows a close-up of the spot indicated by the blue circle.
Supplementary Figure 7 X-ray and corresponding MicroED diffraction pattern from xylanase.
The X-ray diffraction pattern displays several multiple lattices. No optimization of crystal growth was done; instead crystals were sonicated and probed by MicroED. The obtained resolution was as good as what was obtained from the parent crystal. For the MicroED pattern the inset shows a close-up of the spot indicated by the blue circle.
Supplementary Figure 8 X-ray and corresponding MicroED diffraction pattern from proteinase K.
The X-ray diffraction pattern displays multiple lattices. No optimization of crystal growth was done; instead crystals were sonicated and probed by MicroED. The obtained resolution was better than what was obtained from the parent crystal. For the MicroED pattern the inset shows a close-up of the spot indicated by the blue circle.
Supplementary Figure 9 X-ray and corresponding diffraction pattern from thermolysin.
The X-ray diffraction pattern is powder-like. No optimization of crystal growth was done; instead crystals were sonicated and probed by MicroED. The obtained resolution was better than what was obtained from the parent crystal. For the MicroED pattern the inset shows a close-up of the spot indicated by the blue circle.
Supplementary Figure 10 Rfree – Rwork as a function of refinement cycle.
The R-factor gap is always less than 6% and the variation is generally within ±1% after the first few cycles. The variation is greater for TGF-βm–TβRII, thaumatin, and thermolysin, which are the lowest-resolution structures in this study. (Left) tau peptide (dashed black curve), lysozyme (solid orange curve), TGF-βm–TβRII (solid blue curve), and xylanase (dashed green curve). (Right) thaumatin (solid black curve), trypsin (dashed orange curve), proteinase K (solid blue curve), and thermolysin (dashed green curve). All refinements were performed using phenix.refine [Afonine et al. (2012) Acta Crystallogr D Biol Crystallogr 68, 352–367].
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de la Cruz, M., Hattne, J., Shi, D. et al. Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED. Nat Methods 14, 399–402 (2017). https://doi.org/10.1038/nmeth.4178
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DOI: https://doi.org/10.1038/nmeth.4178
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