Modern methods in Structural Biology aim to provide detailed insights into how proteins are able to catalyse and control chemical reactions. In addition, the conformational dynamics of proteins are being intensively studied with regards to their role in the regulation of enzyme catalysis, for example by allosteric phenomena. In recent years, time-resolved (TR) X-ray crystallography and structural enzymology have been revived by the advent of serial crystallography at advanced X-ray sources and by new methods for sample delivery, reaction initiation and data processing, making TR studies increasingly accessible. However, electron density maps resulting from TR studies typically correspond to mixtures of states, thus their interpretation thus presents a serious challenge. We are addressing this challenge by supplementing TR structural data with ultra-high-resolution data from macrocrystals of highly homogenous mechanistically trapped states. Routinely achieving ultra-high resolution requires optimised sample quality, top-hat beams of adjustable size and shape, data collection with high-energy X-rays and CdTe detectors, using optimal data strategies designed and executed through direct control by a Workflow. In this talk, I will present
some of our recent work, highlighting what is now possible for Structural Enzymology using X-ray crystallography, yet currently inaccessible by cryo-EM and Artificial Intelligence methods. I will also highlight some of the challenges faced in the pursuit of accurate models to explain ultra-high resolution diffraction data.