Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Time-resolved cryo-EM using Spotiton

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

Data availability

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

References

  1. Frank, J. Time-resolved cryo-electron microscopy: recent progress. J. Struct. Biol. 200, 303–306 (2017).

    Article  CAS  Google Scholar 

  2. Berriman, J. & Unwin, N. Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets. Ultramicroscopy 56, 241–252 (1994).

    Article  CAS  Google Scholar 

  3. White, H. D., Walker, M. L. & Trinick, J. A computer-controlled spraying-freezing apparatus for millisecond time-resolution electron cryomicroscopy. J. Struct. Biol. 121, 306–313 (1998).

    Article  CAS  Google Scholar 

  4. Unwin, N. & Fujiyoshi, Y. Gating movement of acetylcholine receptor caught by plunge-freezing. J. Mol. Biol. 422, 617–634 (2012).

    Article  CAS  Google Scholar 

  5. Subramaniam, S. et al. Protein conformational changes in the bacteriorhodopsin photocycle. J. Mol. Biol. 287, 145–161 (1999).

    Article  CAS  Google Scholar 

  6. Lu, Z. et al. Gas-assisted annular microsprayer for sample preparation for time-resolved cryo-electron microscopy. J. Micromech. Microeng. 24, 115001 (2014).

    Article  CAS  Google Scholar 

  7. Lu, Z. et al. Passive microfluidic device for sub millisecond mixing. Sens. Actuators B Chem. 144, 301–309 (2010).

    Article  CAS  Google Scholar 

  8. Fu, Z. et al. Key intermediates in ribosome recycling visualized by time-resolved cryo-electron microscopy. Structure 24, 2092–2101 (2016).

    Article  CAS  Google Scholar 

  9. Jain, T., Sheehan, P., Crum, J., Carragher, B. & Potter, C. S. Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. J. Struct. Biol. 179, 68–75 (2012).

    Article  Google Scholar 

  10. Dandey, V. P. et al. Spotiton: new features and applications. J. Struct. Biol. 202, 161–169 (2018).

    Article  CAS  Google Scholar 

  11. Wei, H. et al. Optimizing ‘self-wicking’ nanowire grids. J. Struct. Biol. 202, 170–174 (2018).

    Article  CAS  Google Scholar 

  12. Scapin, G. et al. Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis. Nature 556, 122–125 (2018).

    Article  CAS  Google Scholar 

  13. Zhang, Z. et al. Ensemble cryo-EM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme. eLife 7, e33572 (2018).

  14. Han, H. et al. Structure of Vps4 with circular peptides and implications for translocation of two polypeptide chains by AAA+ ATPases. eLife 8, e44071 (2019).

  15. Liu, Y. et al. FACT caught in the act of manipulating the nucleosome. Nature 577, 426–431 (2020).

    Article  CAS  Google Scholar 

  16. Noble, A. J. et al. Routine single-particle Cryo-EM sample and grid characterization by tomography. eLife 7, e34257 (2018).

    Article  Google Scholar 

  17. Wu, J. L. Y., Tellkamp, F., Khajehpour, M., Robertson, W. D. & Miller, R. J. D. Rapid mixing of colliding picoliter liquid droplets delivered through-space from piezoelectric-actuated pipettes characterized by time-resolved fluorescence monitoring. Rev. Sci. Instrum. 90, 055109 (2019).

    Article  CAS  Google Scholar 

  18. Lu, Z. et al. Monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy. J. Struct. Biol. 168, 388–395 (2009).

    Article  Google Scholar 

  19. Chen, B. et al. Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy. Structure 23, 1097–1105 (2015).

    Article  CAS  Google Scholar 

  20. Zadek, B. & Nimigean, C. M. Calcium-dependent gating of MthK, a prokaryotic potassium channel. J. Gen. Physiol. 127, 673–685 (2006).

    Article  CAS  Google Scholar 

  21. Posson, D. J., Rusinova, R., Andersen, O. S. & Nimigean, C. M. Calcium ions open a selectivity filter gate during activation of the MthK potassium channel. Nat. Commun. 6, 8342 (2015).

    Article  CAS  Google Scholar 

  22. Fan, C. et al. Ball-and-chain inactivation in a calcium-gated potassium channel. Nature 580, 288–293 (2020).

  23. Ruff, E. F., Record, M. T. J. & Artsimovitch, I. Initial events in bacterial transcription initiation. Biomolecules 5, 1035–1062 (2015).

    Article  CAS  Google Scholar 

  24. Mazumder, A. & Kapanidis, A. N. Recent advances in understanding σ70-dependent transcription initiation mechanisms. J. Mol. Biol. 431, 3947–3959 (2019).

  25. Saecker, R. M., Record, M. T. J. & Dehaseth, P. L. Mechanism of bacterial transcription initiation: RNA polymerase-promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. J. Mol. Biol. 412, 754–771 (2011).

    Article  CAS  Google Scholar 

  26. Sundborger, A. C. et al. A dynamin mutant defines a superconstricted prefission state. Cell Rep. 8, 734–742 (2014).

    Article  CAS  Google Scholar 

  27. Kong, L. et al. Cryo-EM of the dynamin polymer assembled on lipid membrane. Nature 560, 258–262 (2018).

    Article  CAS  Google Scholar 

  28. Johansson, M., Bouakaz, E., Lovmar, M. & Ehrenberg, M. The kinetics of ribosomal peptidyl transfer revisited. Mol. Cell 30, 589–598 (2008).

    Article  CAS  Google Scholar 

  29. Chen, J. et al. E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation. eLife 8, e49375 (2019).

  30. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    Article  CAS  Google Scholar 

  31. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  32. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

  33. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  Google Scholar 

  34. Sorzano, C. O. S. et al. A clustering approach to multireference alignment of single-particle projections in electron microscopy. J. Struct. Biol. 171, 197–206 (2010).

    Article  CAS  Google Scholar 

  35. Lander, G. C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009).

    Article  CAS  Google Scholar 

  36. Roseman, A. M. FindEM—a fast, efficient program for automatic selection of particles from electron micrographs. J. Struct. Biol. 145, 91–99 (2004).

    Article  CAS  Google Scholar 

  37. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  Google Scholar 

  38. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

Download references

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.).

Author information

Authors and Affiliations

Authors

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.

Ethics declarations

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 14 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.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-020-0925-6

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing