Speaker
Description
There is increasing interest in obtaining room temperature, time resolved crystal structures of
proteins carrying out their biological functions. The transition between conventional cryogenic
macromolecular crystallography and serial crystallography involving microcrystals remains chal-
lenging for many projects. We have recently demonstrated the capability to measure good quality,
low dose serial crystallography data from microcrystals within crystallisation plates [1]. This capa-
bility is available in the standard operation mode of the beamline and does not require any specific
serial crystallography apparatus. In this approach, microcrystals are transferred into a crystallisa-
tion plate (typically 100 nL per drop) and each droplet is subjected to raster scanning with a still
diffraction image measured every 10 µm. The resulting images are processed using standard serial
data processing software such as xia2.multiplex. This approach enables straightforward structure
determination and analysis of crystal quality and unit cell parameters from non-optimised crys-
tallisation conditions, guiding users in their optimisation efforts. Very small quantities of protein
are required, and the determination of a human peroxidase structure to 1.88 Angstrom resolution
using only 1.2 µL microcrystal suspension.
Several approaches to sample delivery for time resolved crystallography have been developed in-
cluding droplet-on-demand tape drive-based systems developed for XFEL experiments that have
been combined with X-ray emission spectroscopy (XES) to monitor the redox and spin state of
metal -containing cofactors within the proteins [2]. However, currently available systems require
a large quantity of microcrystal sample as well as requiring multiple skilled staff to operate. A
new system for serial crystallography at VMXi is currently under development. This incorporates
a picolitre droplet-on-demand tape drive system capable of anaerobic operation together with an
XES von Hamos spectrometer to enable spectroscopic validation in time resolved experiments of
metalloproteins. A compact design was required due to the tight spatial constraints of the VMXi
end station that was built for highly automated data collection from crystallisation plates, and the
design incorporates automation to reduce the number of personnel required to more closely ap-
proach a typical synchrotron experiment.
Proof of concept data obtained during the development process of the tape drive and XES spec-
trometer will be presented, including a high-resolution protein structure determined using the tape
drive system and XES data obtained from microcrystals of the copper enzyme nitrite reductase.
[1] A.J. Thompson, J. Sanchez-Weatherby, L.J. Williams, H. Mikolajek, J. Sandy, J.A.R. Worrall and
M.A. Hough (2024) Efficient in situ screening of and data collection from microcrystals in crystal-
lization plates Acta Cryst.D80, 279-288
[2] Butyrin, A. et al (2021) An on-demand, drop-on-drop method for studying enzyme catalysis by
serial crystallography. Nature Methods 12, 4461.
Authors:
Juan Sanchez Weatherby, Pierre Aller, Amy Thompson, Abby Telfer, John Sutter, James Sandy, Halina Mikolajek, Matthew Rodrigues, Mike Hough, Allen Orville
