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- EMDB-27407: Group A streptococcus Enolase K252A, K255A, K434A, K435A mutant -

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Basic information

Entry
Database: EMDB / ID: EMD-27407
TitleGroup A streptococcus Enolase K252A, K255A, K434A, K435A mutant
Map dataGAS enolase with K252A, K255A, K434A, and K435 mutations made full map
Sample
  • Complex: Octameric Structure of Enolase from Streptococcus Pyogenes
    • Protein or peptide: Enolase
Keywordsmetalloenzyme / hPg-receptor / LYASE
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) / Streptococcus sp. 'group A' (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.12 Å
AuthorsTjia-Fleck SC / Readnour BM / 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
DepositionJun 22, 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_27407.png

Downloads & links

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Map

FileDownload / File: emd_27407.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGAS enolase with K252A, K255A, K434A, and K435 mutations made full map
Voxel sizeX=Y=Z: 1.29 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.7757171 - 1.2611233
Average (Standard dev.)0.00090966315 (±0.042166192)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 330.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27407_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: GAS enolase with K252A, K255A, K434A, and K435...

Fileemd_27407_half_map_1.map
AnnotationGAS enolase with K252A, K255A, K434A, and K435 mutations made first half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: GAS enolase with K252A, K255A, K434A, and K435...

Fileemd_27407_half_map_2.map
AnnotationGAS enolase with K252A, K255A, K434A, and K435 mutations made second half map
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 Streptococcus Pyogenes

EntireName: Octameric Structure of Enolase from Streptococcus Pyogenes
Components
  • Complex: Octameric Structure of Enolase from Streptococcus Pyogenes
    • Protein or peptide: Enolase

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Supramolecule #1: Octameric Structure of Enolase from Streptococcus Pyogenes

SupramoleculeName: Octameric Structure of Enolase from Streptococcus 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: Enolase

MacromoleculeName: Enolase / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
Source (natural)Organism: Streptococcus sp. 'group A' (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 YDAERAVYDY TKFEGEGAAV RTSAEQVDYL EELVNKYPII TIEDGMDEND WDGWKVLTER LGKRVQLVGD DFFVTNTEYL ARGIKENAAN SILIKVNQIG TLTETFEAIE MAKEAGYTAV VSHRSGETED STIADIAVAT NAGQIKTGSL SRTDRIAKYN QLLRIEDQLG EVAQYKGIKS FYNLAA

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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

BufferpH: 7.4 / Component - Concentration: 0.05 mM / Component - Formula: NaH2PO4 / Component - Name: Sodium Phosphate
GridModel: Quantifoil R1.2/1.3 / 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

MicroscopeTFS KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Number grids imaged: 1 / Number real images: 2756 / Average electron dose: 61.37 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.1 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2000000
Startup modelType of model: EMDB MAP
EMDB ID:

26406

Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.12 Å / Resolution method: FSC 0.33 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 813556
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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