Using Cryo-Em To Untangle The Conformational Landscape Of A Small Allosterically-Regulated Complex

Use of direct-electron-detectors is driving an unprecedented increase in the resolution of protein complex structures solved by cryo-EM. Computer driven image acquisition combined with streamlined image processing pipelines offer the opportunity to determine structures relatively automated way. Here, we combine these developments to characterize the binding of small ligands on glutamate dehydrogenase (GDH), a clinically significant 336 kDa homohexameric enzyme that is a relevant pharmaceutical target for cancer, Parkinsonu0027s, and diabetes. Images from single specimens collected in a single session provided enough information to localize nucleotides in a complex at ∼3.2 A resolution. We first confirmed the validity of the approach by determining the structure of GDH complexes for which comparable crystallographic coordinates are available. We then found that binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture that can be resolved into two ∼3.3 A structures for which the enzyme is in either an “open” or “closed” state. The work-flow used in this work provides a streamlined path to rapidly solve the structure of macromolecular complexes, to image the binding target of drug molecules and to resolve conformers at near atomic resolution.