Recognizing and validating ligands with CheckMyBlob

Brzeziński, Dariusz; Porebski, P.J.; Kowiel, Marcin; Macnar, Joanna M.; Minor, Władek

doi:10.1093/nar/gkab296

Cited by 10 publications

(7 citation statements)

References 31 publications

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“…The remaining missing segments of the structure of the complex were then progressively added and improved using Coot, phenix.refine, and ISoLDE ( 62 ). Cations were identified using the CheckMyBlob server ( 63 ) and inserted with solvent molecules using Coot and refined using Phenix. The final structural model consisted of 493 and 145 residues of eEF-2K TR (92.8%) and CaM (97.9%), respectively, together with 1 Zn 2+ , 2 Ca 2+ , and 5 Mg 2+ ions and 145 water molecules.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

et al. 2022

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Translation is a tightly regulated process that ensures optimal protein quality and enables adaptation to energy/nutrient availability. The α-kinase eukaryotic elongation factor 2 kinase (eEF-2K), a key regulator of translation, specifically phosphorylates the guanosine triphosphatase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and autophosphorylation at the primary stimulatory site, T348. Biochemical studies predict a calmodulin-mediated activation mechanism for eEF-2K distinct from other calmodulin-dependent kinases. Here, we resolve the atomic details of this mechanism through a 2.3-Å crystal structure of the heterodimeric complex of calmodulin and the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights an intimate association of the kinase with the calmodulin C-lobe, creating an “activation spine” that connects its amino-terminal calmodulin-targeting motif to its active site through a conserved regulatory element.

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Section: Methodsmentioning

confidence: 99%

Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

et al. 2022

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“…The authors of http://firstglance.jmol.org/fg.htm?mol=3O4P confirmed that they used http://firstglance.jmol.org/fg.htm?mol=1PJX as an input for their hydrogen refinements. Closer inspection and validation with CheckMyBlob 16 suggested that it is possible that places where MES was placed could be occupied by a different ligand or water. Our refinement did not give unequivocal results which can suggest that part of the crystal contains MES and partly something else.…”

Section: Resultsmentioning

confidence: 99%

“… 32 Finally, a careful check of the final structures was done using Molprobity. 33 If the resulting ligand conformations and corresponding density looked suspicious, we used the CheckMyBlob web‐server 16 , 34 to check the possibility of HEPES or MES being substituted by other molecules.…”

Section: Methodsmentioning

confidence: 99%

“…Unfortunately, this information is not always available, even for the author of the deposit, as crystallization is frequently performed long before diffraction experiments and often by a different researcher. For example, it was shown in several papers 15 , 16 that authors associated a low‐resolution electron density blob with a functional ligand that was used during soaking instead of a HEPES molecule that was present in crystallization buffer.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of protein structures containing HEPES and MES molecules

et al. 2022

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X‐ray crystallography is the main experimental method behind ligand–macromolecule complexes found in the Protein Data Bank (PDB). Applying bioinformatics methods to such structural data can fuel drug discovery, albeit under the condition that the information is correct. Regrettably, a small number of structures in the PDB are of suboptimal quality due to incorrectly identified and modeled ligands in protein–ligand complexes. In this paper, we combine a theoretical‐graph approach, nuclear density estimates, bioinformatics methods, and prior chemical knowledge to analyze two non‐physiological ligands, HEPES and MES, that are frequent components of crystallization and purifications buffers. Our analysis includes quantum mechanics calculations and Cambridge Structure Database (CSD) queries to define the ideal conformation of these ligands, geometry analysis of PDB deposits regarding several quality factors, and a search for homologous structures to identify other small molecules that could bind in place of the parasitic ligand. Our results highlight the need for careful refinement of macromolecule–ligand complexes and better validation tools that integrate results from all relevant resources.

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“…41) -44) The validation of ligands is still challenging, and many X-ray deposits contain misidentified and/or poorly refined ligands. 45) ,46) The use of an artificial intelligence(AI) approach for ligand identifications recently became successful 47) but not as successful as we would like to see.…”

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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Recognizing and validating ligands with CheckMyBlob

Cited by 10 publications

References 31 publications

Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

Structural basis for the calmodulin-mediated activation of eukaryotic elongation factor 2 kinase

Analysis of protein structures containing HEPES and MES molecules

Continuous Validation Across Macromolecular Structure Determination Process

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