<scp>CMM</scp>—An enhanced platform for interactive validation of metal binding sites

Gucwa, Michał; Lenkiewicz, Joanna; Zheng, Heping; Cymborowski, M.; Cooper, David R.; Murzyn, Krzysztof; Minor, Władek

doi:10.1002/pro.4525

Cited by 51 publications

(34 citation statements)

References 37 publications

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“…Starting from the previously reported model [5] of μOR with 1b, Mg 2+ was added to the center of the unexplained density with sphere refinement using Coot [45] in CCP4i2 [46], and uploaded with the original structure factors to PDB-REDO server [47] for automated refinement. The resulting complex was submitted to CheckMyMetal [13] for validation; all suggested alternative metals were also resubmitted for validation.…”

Section: Methodsmentioning

confidence: 99%

“…Additionally, note the highly asymmetrical geometry required, with a bond angle of 105°, compared to 90° for the N atoms; magnesium complexes are symmetrical [10]. Other evidence against Mg 2+ was revealed by CheckMyMetal [13]. The values of five of the eight parameters evaluated were classed as dubious, including three that strongly suggest a misidentified element:…”

Section: His54 3σmentioning

confidence: 99%

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The μ opioid receptor crystal structure with BU72 is a covalent adduct

Munro

2022

Preprint

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Background The first crystal structure of the active μ opioid receptor (μOR) exhibited several unexplained features. The ligand BU72 exhibited many extreme deviations from ideal geometry, along with unexplained electron density around the benzylic carbon. I previously showed that inverting the benzylic configuration resolved these problems, establishing revised stereochemistry of BU72 and its analog BU74. However, another problem remains unresolved: additional unexplained electron density contacts both BU72 and a histidine residue in the N-terminus. Results Here I show that these short contacts and uninterrupted density are inconsistent with non-covalent interactions. Therefore, BU72 and μOR form a covalent adduct through an unmodeled atom, and the published model as two separate entities is incorrect. A subsequently proposed magnesium complex is also inconsistent with multiple lines of evidence. However, oxygen fits the unexplained density well. While the structure I propose is tentative, similar oxygen-bridged adducts have been reported previously in the presence of reactive oxygen species. Moreover, known sources of reactive oxygen species were present: HEPES buffer, nickel ions, and a sequence motif that forms redox-active nickel complexes. This motif contacts the unexplained density. The adduct exhibits severe strain, and the tethered N-terminus forms contacts with adjacent residues. These forces, along with the nanobody used as a G protein substitute, would be expected to influence the receptor conformation. Consistent with this, the intracellular end of the structure differs markedly from subsequent structures of active μOR bound to Gi protein. Conclusions Later Gi-bound structures are likely to be more accurate templates for docking and molecular dynamics simulations of active μOR. The possibility of reactions like this should be considered in the choice of protein truncation sites and purification conditions, and in the interpretation of excess or unexplained density.

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Section: Methodsmentioning

confidence: 99%

Section: His54 3σmentioning

confidence: 99%

The μ opioid receptor crystal structure with BU72 is a covalent adduct

Munro

2022

Preprint

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“…Metal ion selection and placement was made based upon electron density, coordination and was limited to elements found in crystallisation conditions. Metal ion placement was verified by refinement and using the online server CheckMyMetal [51,52]. Crystallographic data are shown in Table S1.…”

Section: Structure Determinationmentioning

confidence: 99%

Unravelling the mechanism of autoinhibition by Pseudomonas aeruginosa aminopeptidase enables the development of a potent antibiofilm cyclic peptide

Harding

Bischoff

Bergkessel

et al. 2023

Preprint

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Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals, including those with cystic fibrosis. P. aeruginosa extensively forms biofilms which significantly contribute to its growth and persistence in a wide range of environments. In addition, its success can be attributed to several secreted proteases. Here, we investigated the aminopeptidase PaAP from P. aeruginosa, which is one of the most abundant extracellular proteins in the biofilm matrix. Increasing evidence suggests this quorum-sensing-regulated peptidase is associated with biofilm development and contributes to nutrient recycling by complementing the activity of other secreted proteases. We confirmed that post translational processing was required for activation, as a C-terminal truncation mutant possessed ~100-fold higher activity than the full-length enzyme. We determined that PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. High-resolution crystal structures of wild type enzyme and mutants revealed the mechanism of autoinhibition, whereby the Cterminal pro-peptide locks the protease-associated (PA) domain and the catalytic peptidase domain, into a closed, inhibited conformation. Inspired by this selfregulatory mechanism and guided by structural data, we designed a highly potent small cyclic-peptide inhibitor (KI in the nM range). This inhibitor recapitulates the deleterious phenotype observed with a PaAP deletion mutant in liquid cultures and biofilm assays and presents a path towards targeting secreted proteins in a biofilm context.

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“…49) There are others, like PIXE. 50) CheckMyMetal (CMM) 51) analyze parameters of the binding site like ligand distances, the geometry of coordinating sphere, valence, and the metal ion-environment B-factor. CMM, especially its new version, became a potent tool for identifying and refining metal ion binding sites.…”

Section: ．Ligands Validationmentioning

confidence: 99%

Continuous Validation Across Macromolecular Structure Determination Process

Bijak

Gucwa

Lenkiewicz

et al. 2023

Journal of the Crystallographic Society of Japan

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The overall quality of the experimentally determined structures contained in the PDB is exceptionally high, mainly due to the continuous improvement of model building and structural validation programs. Improving reproducibility on a large scale requires expanding the concept of validation in structural biology and all other disciplines to include a broader framework that encompasses the entire project. A successful approach to science requires diligent attention to detail and a focus on the future. An earnest commitment to data availability and reuse is essential for scientific progress, be that by human minds or artificial intelligence. 1．Introduction The Protein Data Bank(PDB) 1) was formed over 50 years ago and initially contained only seven macromolecular structures. The PDB founders realized that access to macromolecular models is essential for crystallographers, students, and researchers who might use structural information for their research. Initially, every deposit contained only the coordinates of the atoms. Even then, every new structure was carefully checked to see whether the 3-D model agreed with known chemical and physical properties. Since 2007, every submission to the PDB has had two additional components: 1)information about the sample (crystal in X-ray crystallography) , a rough description of the experimental setup, and other metadata(header) , and 2)intensity amplitudes(usually called structure factors) of all diffraction spots measured during the experiment. The deposit header also contains additional information like authors, connections to other databases, software used to determine the structure, etc. These three components allow for calculating the electron density map and checking whether the macromolecule model, including ligands, nucleic acids, and solvent, agrees with experimental data. This checking procedure is called validation of the macromolecular model and was usually performed by the experimenter at the end of the structure determination process. The PDB also routinely performs validation on all deposited structures. 2) Moreover, scientists who find disagreement between their experiments and PDB deposits can perform validations for themselves. Sometimes their validation triggers a re-refinement and leads to the deposition of a new, improved model to the PDB. 3)Occasionally, new biomedical interpretations arise from improved structural models. 4)

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CMM—An enhanced platform for interactive validation of metal binding sites

Cited by 51 publications

References 37 publications

The μ opioid receptor crystal structure with BU72 is a covalent adduct

The μ opioid receptor crystal structure with BU72 is a covalent adduct

Unravelling the mechanism of autoinhibition by Pseudomonas aeruginosa aminopeptidase enables the development of a potent antibiofilm cyclic peptide

Continuous Validation Across Macromolecular Structure Determination Process

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