Tracking enzymes in action – new 5D crystallography method captures protein dynamics at physiological temperatures

An interdisciplinary research team from Hamburg has developed a novel method that allows for time-resolved structural studies of proteins across a wide temperature range, including physiologically relevant conditions. The new approach, called ‘5D serial synchrotron crystallography (5D-SSX)’, enables the collection of temperature-resolved structural snapshots during enzymatic reactions and provides insights into protein function under near-native conditions. Their work has now been published in Nature Communications.

Temperature plays a key role in modulating enzyme activity and protein dynamics. Yet, the majority of high-resolution protein structures are still obtained at cryogenic temperatures, far from physiological conditions. Time-resolved crystallography, which can reveal the structural basis of catalysis, allostery, and ligand binding, is typically conducted at ambient temperatures, which can miss conformational states that become visible at physiological temperatures.

The team—comprising scientists from the University of Hamburg (UHH), the University Medical Center Hamburg-Eppendorf (UKE), the Max Planck Institute for the Structure and Dynamics of Matter (MPSD), and the European Molecular Biology Laboratory (EMBL) Hamburg—has now established a workflow to overcome these limitations. Their 5D-SSX method allows researchers to perform serial synchrotron crystallography at defined time points and at temperatures ranging from below 10 °C to above 70 °C.

In proof-of-concept experiments, the team demonstrated temperature-dependent enzymatic turnover in both the mesophilic β-lactamase CTX-M-14 and the thermophilic enzyme xylose isomerase. The method reveals how reaction kinetics and conformational landscapes are modulated by temperature and allows researchers to investigate enzyme mechanisms under biologically relevant conditions.

"The most challenging aspect of this project was managing the large volume of water vapor condensation without allowing it to enter the sensitive synchrotron end station located directly beneath it," says Friedjof Tellkamp, head of the Scientific Support Unit Machine Physics at the MPSD, who led the development of the environmental chamber.

Dr. Eike C. Schulz, heading a BMFTR & ERC funded research group at the UKE, adds: “5D-SSX opens up completely new perspectives for biomedical research. Understanding the dynamics of proteins under realistic conditions is crucial, especially when developing antibiotics or elucidating disease-relevant mechanisms.”

The study demonstrates that integrating time and temperature into crystallographic workflows provides a more complete and physiologically meaningful view of protein function—one snapshot at a time.

_{This text is based on the UKE's press release (German).}