@article{0def22e35f644a8ba42f54b95e1e46c5,
title = "Light-coupled cryo-plunger for time-resolved cryo-EM",
abstract = "Proteins are dynamic molecules that can undergo rapid conformational rearrangements in response to stimuli. These structural changes are often critical to protein function, and thus elucidating time-dependent conformational landscapes has been a long-standing goal of structural biology. To harness the power of single particle cryo-EM methods to enable {\textquoteleft}time-resolved{\textquoteright} structure determination, we have developed a light-coupled cryo-plunger that pairs flash-photolysis of caged ligands with rapid sample vitrification. The {\textquoteleft}flash-plunger{\textquoteright} consists of a high-power ultraviolet LED coupled with focusing optics and a motorized linear actuator, enabling the user to immobilize protein targets in vitreous ice within a programmable time window – as short as tens of milliseconds – after stimulus delivery. The flash-plunger is a simple, inexpensive and flexible tool to explore short-lived conformational states previously unobtainable by conventional sample preparation methods.",
keywords = "Cryo-EM, Cryo-electron microscopy, Manual cryo-plunger, Single particle, Time-resolved",
author = "Nate Yoder and Farzad Jalali-Yazdi and Sigrid Noreng and Alexandra Houser and Isabelle Baconguis and Eric Gouaux",
note = "Funding Information: We thank L. Vaskalis for help with figures, H. Owen for manuscript preparation and all Gouaux lab members for their support. Additionally, we thank Thomas Braun and Lucas Rima for helpful instruction regarding manual plunger design and Hans-J{\"u}rgen Apell for advice concerning the selection, synthesis and use of caged proton compounds. This research was supported by the National Institute of Diabetes and Digestive Kidney Diseases (5T32DK007680), the National Institute of Neurological Disorders and Stroke (5F31NS096782 to N.Y., 5F32MH115595 to F.J.-Y, and 5R01NS038631 to E.G.), the American Heart Association (18PRE33990205 to S.N.), the National Science Foundation (DGE-1937961 to A.H.) and the National Institutes of Health (DP5OD017871 to I.B.). Initial electron microscopy work was performed at the Multiscale Microscopy Core at OHSU. A portion of this research was performed at the National Center for Cryo-EM Access and Training and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24GM129539) and by grants from the Simons Foundation (SF349247) and New York State. Subsequent research was supported by NIH grant U24GM129547 and performed at the Pacific Northwest Cryo-EM Center at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. Additional support was provided by ARCS Foundation and Tartar Trust fellowships. E.G. is an Investigator with the Howard Hughes Medical Institute. Funding Information: We thank L. Vaskalis for help with figures, H. Owen for manuscript preparation and all Gouaux lab members for their support. Additionally, we thank Thomas Braun and Lucas Rima for helpful instruction regarding manual plunger design and Hans-J?rgen Apell for advice concerning the selection, synthesis and use of caged proton compounds. This research was supported by the National Institute of Diabetes and Digestive Kidney Diseases (5T32DK007680), the National Institute of Neurological Disorders and Stroke (5F31NS096782 to N.Y. 5F32MH115595 to F.J.-Y, and 5R01NS038631 to E.G.), the American Heart Association (18PRE33990205 to S.N.), the National Science Foundation (DGE-1937961 to A.H.) and the National Institutes of Health (DP5OD017871 to I.B.). Initial electron microscopy work was performed at the Multiscale Microscopy Core at OHSU. A portion of this research was performed at the National Center for Cryo-EM Access and Training and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24GM129539) and by grants from the Simons Foundation (SF349247) and New York State. Subsequent research was supported by NIH grant U24GM129547 and performed at the Pacific Northwest Cryo-EM Center at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. Additional support was provided by ARCS Foundation and Tartar Trust fellowships. E.G. is an Investigator with the Howard Hughes Medical Institute. N.Y. F.J.-Y. A.H. S.N. I.B. and E.G. designed the project. N.Y. designed and built the apparatus. N.Y. and F.J.-Y. tested the apparatus. N.Y. performed the protein purification and data collection. N.Y. and F.J.-Y. processed the cryo-EM data. S.N. and A.H. performed preliminary biochemistry and electron microscopy experiments. N.Y wrote the manuscript and all authors edited the manuscript. All cryo-EM maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-21385 for 1000 mA-flashed cASIC1a and EMD-21384 for 3000 mA-flashed cASIC1a. Locally refined ECD-only map deposited as an additional map under entry EMD-21384. Technical drawings for most commercial components are available directly from the manufacturer and drawings for custom 3D printed parts will be made available upon request. Publisher Copyright: {\textcopyright} 2020 Elsevier Inc.",
year = "2020",
month = dec,
day = "1",
doi = "10.1016/j.jsb.2020.107624",
language = "English (US)",
volume = "212",
journal = "Journal of Structural Biology",
issn = "1047-8477",
publisher = "Academic Press Inc.",
number = "3",
}