An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography
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 fe...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Springer Nature
2021
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| _version_ | 1826264727700897792 |
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| author | Butryn, A Simon, PS Aller, P Hinchliffe, P Massad, RN Leen, G Tooke, CL Bogacz, I Kim, I-S 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 |
| author_facet | Butryn, A Simon, PS Aller, P Hinchliffe, P Massad, RN Leen, G Tooke, CL Bogacz, I Kim, I-S 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 |
| author_sort | Butryn, A |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-06T20:12:30Z |
| format | Journal article |
| id | oxford-uuid:2b0a4284-ed59-4e6a-b67a-91cad3723d95 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T20:12:30Z |
| publishDate | 2021 |
| publisher | Springer Nature |
| record_format | dspace |
| spelling | oxford-uuid:2b0a4284-ed59-4e6a-b67a-91cad3723d952022-03-26T12:28:48ZAn on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallographyJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:2b0a4284-ed59-4e6a-b67a-91cad3723d95EnglishSymplectic ElementsSpringer Nature2021Butryn, ASimon, PSAller, PHinchliffe, PMassad, RNLeen, GTooke, CLBogacz, IKim, I-SBhowmick, ABrewster, ASDevenish, NEBrem, JKamps, JJAGLang, PARabe, PAxford, DBeale, JHDavy, BEbrahim, AOrlans, JStorm, SLSZhou, TOwada, STanaka, RTono, KEvans, GOwen, RLHoule, FASauter, NKSchofield, CJSpencer, JYachandra, VKYano, JKern, JFOrville, AMSerial 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. |
| spellingShingle | Butryn, A Simon, PS Aller, P Hinchliffe, P Massad, RN Leen, G Tooke, CL Bogacz, I Kim, I-S 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 An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography |
| title | An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography |
| title_full | An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography |
| title_fullStr | An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography |
| title_full_unstemmed | An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography |
| title_short | An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography |
| title_sort | on demand drop on drop method for studying enzyme catalysis by serial crystallography |
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