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Growing and making nano- and microcrystals

Nature Protocols volume 18, pages 854–882 (2023)Cite this article

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Abstract

Thanks to recent technological advances in X-ray and micro-electron diffraction and solid-state NMR, structural information can be obtained by using much smaller crystals. Thus, microcrystals have become a valuable commodity rather than a mere stepping stone toward obtaining macroscopic crystals. Microcrystals are particularly useful for structure determination using serial data collection approaches at synchrotrons and X-ray free-electron lasers. The latter’s enormous peak brilliance and short X-ray pulse duration mean that structural information can be obtained before the effects of radiation damage are seen; these properties also facilitate time-resolved crystallography. To establish defined reaction initiation conditions, microcrystals with a desired and narrow size distribution are critical. Here, we describe milling and seeding techniques as well as filtration approaches for the reproducible and size-adjustable preparation of homogeneous nano- and microcrystals. Nanocrystals and crystal seeds can be obtained by milling using zirconium beads and the BeadBug homogenizer; fragmentation of large crystals yields micro- or nanocrystals by flowing crystals through stainless steel filters by using an HPLC pump. The approaches can be scaled to generate micro- to milliliter quantities of microcrystals, starting from macroscopic crystals. The procedure typically takes 3–5 d, including the time required to grow the microcrystals.

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Fig. 1: Crystallization phase diagram.
Fig. 2: Schematic representation of seeding with the BeadBug.
Fig. 3: Microcrystals of Scaf8.
Fig. 4: Crystal damage during imaging.
Fig. 5: Manual crystal filtration system.
Fig. 6: Analysis of seed preparation.
Fig. 7: Equipment setup, flow diagram and decision tree for the crystal-sizing procedure.
Fig. 8: Equipment setup, flow diagram and decision tree for the crystal fragmentation procedure.
Fig. 9: Microcrystals of dimensions suitable for optical pump X-ray probe experiments can often be grown from seeds.
Fig. 10: Fragmentation of large crystals yields microcrystals suitable for pump probe experiments using GDVN injection (or any other sample-delivery method).

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Data availability

Data and coordinates for the example proteins have been submitted to the Protein Data Bank; the PDB codes are given in Supplementary Table 1 (8A9E, 4N5R, 8A9F, 5FGT; e.g., https://www.rcsb.org/structure/4N5R). Other data are available from the authors upon reasonable request.

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Acknowledgements

We are grateful to T. Barends for making the initial versions of Figs. 1, 2, 7 and 8. We thank M. Kloos for sharing the preliminary data on fracturing LCP-grown bacteriorhodopsin crystals by filtration. We thank the Heidelberg FEL group for discussions and detailed feedback on the protocol during many XFEL beamtimes and the prior preparations. The work was supported by the Max Planck Society.

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  1. Max Planck Institute for Medical Research, Heidelberg, Germany

    Robert L. Shoeman, Elisabeth Hartmann & Ilme Schlichting

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  1. Robert L. Shoeman
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R.L.S. and I.S. conceived and established the protocols, and E.H. applied and fine-tuned the protocols for different systems. R.L.S. and I.S. wrote the manuscript with the help of E.H.

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Correspondence to Ilme Schlichting.

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Key references using this protocol

Barends, T. R. et al. Science 350, 445–450 (2015): https://doi.org/10.1126/science.aac5492

Nass, K. et al. IUCrJ 3, 180–191 (2016): https://doi.org/10.1107/S2052252516002980

Coquelle, N. et al. Nat. Chem. 10, 31–37 (2018): https://doi.org/10.1038/nchem.2853

Nass, K. et al. Nat. Commun. 11, 1814 (2020): https://doi.org/10.1038/s41467-020-15610-4

Sorigue, D. et al. Science 372, eabd5687 (2021): https://doi.org/10.1126/science.abd5687

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Supplementary Results 1–3, Methods, Note, Table 1 and Figs. 1–10.

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Shoeman, R.L., Hartmann, E. & Schlichting, I. Growing and making nano- and microcrystals. Nat Protoc 18, 854–882 (2023). https://doi.org/10.1038/s41596-022-00777-5

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