Background

We solved a number of X-ray crystal structures of glutaminase C (GAC) bound to different BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide)-class allosteric inhibitors under cryogenic crystallographic conditions (Huang et al., 2018; Milano et al., 2022). Although they vary in potency for GAC, the structures show that the contacts between the small molecule inhibitors and GAC are largely conserved. In order to visualize differences at the binding interface between GAC and the inhibitors, we employed fixed-target serial room temperature synchrotron crystallography (Wierman et al., 2019; Milano et al., 2022). An advantage of using serial room temperature X-ray crystallography is to help overcome any cryo-induced structural biases, such as “trapping” the protein/ligand in a distinct conformation as a result from crystal freezing. Although this technique requires substantially more crystals than cryogenic crystallography due to radiation damage to the sample, it can yield structures that are more dynamic and fluid. Consequently, structures solved using room temperature X-ray crystallography are capable of revealing differences in proteins and protein/ligand complexes that perhaps are more physiologically relevant. We recently used this technique to solve the structure of apo GAC (Illava et al., 2021). We hypothesized that structures of the GAC/inhibitor complexes at room temperature would make it possible to visualize subtle changes in the binding characteristics of the small molecules that were not evident from the low-temperature cryo-structures. Indeed, we were able for the first time to observe clear structural differences in the binding poses between two allosteric inhibitors of widely different potencies in the GAC/inhibitor complexes solved at room temperature. Here, we describe a detailed protocol for crystal handling, data collection, and data processing when employing serial room temperature crystallography (Figure 1).