An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

Agata Butryn; Philipp S. Simon; Pierre Aller; Philip Hinchliffe; Ramzi N. Massad; Gabriel Leen; Catherine L. Tooke; Isabel Bogacz; In Sik Kim; Asmit Bhowmick; Aaron S. Brewster; Nicholas E. Devenish; Jürgen Brem; Jos J.A.G. Kamps; Pauline A. Lang; Patrick Rabe; Danny Axford; John H. Beale; Bradley Davy; Ali Ebrahim; Julien Orlans; Selina L.S. Storm; Tiankun Zhou; Shigeki Owada; Rie Tanaka; Kensuke Tono; Gwyndaf Evans; Robin L. Owen; Frances A. Houle; Nicholas K. Sauter; Christopher J. Schofield; James Spencer; Vittal K. Yachandra; Junko Yano; Jan F. Kern; Allen M. Orville

doi:10.1038/s41467-021-24757-7

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

Agata Butryn, Philipp S. Simon, Pierre Aller, Philip Hinchliffe, Ramzi N. Massad, Gabriel Leen, Catherine L. Tooke, Isabel Bogacz, In Sik Kim, Asmit Bhowmick, Aaron S. Brewster, Nicholas E. Devenish, Jürgen Brem, Jos J.A.G. Kamps, Pauline A. Lang, Patrick Rabe, Danny Axford, John H. Beale, Bradley Davy, Ali EbrahimJulien Orlans, Selina L.S. Storm, Tiankun Zhou, Shigeki Owada, Rie Tanaka, Kensuke Tono, Gwyndaf Evans, Robin L. Owen, Frances A. Houle, Nicholas K. Sauter, Christopher J. Schofield, James Spencer, Vittal K. Yachandra, Junko Yano, Jan F. Kern, Allen M. Orville

Department of Electronics & Computer Engineering

Research output: Contribution to journal › Article › peer-review

Abstract

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

Original language	English
Article number	4461
Journal	Nature Communications
Volume	12
Issue number	1
DOIs	https://doi.org/10.1038/s41467-021-24757-7
Publication status	Published - 1 Dec 2021

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Butryn, A., Simon, P. S., Aller, P., Hinchliffe, P., Massad, R. N., Leen, G., Tooke, C. L., Bogacz, I., Kim, I. S., Bhowmick, A., Brewster, A. S., Devenish, N. E., Brem, J., Kamps, J. J. A. G., Lang, P. A., Rabe, P., Axford, D., Beale, J. H., Davy, B., ... Orville, A. M. (2021). An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography. Nature Communications, 12(1), Article 4461. https://doi.org/10.1038/s41467-021-24757-7

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title = "An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography",

abstract = "Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.",

author = "Agata Butryn and Simon, {Philipp S.} and Pierre Aller and Philip Hinchliffe and Massad, {Ramzi N.} and Gabriel Leen and Tooke, {Catherine L.} and Isabel Bogacz and Kim, {In Sik} and Asmit Bhowmick and Brewster, {Aaron S.} and Devenish, {Nicholas E.} and J{\"u}rgen Brem and Kamps, {Jos J.A.G.} and Lang, {Pauline A.} and Patrick Rabe and Danny Axford and Beale, {John H.} and Bradley Davy and Ali Ebrahim and Julien Orlans and Storm, {Selina L.S.} and Tiankun Zhou and Shigeki Owada and Rie Tanaka and Kensuke Tono and Gwyndaf Evans and Owen, {Robin L.} and Houle, {Frances A.} and Sauter, {Nicholas K.} and Schofield, {Christopher J.} and James Spencer and Yachandra, {Vittal K.} and Junko Yano and Kern, {Jan F.} and Orville, {Allen M.}",

note = "Publisher Copyright: {\textcopyright} 2021, Crown.",

year = "2021",

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doi = "10.1038/s41467-021-24757-7",

language = "English",

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Butryn, A, Simon, PS, Aller, P, Hinchliffe, P, Massad, RN, Leen, G, Tooke, CL, Bogacz, I, Kim, IS, Bhowmick, A, Brewster, AS, Devenish, NE, Brem, J, Kamps, JJAG, Lang, PA, Rabe, P, Axford, D, Beale, JH, Davy, B, Ebrahim, A, Orlans, J, Storm, SLS, Zhou, T, Owada, S, Tanaka, R, Tono, K, Evans, G, Owen, RL, Houle, FA, Sauter, NK, Schofield, CJ, Spencer, J, Yachandra, VK, Yano, J, Kern, JF & Orville, AM 2021, 'An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography', Nature Communications, vol. 12, no. 1, 4461. https://doi.org/10.1038/s41467-021-24757-7

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T1 - An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

AU - Butryn, Agata

AU - Simon, Philipp S.

AU - Aller, Pierre

AU - Hinchliffe, Philip

AU - Massad, Ramzi N.

AU - Leen, Gabriel

AU - Tooke, Catherine L.

AU - Bogacz, Isabel

AU - Kim, In Sik

AU - Bhowmick, Asmit

AU - Brewster, Aaron S.

AU - Devenish, Nicholas E.

AU - Brem, Jürgen

AU - Kamps, Jos J.A.G.

AU - Lang, Pauline A.

AU - Rabe, Patrick

AU - Axford, Danny

AU - Beale, John H.

AU - Davy, Bradley

AU - Ebrahim, Ali

AU - Orlans, Julien

AU - Storm, Selina L.S.

AU - Zhou, Tiankun

AU - Owada, Shigeki

AU - Tanaka, Rie

AU - Tono, Kensuke

AU - Evans, Gwyndaf

AU - Owen, Robin L.

AU - Houle, Frances A.

AU - Sauter, Nicholas K.

AU - Schofield, Christopher J.

AU - Spencer, James

AU - Yachandra, Vittal K.

AU - Yano, Junko

AU - Kern, Jan F.

AU - Orville, Allen M.

PY - 2021/12/1

Y1 - 2021/12/1

N2 - Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

AB - Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

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DO - 10.1038/s41467-021-24757-7

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AN - SCOPUS:85111135575

SN - 2041-1723

VL - 12

JO - Nature Communications

JF - Nature Communications

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M1 - 4461

ER -

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

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