Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server

Abstract

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.

Data mining of iron(II) and iron(III) bond-valence parameters, and their relevance for macromolecular crystallography

“…Using the updated set of bond-valence parameters derived here, we have recently launched a web-based service to provide metal-binding site validation in macromolecular structures (Zheng et al , 2014). The bond-valence sum method has previously been shown to be applicable to metal-binding sites present in macromolecular crystal structures (Müller et al , 2003).…”

“…During the investigation of metal ion-binding architectures in proteins (Zheng et al , 2008, 2014), we successfully employed the bond-valence model to check the quality of metal-binding site modeling in low-resolution structures. Initially, we used reference literature values for bond-valence ( R 0 ) parameters, which were derived two decades ago from manually curated structures and extrapolated linear relationships between bond-valence contributions (Brese & O’Keeffe, 1991; Brown & Altermatt, 1985).…”

Structure of the Open Reading Frame 49 Protein Encoded by Kaposi's Sarcoma-Associated Herpesvirus

“…Sulfate ions were modeled manually into the positive electron densities using Coot and refined with phenix.refine (39)(40)(41). Further validation with a metal binding site server could not identify any binding of metal ions to the positive electron densities (42).…”

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

“…An initial model was built using Autobuild (24). The final model was manually built using Coot and CheckMyMetal and refined using Phenix.Refine (25)(26)(27) To obtain crystals of the CNB-A⅐cGMP complex, cGMP powder (Sigma-Aldrich) was added to the purified PKG II CNB-A and the mixture was concentrated to reach final concentrations of 55 mg/ml for protein and 33 mM for cGMP using a 10 kDa-cutoff Amicon Ultra (Millipore). Co-crystals of the CNB-A⅐cGMP complex were obtained using the vapor diffusion method in 200 mM sodium malonate at pH 5.0, 5-10% ethylene glycol, and 20% PEG, after 1 day incubation at 22°C.…”