Metalloprotein catalysis: structural and mechanistic insights into oxidoreductases from neutron protein crystallography

Metalloproteins catalyze a range of reactions, with enhanced chemical functionality due to their metal cofactor. The reaction mechanisms of metalloproteins have been experimentally characterized by spectroscopy, macromolecular crystallography and cryo-electron microscopy. An important caveat in structural studies of metalloproteins remains the artefacts that can be introduced by radiation damage. Photoreduction, radiolysis and ionization deriving from the electromagnetic beam used to probe the structure complicate structural and mechanistic interpretation. Neutron protein diffraction remains the only structural probe that leaves protein samples devoid of radiation damage, even when data are collected at room temperature. Additionally, neutron protein crystallography provides information on the positions of light atoms such as hydrogen and deuterium, allowing the characterization of protonation states and hydrogen-bonding networks. Neutron protein crystallography has further been used in conjunction with experimental and computational techniques to gain insight into the structures and reaction mechanisms of several transition-state metal oxidoreductases with iron, copper and manganese cofactors. Here, the contribution of neutron protein crystallography towards elucidating the reaction mechanism of metalloproteins is reviewed.

Seven quick tips for beginners in protein crystallography

The aim of this brief review is to provide a roadmap for beginning crystallographers who have little or no experience in structural biology and yet are keen to produce protein crystals and analyze their 3D structures to understand their biological roles. To achieve this goal it is crucial to perform crystallization, structure determination, visualization and analysis of the protein’s structural features related to its biological function. Keeping that objective in mind, tips presented herein cover the most important steps in a crystallographic endeavor and present a selection of databases and software which can aid and accelerate the whole process. We hope that this short overview will help novices coming from different disciplines to navigate a protein crystallography project and, hopefully, allow avoiding some costly mistakes, even though being a crystallographer means learning by trial and error.