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Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

Author

Listed:
  • Pedram Mehrabi

    (Institute for Nanostructure and Solid-State Physics, Universität Hamburg
    Max Planck Institute for the Structure and Dynamics of Matter)

  • Sihyun Sung

    (European Molecular Biology Laboratory, Hamburg Unit)

  • David Stetten

    (European Molecular Biology Laboratory, Hamburg Unit)

  • Andreas Prester

    (University Medical Center Hamburg-Eppendorf (UKE))

  • Caitlin E. Hatton

    (Institute for Nanostructure and Solid-State Physics, Universität Hamburg)

  • Stephan Kleine-Döpke

    (Institute for Nanostructure and Solid-State Physics, Universität Hamburg)

  • Alexander Berkes

    (Institute for Nanostructure and Solid-State Physics, Universität Hamburg)

  • Gargi Gore

    (Institute for Nanostructure and Solid-State Physics, Universität Hamburg)

  • Jan-Philipp Leimkohl

    (Max Planck Institute for the Structure and Dynamics of Matter)

  • Hendrik Schikora

    (Max Planck Institute for the Structure and Dynamics of Matter)

  • Martin Kollewe

    (Max Planck Institute for the Structure and Dynamics of Matter)

  • Holger Rohde

    (University Medical Center Hamburg-Eppendorf (UKE))

  • Matthias Wilmanns

    (European Molecular Biology Laboratory, Hamburg Unit
    University Medical Center Hamburg-Eppendorf (UKE))

  • Friedjof Tellkamp

    (Max Planck Institute for the Structure and Dynamics of Matter)

  • Eike C. Schulz

    (Institute for Nanostructure and Solid-State Physics, Universität Hamburg
    Max Planck Institute for the Structure and Dynamics of Matter
    University Medical Center Hamburg-Eppendorf (UKE))

Abstract

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging. The SPINE-standard sample holder is directly plunged into a storage puck, enabling compatibility with high-throughput infrastructure. Here we demonstrate binding of glucose and 2,3-butanediol in microcrystals of xylose isomerase, and of avibactam and ampicillin in microcrystals of the extended spectrum beta-lactamase CTX-M-14. We also trap reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase to demonstrate that the spitrobot enables insight into catalytic events.

Suggested Citation

  • Pedram Mehrabi & Sihyun Sung & David Stetten & Andreas Prester & Caitlin E. Hatton & Stephan Kleine-Döpke & Alexander Berkes & Gargi Gore & Jan-Philipp Leimkohl & Hendrik Schikora & Martin Kollewe & H, 2023. "Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography," Nature Communications, Nature, vol. 14(1), pages 1-9, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-37834-w
    DOI: 10.1038/s41467-023-37834-w
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    References listed on IDEAS

    as
    1. Max O. Wiedorn & Dominik Oberthür & Richard Bean & Robin Schubert & Nadine Werner & Brian Abbey & Martin Aepfelbacher & Luigi Adriano & Aschkan Allahgholi & Nasser Al-Qudami & Jakob Andreasson & Steve, 2018. "Megahertz serial crystallography," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
    2. Lars V. Bock & Helmut Grubmüller, 2022. "Effects of cryo-EM cooling on structural ensembles," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    Full references (including those not matched with items on IDEAS)

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