Serial Room Temperature Crystal Loading, Data Collection, and Data Processing of Human Glutaminase C in Complex with Allosteric Inhibitors.

Abstract

Cancer cells often overexpress glutaminase enzymes, in particular glutaminase C (GAC). GAC resides in the mitochondria and catalyzes the hydrolysis of glutamine to glutamate. High levels of GAC have been observed in aggressive cancers and the inhibition of its enzymatic activity has been shown to reduce their growth and survival. Numerous GAC inhibitors have been reported, the most heavily investigated being a class of compounds derived from the small molecule BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide). X-ray structure determination under cryo-cooled conditions showed that the binding contacts for the different inhibitors were largely conserved despite their varying potencies. However, using the emerging technique serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors. Here, we describe a step-by-step protocol for crystal handling, data collection, and data processing of GAC in complex with allosteric inhibitors using serial room temperature crystallography. Graphical abstract: Figure 1. Workflow for serial room temperature crystallography. Diagram showing the processing and scaling routine for crystals analyzed using serial room temperature crystallography.