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 (over 90%) of the 184,000+ experimentally determined protein/macromolecular structures to date (see Protein Data Bank). Notably, of all experimental methods, MX is responsible for 99 % of structures with resolutions better than 3 Å (and even higher fractions in higher resolution bins). However, MX requires sufficiently large well-diffracting crystals, most structures are determined at cryo temperatures, and only static structures are determined, representing an average over the crystal. 

The next frontier in structural biology is not a jump in resolution, but an extra dimension: time. The invention of XFEL crystallography was revolutionary in that it enables us to see smaller and faster: 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 (e.g. structural changes responsible for energy dissipation from photon absorption) [Tenboer et al. 2014 Science, Pande et al. 2016 Science], as well as structural changes at multiple time points in Photosystem II, a large membrane protein complex responsible for producing oxygen during photosynthesis [Kupitz et al. 2014 Nature]. For reactions initiated by chemical mixing and small molecule binding, mix-and-inject serial crystallography (MISC) is the leading method for real time binding studies. In our pioneering MISC experiments, we have observed ligand binding to an adenine RNA riboswitch, which is involved in regulation of gene expression [Stagno et al. 2017 Nature] and the catalytic reaction of an enzyme (β-lactamase) that is reponsible for resistance to common antibacterials in numerous bacteria (Olmos et al. BMC Biology 2018). 

In the Zatsepin lab, we are developing serial micro-crystallography methods to structurally and dynamically probe proteins and other macromolecules with X-rays. Serial snapshot micro-crystallography differs from conventional goniometer (rotation-based) crystallography in that only a single snapshot diffraction pattern is collected from each randomly oriented micro-crystal and the data are assembled from thousands of individual nano/microcrystals. By using extremely bright and brief X-ray pulses (such as those generated by an X-ray free-electron laser, or XFEL) allows us to collect diffraction data before inevitable radiation damage manifests as structural changes in the crystals, despite the tiny size and room temperature data collection. This enables us to carry out time-resolved experiments studying reversible and irreversible reactions, as the crystals are exposed only once. The snapshot diffraction data are then indexed and merged (e.g. using CrystFEL) to yield a crystallographic reflection list usable for phasing with conventional software. 

We have adapted sample delivery and data analysis methods developed for serial crystallography with XFELs (serial femtosecond crystallography: SFX) for use at modern microfocus beamlines, conferring many of the advantages of SFX to synchrotron beamlines, which are much more readily accessible. 

The Zatsepin lab specialises in serial crystallography data analysis and experimental design. We are in the process of obtaining a high-viscosity microfluidic injection system for injector-based synchrotron serial crystallography. This injector, invented in the Spence and Weierstall lab at Arizona State University, has enabled room temperature structure determination from membrane proteins (see the publication list), in particular G protein-coupled receptor microcrystals delivered in their favoured crystal growth medium (lipidic cubic phase). We are also working in collaboration with the MX3 team at the Australian Synchrotron to make serial micro-crystallography and time-resolved studies feasible and accessible to the broader structural biology community in Australia. 

We welcome new collaborations! If you are interested in any aspects of serial crystallography, with XFELs or synchrotrons, for static or time-resolved studies, or just have questions about the technique and whether it may be right for your sample, please reach out: n.zatsepin at latrobe.edu.au

Before joining La Trobe University in 2019, the Zatsepin lab led the serial femtosecond crystallography data analysis for the US NSF BioXFEL Science and Technology Center (from 2013 to 2019). BioXFEL is a multi-university consortium, leading the development and application of XFELs in structural biology. Visit the BioXFEL website to learn more.