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- EMDB-30989: Structure of PEP bound Enolase from Mycobacterium tuberculosis -

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Basic information

Entry
Database: EMDB / ID: EMD-30989
TitleStructure of PEP bound Enolase from Mycobacterium tuberculosis
Map dataThis is the final combined B factor sharpened map
Sample
  • Organelle or cellular component: Octameric Enolase enzyme
    • Protein or peptide: Enolase
  • Ligand: PHOSPHOENOLPYRUVATE
  • Ligand: MAGNESIUM ION
KeywordsMETAL BINDING PROTEIN
Function / homology
Function and homology information


phosphopyruvate hydratase / phosphopyruvate hydratase complex / phosphopyruvate hydratase activity / 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 speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsBose S / Vinothkumar KR
Funding support India, 2 items
OrganizationGrant numberCountry
Science and Engineering Research Board (SERB) India
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
CitationJournal: IUCrJ / Year: 2023
Title: Structural snapshots of Mycobacterium tuberculosis enolase reveal dual mode of 2PG binding and its implication in enzyme catalysis.
Authors: Mohammed Ahmad / Bhavya Jha / Sucharita Bose / Satish Tiwari / Abhisek Dwivedy / Deepshikha Kar / Ravikant Pal / Richard Mariadasse / Tanya Parish / Jeyaraman Jeyakanthan / Kutti R ...Authors: Mohammed Ahmad / Bhavya Jha / Sucharita Bose / Satish Tiwari / Abhisek Dwivedy / Deepshikha Kar / Ravikant Pal / Richard Mariadasse / Tanya Parish / Jeyaraman Jeyakanthan / Kutti R Vinothkumar / Bichitra Kumar Biswal /
Abstract: Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The ...Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of Mg is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.
History
DepositionFeb 16, 2021-
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateNov 8, 2023-
Current statusNov 8, 2023Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0415
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0415
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7e51
  • Surface level: 0.0415
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7e51
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30989.png

Downloads & links

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Map

FileDownload / File: emd_30989.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the final combined B factor sharpened map
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0415 / Movie #1: 0.0415
Minimum - Maximum-0.105409294 - 0.23666893
Average (Standard dev.)-0.000008118909 (±0.006405862)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 342.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z342.400342.400342.400
α/β/γ90.00090.00090.000
start NX/NY/NZ535455
NX/NY/NZ134138134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1050.237-0.000

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Supplemental data

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Additional map: This is one of the unsharpened half maps

Fileemd_30989_additional_1.map
AnnotationThis is one of the unsharpened half maps
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: This is the other unsharpened half map

Fileemd_30989_additional_2.map
AnnotationThis is the other unsharpened 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 Enolase enzyme

EntireName: Octameric Enolase enzyme
Components
  • Organelle or cellular component: Octameric Enolase enzyme
    • Protein or peptide: Enolase
  • Ligand: PHOSPHOENOLPYRUVATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Octameric Enolase enzyme

SupramoleculeName: Octameric Enolase enzyme / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 370 KDa

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Macromolecule #1: Enolase

MacromoleculeName: Enolase / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: phosphopyruvate hydratase
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 45.962355 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis (bacteria)
SequenceString: MHHHHHHMPI IEQVRAREIL DSRGNPTVEV EVALIDGTFA RAAVPSGAST GEHEAVELRD GGDRYGGKGV QKAVQAVLDE IGPAVIGLN ADDQRLVDQA LVDLDGTPDK SRLGGNAILG VSLAVAKAAA DSAELPLFRY VGGPNAHILP VPMMNILNGG A HADTAVDI ...String:
MHHHHHHMPI IEQVRAREIL DSRGNPTVEV EVALIDGTFA RAAVPSGAST GEHEAVELRD GGDRYGGKGV QKAVQAVLDE IGPAVIGLN ADDQRLVDQA LVDLDGTPDK SRLGGNAILG VSLAVAKAAA DSAELPLFRY VGGPNAHILP VPMMNILNGG A HADTAVDI QEFMVAPIGA PSFVEALRWG AEVYHALKSV LKKEGLSTGL GDEGGFAPDV AGTTAALDLI SRAIESAGLR PG ADVALAL DAAATEFFTD GTGYVFEGTT RTADQMTEFY AGLLGAYPLV SIEDPLSEDD WDGWAALTAS IGDRVQIVGD DIF VTNPER LEEGIERGVA NALLVKVNQI GTLTETLDAV TLAHHGGYRT MISHRSGETE DTMIADLAVA IGSGQIKTGA PARS ERVAK YNQLLRIEEA LGDAARYAGD LAFPRFACET K

UniProtKB: Enolase

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Macromolecule #2: PHOSPHOENOLPYRUVATE

MacromoleculeName: PHOSPHOENOLPYRUVATE / type: ligand / ID: 2 / Number of copies: 8 / Formula: PEP
Molecular weightTheoretical: 168.042 Da
Chemical component information

ChemComp-PEP:
PHOSPHOENOLPYRUVATE / Phosphoenolpyruvic acid

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 16 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration3.5 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMTris
150.0 mMNaClSodium chloridesodium chloride
25.0 mMImidazoleImidazole
1.0 mMMgSO4magnesium sulphate
GridModel: Quantifoil / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 130841 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.813 µm / Nominal defocus min: 0.931 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 27.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 147184
Startup modelType of model: OTHER
Details: Initial model was generated using SGD built in Relion
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 5 / Resolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 61261
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7cll


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 59.41
Output model

PDB-7e51:
Structure of PEP bound Enolase from Mycobacterium tuberculosis

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