Format

Send to

Choose Destination
Nat Protoc. 2018 May;13(5):1062-1090. doi: 10.1038/nprot.2018.018. Epub 2018 Apr 19.

Characterizing metal-binding sites in proteins with X-ray crystallography.

Author information

1
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA.
2
Center for Structural Genomics of Infectious Diseases (CSGID), University of Virginia, Charlottesville, Virginia, USA.
3
Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Krakow, Poland.
4
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA.

Abstract

Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7-190 d, most of which (2-180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center