Antimicrobial resistance is a major and growing cause of death worldwide. Antivirulence agents are an attractive alternative because they disarm pathogens without the selective pressure that drives resistance to conventional antibiotics. Bacterial disulfide bond protein A (DsbA) folds the virulence factors essential for Gram-negative invasion, so inhibiting DsbA should attenuate virulence without inducing detectable resistance.
Yet twenty years of structure-guided effort on E. coli DsbA have produced inhibitor candidates that stall at the pre-clinical stage, guided almost entirely by crystal structures determined at cryogenic temperature. Cryocooling can remodel sidechain rotamers, reshuffle water networks and occlude alternative conformational states, while warming crystals has elsewhere exposed cryptic pockets and ligand binding that cryogenic data missed [1]. This matters for DsbA: its most promising inhibitor-binding site includes a cryptic pocket that opens only when the protein is dynamic [2].
This talk will present the first serial synchrotron crystallography (SSX) structures determined at the Australian Synchrotron MX3 microfocus beamline [3], using in-tray SSX data collection. The workflow proved reproducible from the first attempt in late 2025, adapting in-tray screening into in-tray SSX collection of complete room-temperature (RT) structures of apo E. coli DsbA and lysozyme. We acquired serial data directly from raster scans of microcrystal showers in 96-well MiTeGen in-situ plates, without batch micro-crystallisation optimisation, bespoke scripting, or human sample handling beyond setting up the trays.
SSX spreads dose across thousands of crystals, limiting global damage at RT, but disulfides are damaged roughly 5-fold faster than the rest of the structure [4], and worse still in arsenic-containing liquors. Comparing our high-resolution DsbA structures from SSX data collected at 3%, 5% and 20% transmission, the catalytic disulfide stayed intact even in sodium cacodylate, while Cu-site damage, seen as loss of density near the site, increased with dose.
We will also highlight hot-off-the-press results on how one DsbA-inhibitor complex differs between cryogenic and RT structures, as we work towards capturing transient states during binding to inform structure-based development. Finally, we present a practical guide to which drops are worth collecting from (crystal size and concentration), how many drops are needed for a complete dataset, and what orientational sampling to expect, as a potential structure-solution route for crystallisation campaigns that stall at microcrystal showers.