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

Nat Commun. 2021 Jul 22;12(1):4461. doi: 10.1038/s41467-021-24757-7.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Avian Proteins / chemistry
  • Avian Proteins / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism
  • Biocatalysis
  • Catalytic Domain
  • Chickens
  • Crystallography, X-Ray / instrumentation
  • Crystallography, X-Ray / methods*
  • Enzymes / chemistry*
  • Enzymes / metabolism*
  • Equipment Design
  • Models, Molecular
  • Muramidase / chemistry
  • Muramidase / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • beta-Lactamases / chemistry
  • beta-Lactamases / metabolism


  • Avian Proteins
  • Bacterial Proteins
  • Enzymes
  • Recombinant Proteins
  • hen egg lysozyme
  • Muramidase
  • beta-lactamase CTX-M-15
  • beta-Lactamases