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- EMDB-26406: Structure of enolase from Streptococcus Pyogenes -

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Basic information

Entry
Database: EMDB / ID: EMD-26406
TitleStructure of enolase from Streptococcus Pyogenes
Map dataImage of Cryo-Em Map of Octameric streptococcal Enolase
Sample
  • Complex: Octameric structure of Enolase from Streoptococcus Pyogenes
    • Protein or peptide: Streptococcal Surface Enolase
Keywordsmetalloenzyme / lyase / hPg-Receptor
Function / homology
Function and homology information


phosphopyruvate hydratase / phosphopyruvate hydratase complex / phosphopyruvate hydratase activity / peptidoglycan-based cell wall / glycolytic process / magnesium ion binding / cell surface / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsTjia-Fleck S / Readnour B / Castellino FJ
Funding support United States, 1 items
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 11, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateJun 28, 2023-
Current statusJun 28, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26406.png

Downloads & links

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Map

FileDownload / File: emd_26406.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationImage of Cryo-Em Map of Octameric streptococcal Enolase
Voxel sizeX=Y=Z: 1.29052 Å
Density
Contour LevelBy AUTHOR: 0.246
Minimum - Maximum-1.8541595 - 2.5878444
Average (Standard dev.)0.000019469257 (±0.0656735)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 330.372 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26406_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Cryo-Em Map 1 of Octameric streptococcal Enolase

Fileemd_26406_half_map_1.map
AnnotationHalf Cryo-Em Map 1 of Octameric streptococcal Enolase
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Cryo-Em Map 2 of Octameric streptococcal Enolase

Fileemd_26406_half_map_2.map
AnnotationHalf Cryo-Em Map 2 of Octameric streptococcal Enolase
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Octameric structure of Enolase from Streoptococcus Pyogenes

EntireName: Octameric structure of Enolase from Streoptococcus Pyogenes
Components
  • Complex: Octameric structure of Enolase from Streoptococcus Pyogenes
    • Protein or peptide: Streptococcal Surface Enolase

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Supramolecule #1: Octameric structure of Enolase from Streoptococcus Pyogenes

SupramoleculeName: Octameric structure of Enolase from Streoptococcus Pyogenes
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Streptococcus pyogenes (bacteria) / Strain: AP53
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: Streptococcal Surface Enolase

MacromoleculeName: Streptococcal Surface Enolase / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Streptococcus pyogenes (bacteria) / Strain: AP53
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HMSIITDVYA REVLDSRGNP TLEVEVYTES GAFGRGMVPS GASTGEHEAV ELRDGDKSRY LGLGTQKAVD NVNNIIAEAI IGYDVRDQQA IDRAMIALDG TPNKGKLGAN AILGVSIAVA RAAADYLEVP LYTYLGGFNT KVLPTPMMNI INGGSHSDAP IAFQEFMIMP ...String:
HMSIITDVYA REVLDSRGNP TLEVEVYTES GAFGRGMVPS GASTGEHEAV ELRDGDKSRY LGLGTQKAVD NVNNIIAEAI IGYDVRDQQA IDRAMIALDG TPNKGKLGAN AILGVSIAVA RAAADYLEVP LYTYLGGFNT KVLPTPMMNI INGGSHSDAP IAFQEFMIMP VGAPTFKEGL RWGAEVFHAL KKILKERGLV TAVGDEGGFA PKFEGTEDGV ETILKAIEAA GYEAGENGIM IGFDCASSEF YDKERKVYDY TKFEGEGAAV RTSAEQVDYL EELVNKYPII TIEDGMDEND WDGWKVLTER LGKRVQLVGD DFFVTNTEYL ARGIKENAAN SILIKVNQIG TLTETFEAIE MAKEAGYTAV VSHRSGETED STIADIAVAT NAGQIKTGSL SRTDRIAKYN QLLRIEDQLG EVAQYKGIKS FYNLKK

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.4 / Component - Concentration: 0.05 mM / Component - Formula: NaH2PO4 / Component - Name: Sodium phospate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 105000
Specialist opticsPhase plate: VOLTA PHASE PLATE
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number grids imaged: 1 / Number real images: 2756 / Average electron dose: 61.37 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 813556
Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionNumber classes used: 17 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 442256
FSC plot (resolution estimation)

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