Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography

Jos J. A. G. Kamps; Philip Hinchliffe; Johan Glerup; Emily I. Freeman; Pauline A. Lang; Catherine L. Tooke; Michael Beer; Laura Parkinson; Do Heon Gu; Sehan Park; Nicholas Devenish; Tiankun Zhou; Anastasya Shilova; Samanpreet Kaur; Patrick Rabe; Christopher J. Schofield; James Spencer; Jaehyun Park; Robin L. Owen; Allen M. Orville; Pierre Aller

doi:10.1107/S2052252526003489

Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography

Jos J. A. G. Kamps
, Philip Hinchliffe
, Johan Glerup
, Emily I. Freeman
, Pauline A. Lang
, Catherine L. Tooke
, Michael Beer
, Laura Parkinson
, Do Heon Gu
, Sehan Park
, Nicholas Devenish
, Tiankun Zhou
, Anastasya Shilova
, Samanpreet Kaur
, Patrick Rabe
, Christopher J. Schofield
, James Spencer
, Jaehyun Park
, Robin L. Owen
, Allen M. Orville

Pierre Aller

Diamond Light Source Ltd
Rutherford Appleton Laboratory
University of Bristol
University of Oxford
POSTECH

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

Abstract

We describe the design and implementation of a drop-on-fixed-target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume aqueous droplets (∼40-90 pl; ∼40-55 μm diameter sphere), containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals previously loaded into the trapezoidal wells of an etched crystalline silicon fixed-target chip containing 25 600 wells in a high-density, square grid with 125 μm centre-to-centre well spacing. These features demand exquisite accuracy and thereby constrain motion controls to enable robust time-resolved crystallographic studies. The system was tested with three enzyme systems, comprising lysozyme and two β-lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free-electron laser facilities was evaluated. This drop-on-fixed-target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.

Original language	English
Pages (from-to)	471-484
Number of pages	14
Journal	IUCrJ
Volume	13
Early online date	5 Jun 2026
DOIs	https://doi.org/10.1107/S2052252526003489
Publication status	Published - 1 Jul 2026

Acknowledgements

We thank the staff supporting our beamtimes (2022-2nd-NCI-008, 2023-2nd-NCI-014) at NCI, PAL-XFEL. The authors also thank the Global Science experimental Data Hub Center (GSDC) at the Korea Institute of Science and Technology Information (KISTI) for providing computing resources and technical support. We also thank the staff for experiments (MX-25260) at beamline I24, DLS.

Funding

AMO, RLO, PA, JG and JJAGK received support from DLS and UK Science and Technology Facilities Council (STFC). AMO is the recipient of a Royal Society Wolfson Fellowship RSWF\R2\182017 and a Wellcome Investigator Award 210734/Z/18/Z. CLT thanks the University of Bath Prize Fellowship Scheme and the UK Medical Research Council for fellowship funding (UKRI330). Research was supported by the BBSRC-funded South West Biosciences Doctoral Training Partnership (training grant reference BB/T008741/1, studentships to LP and MB), and the Interdisciplinary Bioscience Doctoral Training Partnership (grant number BB/T008784/1, studentship to EIF). PAL thanks the National PhD Training Program in Antimicrobial Resistance Research by the Medical Research Foundation (MRF-145-0004-TPG-AVISO) for funding a postgraduate studentship. CLT and JS thank the UK Medical Research Council for support through the grant MR/T016035/1. This work is part of a project that has received funding from the European Research Council under the European Horizon 2020 research and innovation program (PREDACTED Advanced Grant Agreement No. 101021207) to JS. PR thanks the Wellcome Trust (227298/Z/23/Z). EIF and CJS were supported by the Ineos Oxford Institute for Antimicrobial Research.

Keywords

enzyme mechanisms
sample delivery
serial crystallography
structure determination
time-resolved studies

ASJC Scopus subject areas

General Chemistry
Biochemistry
General Materials Science
Condensed Matter Physics

Access to Document

10.1107/S2052252526003489Licence: CC BY

Cite this

Kamps, J. J. A. G., Hinchliffe, P., Glerup, J., Freeman, E. I., Lang, P. A., Tooke, C. L., Beer, M., Parkinson, L., Gu, D. H., Park, S., Devenish, N., Zhou, T., Shilova, A., Kaur, S., Rabe, P., Schofield, C. J., Spencer, J., Park, J., Owen, R. L., ... Aller, P. (2026). Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography. IUCrJ, 13, 471-484. https://doi.org/10.1107/S2052252526003489

Kamps, JJAG, Hinchliffe, P, Glerup, J, Freeman, EI, Lang, PA, Tooke, CL, Beer, M, Parkinson, L, Gu, DH, Park, S, Devenish, N, Zhou, T, Shilova, A, Kaur, S, Rabe, P, Schofield, CJ, Spencer, J, Park, J, Owen, RL, Orville, AM & Aller, P 2026, 'Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography', IUCrJ, vol. 13, pp. 471-484. https://doi.org/10.1107/S2052252526003489

@article{5e86281bf3634c2890b2d73a56c9e7dc,

title = "Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography",

abstract = "We describe the design and implementation of a drop-on-fixed-target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume aqueous droplets (∼40-90 pl; ∼40-55 μm diameter sphere), containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals previously loaded into the trapezoidal wells of an etched crystalline silicon fixed-target chip containing 25 600 wells in a high-density, square grid with 125 μm centre-to-centre well spacing. These features demand exquisite accuracy and thereby constrain motion controls to enable robust time-resolved crystallographic studies. The system was tested with three enzyme systems, comprising lysozyme and two β-lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free-electron laser facilities was evaluated. This drop-on-fixed-target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.",

keywords = "enzyme mechanisms, sample delivery, serial crystallography, structure determination, time-resolved studies",

author = "Kamps, \{Jos J. A. G.\} and Philip Hinchliffe and Johan Glerup and Freeman, \{Emily I.\} and Lang, \{Pauline A.\} and Tooke, \{Catherine L.\} and Michael Beer and Laura Parkinson and Gu, \{Do Heon\} and Sehan Park and Nicholas Devenish and Tiankun Zhou and Anastasya Shilova and Samanpreet Kaur and Patrick Rabe and Schofield, \{Christopher J.\} and James Spencer and Jaehyun Park and Owen, \{Robin L.\} and Orville, \{Allen M.\} and Pierre Aller",

year = "2026",

month = jul,

day = "1",

doi = "10.1107/S2052252526003489",

language = "English",

volume = "13",

pages = "471--484",

journal = "IUCrJ",

issn = "2052-2525",

publisher = "International Union of Crystallography",

}

TY - JOUR

T1 - Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography

AU - Kamps, Jos J. A. G.

AU - Hinchliffe, Philip

AU - Glerup, Johan

AU - Freeman, Emily I.

AU - Lang, Pauline A.

AU - Tooke, Catherine L.

AU - Beer, Michael

AU - Parkinson, Laura

AU - Gu, Do Heon

AU - Park, Sehan

AU - Devenish, Nicholas

AU - Zhou, Tiankun

AU - Shilova, Anastasya

AU - Kaur, Samanpreet

AU - Rabe, Patrick

AU - Schofield, Christopher J.

AU - Spencer, James

AU - Park, Jaehyun

AU - Owen, Robin L.

AU - Orville, Allen M.

AU - Aller, Pierre

PY - 2026/7/1

Y1 - 2026/7/1

N2 - We describe the design and implementation of a drop-on-fixed-target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume aqueous droplets (∼40-90 pl; ∼40-55 μm diameter sphere), containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals previously loaded into the trapezoidal wells of an etched crystalline silicon fixed-target chip containing 25 600 wells in a high-density, square grid with 125 μm centre-to-centre well spacing. These features demand exquisite accuracy and thereby constrain motion controls to enable robust time-resolved crystallographic studies. The system was tested with three enzyme systems, comprising lysozyme and two β-lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free-electron laser facilities was evaluated. This drop-on-fixed-target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.

AB - We describe the design and implementation of a drop-on-fixed-target method for time-resolved serial crystallography at both synchrotron and XFEL facilities. A piezoelectric droplet dispensing pipette is employed for addition of picolitre volume aqueous droplets (∼40-90 pl; ∼40-55 μm diameter sphere), containing (co-)substrate(s) or ligand(s), onto enzyme microcrystals previously loaded into the trapezoidal wells of an etched crystalline silicon fixed-target chip containing 25 600 wells in a high-density, square grid with 125 μm centre-to-centre well spacing. These features demand exquisite accuracy and thereby constrain motion controls to enable robust time-resolved crystallographic studies. The system was tested with three enzyme systems, comprising lysozyme and two β-lactamases, CTX-M-15 and AmpCEC. Mitigation strategies for cross-well contamination, including the implementation of interleaved controls, are described; the overall performance of the system at synchrotron and X-ray free-electron laser facilities was evaluated. This drop-on-fixed-target method is a reliable framework for time-resolved crystallography and will improve the consistency of measurements across facilities.

KW - enzyme mechanisms

KW - sample delivery

KW - serial crystallography

KW - structure determination

KW - time-resolved studies

UR - https://www.scopus.com/pages/publications/105043716266

U2 - 10.1107/S2052252526003489

DO - 10.1107/S2052252526003489

M3 - Article

AN - SCOPUS:105043716266

SN - 2052-2525

VL - 13

SP - 471

EP - 484

JO - IUCrJ

JF - IUCrJ

ER -

Drop-on-fixed-target reaction initiation approach for serial and time-resolved crystallography

Abstract

Acknowledgements

Funding

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this