Research Overview

Every major invention that enables us to see beyond what our eyes allow - further, deeper, smaller - has led to a profound new understanding of, and ability to affect, our world and our place in it. In microscopy, jumps of orders of magnitude in resolution have given us completely new perspectives on biology through the discovery of microbes, then cells, then protein structures at the atomic scale. Proteins and other biomacromolecules perform structural and functional duties. They are the building blocks, biological catalysts and they control all our senses. Uncovering their structures and dynamic interactions, which govern their function, has given us mechanistic insights into biochemical processes, a profound understanding of the molecular underpinnings of disease and bolstered pharmaceutical drug discovery.

Macromolecular X-ray crystallography (MX) is the workhorse of structural biology, responsible for the vast majority of the 220,000+ experimentally determined protein structures in the Protein Data Bank. Notably, MX accounts for 99% of structures with resolutions better than 3 Å. However, MX requires sufficiently large well-diffracting crystals, most structures are determined at cryo temperatures, and only static structures are obtained.

The next frontier is not a jump in resolution, but an extra dimension: time. XFEL crystallography enables us to see smaller and faster, i.e. to watch fundamental biochemical reactions in real time, at ambient temperature, with atomic resolution. We have obtained structural intermediates from light-triggered reactions with femtosecond resolution, observing photon capture leading to chromophore isomerisation (a step common to photon detection by various organisms, including mammalian vision) and subsequent protein dynamics [Tenboer et al. 2014 Science; Pande et al. 2016 Science], as well as structural changes in Photosystem II [Kupitz et al. 2014 Nature]. For chemically triggered reactions, mix-and-inject serial crystallography (MISC), which we pioneered (in BioXFEL), enables real-time observation of ligand binding and catalysis: we have captured ligand binding to an RNA riboswitch [Stagno et al. 2017 Nature] and an antibiotic-resistance enzyme cleaving an antibiotic in real time [Olmos et al. 2018 BMC Biology].

The Zatsepin lab develops serial crystallography methods - sample delivery, data analysis, software and experiment design - for both XFELs and synchrotrons. We have adapted methods from XFEL serial crystallography for use at microfocus synchrotron beamlines, making many of the advantages of SFX accessible at facilities worldwide. We are currently establishing serial and time-resolved crystallography at the Australian Synchrotron, in collaboration with the MX3 team.

Before joining Swinburne (2024), I led serial femtosecond crystallography data analysis for the NSF BioXFEL Science and Technology Center, a US multi-university consortium that pioneered the development and application of XFELs in structural biology.