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Time-resolved cryo-EM using Spotiton

Nature Methods volume 17, pages 897–900 (2020)Cite this article

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Abstract

We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.

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Fig. 1: Apoferritin and 70S ribosomes were used as a proof of principle to illustrate mixing on the nanowire grids.
Fig. 2: Four examples of biological systems in which time-resolved cryo-EM provides answers.

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All data and metadata from these studies are available from the corresponding author upon request.

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Acknowledgements

We are grateful to the staff of the Simons Electron Microscopy Center at the New York Structural Biology Center for help and technical support. We thank I. Fernandez and B. Huang for kindly providing the ribosome subunits. We thank H. He (National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH)) for cryo-EM data collection, NIDDK EM Core Facility and the Hinshaw laboratory for critical comments. The work presented here was conducted at the National Resource for Automated Molecular Microscopy at the New York Structural Biology Center and supported by grants from the NIH (GM103310), the Simons Foundation (SF349247) and the NIDDK NIH Intramural Research Program. Other support included NIH/NIGMS grants R35 GM118130 (S.A.D.) and NIH/RO1GM088352 (C.M.N.).

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Author notes

  1. These authors contributed equally: Venkata P. Dandey, William C. Budell.

Authors and Affiliations

  1. The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA

    Venkata P. Dandey, William C. Budell, Hui Wei, Daija Bobe, Kashyap Maruthi, Mykhailo Kopylov, Edward T. Eng, Clinton S. Potter & Bridget Carragher

  2. Engineering Arts LLC, Tempe, AZ, USA

    Peter A. Kahn

  3. Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

    Jenny E. Hinshaw & Nidhi Kundu

  4. Department of Anesthesiology, Weill Cornell Medical College, New York, NY, USA

    Crina M. Nimigean, Chen Fan & Nattakan Sukomon

  5. Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, USA

    Seth A. Darst, Ruth M. Saecker, James Chen & Brandon Malone

  6. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Clinton S. Potter & Bridget Carragher

Authors
  1. Venkata P. Dandey
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Contributions

V.P.D. and W.C.B. performed all mixing experiments and analyzed data. H.W. assisted with nanowire grid preparation and Spotiton operation. D.B., K.M., M.K. and E.T.E. collected and analyzed cryo-EM data. M.K. assisted with the generation of figures. P.A.K. designed and built the Spotiton system and wrote the operational software. J.E.H. and N.K. contributed sample and biological insights for the dynamin studies. C.M.N., C.F. and N.S. contributed sample and biological insights for the MthK studies. S.A.D., R.M.S., J.C. and B.M. contributed sample and biological insights for the RNAP studies. B.C. and C.S.P. conceived the Spotiton system, designed the experiments and supervised all aspects of the study. V.P.D., W.C.B. and B.C. prepared the manuscript with assistance from all authors.

Corresponding author

Correspondence to Bridget Carragher.

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Competing interests

B.C. and C.S.P. have a commercial relationship with SPT Labtech, a company that produces a commercially available instrument, chameleon, that is based on the Spotiton prototype.

Additional information

Peer review information Arunima Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Specifications of time-resolved Spotiton operation.

a, Diagrammatic overview of the distances (fixed) and elapsed times (variable) relevant to spraying and mixing two samples on a moving grid. Simultaneous dispensing of both samples is triggered after the grid plunge begins. Representative images from the upper and lower cameras are shown directly below the illustrations of each. Sample 1 and sample 2 are indicated in blue and yellow, respectively. b, Magnified view of (green-dashed) boxed area in (a) showing grid and dispensing at specific time-points with corresponding high-speed video captures of the tips and grid below. Elapsed times shown on each image reflect estimates from a video of a grid plunged under Condition 2 (Supplementary Table 1). Objects in (a) and (b) are not drawn to scale. Supplementary Tables 1–4 list values for the following parameters of a grid plunged as depicted in (a) and (b): aaccel, acceleration rate; adecel, deceleration rate; vmax, maximum velocity; t0, plunge start point; tdisp-1, grid leading edge reaches first dispenser; tsamp-1, sample 1 fully applied to grid; tdisp-2, grid leading edge reaches second dispenser; tmix, samples 1 and 2 fully applied to grid; tUC, grid reaches upper camera, tLC, grid reaches lower camera; te, grid plunges into ethane. ‘Spot-to-plunge’ and ‘mix-to-plunge’ in (a) reflect the elapsed times from tdisp-1 or tmix to te, respectively.

Extended Data Fig. 2 Mixing 30 S and 50 S ribosomal subunits to form 70 S complexes.

a, ~20% of particles present were reconstructed to 70 S complex at a resolution of 4.75 Å as indicated by FSC0.5 (b). c, 2D classes of 50 S ribosomal subunit obtained from the control experiment; 2D class of 50S-50S dimer is shown in red. d, 2D classes of the 30 S ribosomal subunit obtained from the control experiment. Both control experiments show no evidence of 70 S ribosomes as observed in the mixed experiment. Scale bars, 20 nm.

Extended Data Fig. 3 Cryo-EM maps of MthK RCK domain with and without Ca2+.

a, The two additional Ca2+ binding sites of MthK either vacant from a control experiment (top row) or occupied after mixing with calcium (bottom row). b, 3D volumes of MthK RCK domains without (top row) and with (bottom row) Ca2+ bound.

Extended Data Fig. 4 Mixing of GTP with dynamin-decorated lipid tubes results in constriction.

Representative cryo-electron micrographs of control dynamin-decorated tubes without GTP (a), with 2 mM GTP (b) and 4 mM GTP (c). Scale bars, 50 nm.

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Dandey, V.P., Budell, W.C., Wei, H. et al. Time-resolved cryo-EM using Spotiton. Nat Methods 17, 897–900 (2020). https://doi.org/10.1038/s41592-020-0925-6

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