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- PDB-7ugu: Structure of enolase from streptococcus pyogenes -

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Basic information

Entry
Database: PDB / ID: 7ugu
TitleStructure of enolase from streptococcus pyogenes
ComponentsEnolase
KeywordsLYASE / metalloenzyme / hPg-receptor
Function / homology
Function and homology information


phosphopyruvate hydratase / phosphopyruvate hydratase complex / phosphopyruvate hydratase activity / peptidoglycan-based cell wall / glycolytic process / cell surface / magnesium ion binding / extracellular region
Similarity search - Function
Enolase / Enolase, conserved site / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal domain / Enolase signature. / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal domain / Enolase-like, N-terminal / Enolase-like, C-terminal domain superfamily
Similarity search - Domain/homology
Biological speciesStreptococcus pyogenes (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsTjia-Fleck, S. / Readnour, B. / Castellino, F.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)HL013423 United States
CitationJournal: Biochemistry / Year: 2023
Title: High-Resolution Single-Particle Cryo-EM Hydrated Structure of Enolase Offers Insights into Its Function as a Plasminogen Receptor.
Authors: Sheiny Tjia-Fleck / Bradley M Readnour / Yetunde A Ayinuola / Francis J Castellino /
Abstract: Cellular plasminogen (Pg) receptors (PgRs) are utilized to recruit Pg; stimulate its activation to the serine protease, plasmin (Pm); and sterically protect the surface Pm from inactivation by host ...Cellular plasminogen (Pg) receptors (PgRs) are utilized to recruit Pg; stimulate its activation to the serine protease, plasmin (Pm); and sterically protect the surface Pm from inactivation by host inhibitors. One such PgR is the moonlighting enzyme, enolase, some of which leaves the cytoplasm and resides at the cell surface to potentially function as a PgR. Since microbes employ conscription of host Pg by PgRs as one virulence mechanism, we explored the structural basis of the ability of enolase (Sen) to function in this manner. Employing single-particle cryo-electron microscopy (cryo-EM), recombinant Sen from was modeled at 2.6 Å as a stable symmetrical doughnut-shaped homooctamer with point group 422 (D4) symmetry, with a monomeric subunit molecular weight of ∼49 kDa. Binding sites for hPg were reported in other studies to include an internal K and the COOH-terminal K residues of Sen. However, in native Sen, the latter are buried within the minor interfaces of the octamer and do not function as a Pg-binding epitope. Whereas Sen and hPg do not interact in solution, when Sen is bound to a surface, hPg interacts with Sen independently of K. PgRs devoid of COOH-terminal lysine utilize lysine isosteres comprising a basic residue, "", and an anionic residue at " + 3" around one turn of an α-helix. We highlight a number of surface-exposed potential hPg-binding lysine isosteres and further conclude that while the octameric structure of Sen is critical for hPg binding, disruption of this octamer without dissociation exposes hPg-binding epitopes.
History
DepositionMar 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 28, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: Enolase
H: Enolase
E: Enolase
F: Enolase
C: Enolase
D: Enolase
A: Enolase
B: Enolase


Theoretical massNumber of molelcules
Total (without water)380,3468
Polymers380,3468
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Enolase / 2-phospho-D-glycerate hydro-lyase / 2-phosphoglycerate dehydratase


Mass: 47543.281 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pyogenes (bacteria)
Gene: eno, E0F66_01300, E0F67_02490, FGO82_08975, SAMEA1711581_01820, SAMEA864267_00487
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A3F8V6, phosphopyruvate hydratase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Octameric Structure of Enolase from Streptococcus Pyogenes
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.40 MDa / Experimental value: YES
Source (natural)Organism: Streptococcus pyogenes (bacteria) / Strain: AP53
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
Buffer componentConc.: 0.050 mM / Name: Sodium Phosphate / Formula: NaH2PO4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3200 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 61.37 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2756
EM imaging opticsPhase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameVersionCategory
2cryoSPARC3.3.1image acquisition
4cryoSPARC3.3.1CTF correction
10cryoSPARC3.3.1initial Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 813556
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 442256 / Num. of class averages: 17 / Symmetry type: POINT

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