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Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED

Nature Methods volume 14, pages 399–402 (2017)Cite this article

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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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Figure 1: Crystals before and after fragmentation and their X-ray and MicroED diffraction patterns.
Figure 2: Eight atomic-resolution structures determined by MicroED from crystal fragments.

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References

  1. Darwin, C.G. Philisophical Magazine Series 6 43, 800–829 (1922).

    Article  CAS  Google Scholar 

  2. Nave, C. Acta Crystallogr. D Biol. Crystallogr. 54, 848–853 (1998).

    Article  CAS  Google Scholar 

  3. Cusack, S. et al. Nat. Struct. Biol. 5 (Suppl.), 634–637 (1998).

    Article  CAS  Google Scholar 

  4. Schlichting, I. IUCrJ 2, 246–255 (2015).

    Article  CAS  Google Scholar 

  5. Shi, D., Nannenga, B.L., Iadanza, M.G. & Gonen, T. eLife 2, e01345 (2013).

    Article  Google Scholar 

  6. Weierstall, U. Phil. Trans. R. Soc. Lond. B 369, 20130337 (2014).

    Article  Google Scholar 

  7. Sierra, R.G. et al. Acta Crystallogr. D Biol. Crystallogr. 68, 1584–1587 (2012).

    Article  CAS  Google Scholar 

  8. Henderson, R. Q. Rev. Biophys. 28, 171–193 (1995).

    Article  CAS  Google Scholar 

  9. Nannenga, B.L., Shi, D., Leslie, A.G.W. & Gonen, T. Nat. Methods 11, 927–930 (2014).

    Article  CAS  Google Scholar 

  10. Nannenga, B.L., Shi, D., Hattne, J., Reyes, F.E. & Gonen, T. eLife 3, e03600 (2014).

    Article  Google Scholar 

  11. Colliex, C. et al. in International Tables for Crystallography, Vol. C (eds. Prince, E. & Wellberry, T.R.) Ch. 4.3 (International Union of Crystallography, 2006).

  12. Yonekura, K., Kato, K., Ogasawara, M., Tomita, M. & Toyoshima, C. Proc. Natl. Acad. Sci. USA 112, 3368–3373 (2015).

    Article  CAS  Google Scholar 

  13. Hattne, J., Shi, D., de la Cruz, M.J., Reyes, F.E. & Gonen, T. J. Appl. Crystallogr. 49, 1029–1034 (2016).

    Article  CAS  Google Scholar 

  14. Rodriguez, J.A. et al. Nature 525, 486–490 (2015).

    Article  CAS  Google Scholar 

  15. Sawaya, M.R. et al. Proc. Natl. Acad. Sci. USA 113, 11232–11236 (2016).

    Article  CAS  Google Scholar 

  16. Von Laue, M. in Nobel Lectures Physics 1901–1921, 347–355 (Elsevier, 1967).

  17. Stevenson, H.P. et al. Acta. Crystallogr. D Struct. Biol. 72, 603–615 (2016).

    Article  CAS  Google Scholar 

  18. Bublitz, M. et al. IUCrJ 2, 409–420 (2015).

    Article  CAS  Google Scholar 

  19. The PyMOL Molecular Graphics System v. 1.8 (Schrödinger, LLC, 2015).

  20. Zúñiga, J.E. et al. J. Mol. Biol. 354, 1052–1068 (2005).

    Article  Google Scholar 

  21. Watanabe, N., Akiba, T., Kanai, R. & Harata, K. Acta Crystallogr. D Biol. Crystallogr. 62, 784–792 (2006).

    Article  Google Scholar 

  22. English, A.C., Done, S.H., Caves, L.S.D., Groom, C.R. & Hubbard, R.E. Proteins 37, 628–640 (1999).

    Article  CAS  Google Scholar 

  23. de la Cruz, M.J. et al. Micro- and nanocrystal preparation for MicroED and XFEL serial crystallography by fragmentation of imperfect crystals. Protocol Exchange http://dx.doi.org/10.1038/protex.2017.010 (2017).

  24. Hattne, J. et al. Acta Crystallogr. A Found. Adv. 71, 353–360 (2015).

    Article  CAS  Google Scholar 

  25. Shi, D. et al. Nat. Protoc. 11, 895–904 (2016).

    Article  CAS  Google Scholar 

  26. Leslie, A.G.W. & Powell, H.R. (eds.) Evolving Methods for Macromolecular Crystallography (Springer, 2007).

  27. Battye, T.G.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G.W. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

    Article  CAS  Google Scholar 

  28. Kabsch, W. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  29. Evans, P.R. & Murshudov, G.N. Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    Article  CAS  Google Scholar 

  30. Vagin, A. & Teplyakov, A. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    Article  CAS  Google Scholar 

  31. Sheldrick, G.M. Acta Crystallogr. A Found. Adv. 71, 3–8 (2015).

    Article  Google Scholar 

  32. Winn, M.D. et al. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  Google Scholar 

  33. Afonine, P.V. et al. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  Google Scholar 

  34. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  35. Brunger, A.T. Nat. Protoc. 2, 2728–2733 (2007).

    Article  CAS  Google Scholar 

  36. Gonen, T. et al. Nature 438, 633–638 (2005).

    Article  CAS  Google Scholar 

  37. Meyer, P.A. et al. Nat. Commun. 7, 10882 (2016).

    Article  CAS  Google Scholar 

Download references

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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Authors and Affiliations

  1. Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA

    M Jason de la Cruz, Johan Hattne, Dan Shi, Francis E Reyes & Tamir Gonen

  2. Departments of Biological Chemistry, Howard Hughes Medical Institute, UCLA-DOE Institute, Chemistry & Biochemistry, and Molecular Biology Institute, UCLA, Los Angeles, California, USA

    Paul Seidler, Jose Rodriguez, Michael R Sawaya, Duilio Cascio & David Eisenberg

  3. Department of Structural Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Simon C Weiss, Sun Kyung Kim, Cynthia S Hinck, Andrew P Hinck & Guillermo Calero

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  1. M Jason de la Cruz
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  2. Johan Hattne
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Contributions

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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Correspondence to Tamir Gonen.

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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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