Analysis of metal-protein interaction distances, coordination numbers, B-factors (displacement parameters), and occupancies of metal binding sites in protein structures determined by X-ray crystallography and deposited in the PDB shows many unusual values and unexpected correlations. By measuring the frequency of each amino acid in metal ion binding sites, the positive or negative preferences of each residue for each type of cation were identified. Our approach may be used for fast identification of metal-binding structural motifs that cannot be identified on the basis of sequence similarity alone. The analysis compares data derived separately from high and medium resolution structures from the PDB with those from very high resolution small-molecule structures in the Cambridge Structural Database (CSD). For high resolution protein structures, the distribution of metal-protein or metal-water interaction distances agrees quite well with data from CSD, but the distribution is unrealistically wide for medium (2.0 -2.5 Å) resolution data. Our analysis of cation B-factors versus average B-factors of atoms in the cation environment reveals substantial numbers of structures contain either an incorrect metal ion assignment or an unusual coordination pattern. Correlation between data resolution and completeness of the metal coordination spheres is also found.

Metals play vital roles in both the mechanism and architecture of biological macromolecules. Yet structures of metal-containing macromolecules where metals are misidentified and/or suboptimally modeled are abundant in the Protein Data Bank (PDB). This shows the need for a diagnostic tool to identify and correct such modeling problems with metal binding environments. The "CheckMyMetal" (CMM) web server (http://csgid.org/csgid/metal_sites/) is a sophisticated, user-friendly web-based method to evaluate metal binding sites in macromolecular structures in respect to 7350 metal binding sites observed in a benchmark dataset of 2304 high resolution crystal structures. The protocol outlines how the CMM server can be used to detect geometric and other irregularities in the structures of metal binding sites and alert researchers to potential errors in metal assignment. The protocol also gives practical guidelines for correcting problematic sites by modifying the metal binding environment and/or redefining metal identity in the PDB file. Several examples where this has led to meaningful results are described in the anticipated results section. CMM was designed for a broad audience—biomedical researchers studying metal-containing proteins and nucleic acids—but is equally well suited for structural biologists to validate new structures during modeling or refinement. The CMM server takes the coordinates of a metal-containing macromolecule structure in the PDB format as input and responds within a few seconds for a typical protein structure modeled with a few hundred amino acids.

Metals are essential in many biological processes, and metal ions are modeled in roughly 40% of the macromolecular structures in the Protein Data Bank (PDB). However, a significant fraction of these structures contain poorly modeled metal-binding sites. CheckMyMetal (CMM) is an easy-to-use metal-binding site validation server for macromolecules that is freely available at http://csgid.org/ csgid/metal_sites. The CMM server can detect incorrect metal assignments as well as geometrical and other irregularities in the metal-binding sites. Guidelines for metal-site modeling and validation in macromolecules are illustrated by several practical examples grouped by the type of metal. These examples show CMM users (and crystallographers in general) problems they may encounter during the modeling of a specific metal ion.