Droplet microfluidics for time-resolved serial crystallography
Droplet microfluidics for time-resolved serial crystallography
Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here we report the use of droplet miniaturisation for the controlled production of uniform crystals, providing an avenue for controlled diffusion and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3-µm lysozyme crystals and 2-µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of
Stubbs, Jack
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Hornsey, Theo
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Hanrahan, Niall
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Blay Esteban, Luis
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Bolton, Rachel
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Malý, Martin
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Basu, Shibom
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Orlans, Julien
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de Sanctis, Daniele
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Shim, Jung-uk
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Shaw Stewart, Patrick D.
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Orville, Allen M.
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Tews, Ivo
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West, Jonathan
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5 March 2024
Stubbs, Jack
ed13c87e-6ffc-45cd-96d3-7b3c12569a5d
Hornsey, Theo
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Hanrahan, Niall
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Blay Esteban, Luis
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Bolton, Rachel
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Malý, Martin
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Basu, Shibom
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Orlans, Julien
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de Sanctis, Daniele
d59b55e7-fc9a-4f09-a9f7-d2691afd111f
Shim, Jung-uk
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Shaw Stewart, Patrick D.
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Orville, Allen M.
6315216e-d496-43f3-a97b-a07fc45e8b56
Tews, Ivo
9117fc5e-d01c-4f8d-a734-5b14d3eee8dd
West, Jonathan
f1c2e060-16c3-44c0-af70-242a1c58b968
Stubbs, Jack, Hornsey, Theo, Hanrahan, Niall, Blay Esteban, Luis, Bolton, Rachel, Malý, Martin, Basu, Shibom, Orlans, Julien, de Sanctis, Daniele, Shim, Jung-uk, Shaw Stewart, Patrick D., Orville, Allen M., Tews, Ivo and West, Jonathan
(2024)
Droplet microfluidics for time-resolved serial crystallography.
IUCrJ, 11.
(doi:10.1101/2024.01.12.575388).
Abstract
Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here we report the use of droplet miniaturisation for the controlled production of uniform crystals, providing an avenue for controlled diffusion and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3-µm lysozyme crystals and 2-µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of
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Accepted/In Press date: 21 February 2024
Published date: 5 March 2024
Additional Information:
Funding information: the research was funded by a Diamond Doctoral Studentship Programme (JS, RB), a South Coast Biosciences Doctoral Training Partnership SoCoBio DTP BBSRC Studentship BB/T008768/1 (JS), Wellcome Investigator Award 210734/Z/18/Z (AMO), Royal Society Wolfson Fellowship RSWF\R2\182017 (AMO), EPSRC Impact Acceleration Account and EPSRC Transformative Healthcare Grant EP/T020997/1 (NH), University of Southampton Seed Enterprise Development Fund (NH), BBSRC BB/S008470/1 (IT) and STFC “Serial Data Processing and Analysis in CCP4i2 (IT, MM).
Identifiers
Local EPrints ID: 487615
URI: http://eprints.soton.ac.uk/id/eprint/487615
ISSN: 2052-2525
PURE UUID: c48a7b59-b3d9-41c1-a308-ba1850e0da81
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Date deposited: 29 Feb 2024 17:36
Last modified: 23 Mar 2024 05:01
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Contributors
Author:
Theo Hornsey
Author:
Niall Hanrahan
Author:
Luis Blay Esteban
Author:
Rachel Bolton
Author:
Martin Malý
Author:
Shibom Basu
Author:
Julien Orlans
Author:
Daniele de Sanctis
Author:
Jung-uk Shim
Author:
Patrick D. Shaw Stewart
Author:
Allen M. Orville
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