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- EMDB-11600: Cubic core of the dihydrolipoamide acyltransferase (E2b) componen... -

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Basic information

Entry
Database: EMDB / ID: EMD-11600
TitleCubic core of the dihydrolipoamide acyltransferase (E2b) component of the branched-chain alpha-ketoacid dehydrogenase complex (BCKDH) from M. tuberculosis
Map dataCryoEM sharpened map obtained from Crysoparc
Sample
  • Complex: Catalytic domain of M. tuberculosis E2b (acyltransferase from branched-chain alpha-ketoacid dehydrogenase complex)
    • Protein or peptide: Dihydrolipoyllysine-residue acyltransferase
Function / homology
Function and homology information


dihydrolipoyllysine-residue (2-methylpropanoyl)transferase / dihydrolipoyllysine-residue (2-methylpropanoyl)transferase activity / lipoic acid binding / acetyltransferase activity / peptidoglycan-based cell wall / plasma membrane / cytoplasm
Similarity search - Function
Peripheral subunit-binding domain / e3 binding domain / Peripheral subunit-binding (PSBD) domain profile. / E3-binding domain superfamily / 2-oxoacid dehydrogenase acyltransferase, catalytic domain / 2-oxoacid dehydrogenases acyltransferase (catalytic domain) / Biotin-requiring enzyme / Biotinyl/lipoyl domain profile. / Biotin/lipoyl attachment / Single hybrid motif / Chloramphenicol acetyltransferase-like domain superfamily
Similarity search - Domain/homology
Dihydrolipoyllysine-residue acyltransferase component of branched-chain alpha-ketoacid dehydrogenase complex
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.92 Å
AuthorsBruch EM / Vilela P / Bellinzoni M
Funding support France, 1 items
OrganizationGrant numberCountry
French National Research AgencyANR-18-CE92-0003 France
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Actinobacteria challenge the paradigm: A unique protein architecture for a well-known, central metabolic complex.
Authors: Eduardo M Bruch / Pierre Vilela / Lu Yang / Alexandra Boyko / Norik Lexa-Sapart / Bertrand Raynal / Pedro M Alzari / Marco Bellinzoni /
Abstract: α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, ...α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high-molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.
History
DepositionAug 6, 2020-
Header (metadata) releaseAug 18, 2021-
Map releaseAug 18, 2021-
UpdateDec 15, 2021-
Current statusDec 15, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.75
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11600.png

Downloads & links

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Map

FileDownload / File: emd_11600.map.gz / Format: CCP4 / Size: 236.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM sharpened map obtained from Crysoparc
Voxel sizeX=Y=Z: 0.97 Å
Density
Contour LevelBy AUTHOR: 0.75 / Movie #1: 0.75
Minimum - Maximum-1.730791 - 3.323458
Average (Standard dev.)-0.0013109574 (±0.107398376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions396396396
Spacing396396396
CellA=B=C: 384.12003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.970.970.97
M x/y/z396396396
origin x/y/z0.0000.0000.000
length x/y/z384.120384.120384.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS396396396
D min/max/mean-1.7313.323-0.001

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Supplemental data

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Additional map: Map after density modification using ResolveEM in Phenix suite

Fileemd_11600_additional_1.map
AnnotationMap after density modification using ResolveEM in Phenix suite
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Catalytic domain of M. tuberculosis E2b (acyltransferase from bra...

EntireName: Catalytic domain of M. tuberculosis E2b (acyltransferase from branched-chain alpha-ketoacid dehydrogenase complex)
Components
  • Complex: Catalytic domain of M. tuberculosis E2b (acyltransferase from branched-chain alpha-ketoacid dehydrogenase complex)
    • Protein or peptide: Dihydrolipoyllysine-residue acyltransferase

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Supramolecule #1: Catalytic domain of M. tuberculosis E2b (acyltransferase from bra...

SupramoleculeName: Catalytic domain of M. tuberculosis E2b (acyltransferase from branched-chain alpha-ketoacid dehydrogenase complex)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 592 KDa

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Macromolecule #1: Dihydrolipoyllysine-residue acyltransferase

MacromoleculeName: Dihydrolipoyllysine-residue acyltransferase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
EC number: dihydrolipoyllysine-residue (2-methylpropanoyl)transferase
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSPDVRPVHG VHARMAEK M TLSHKEIPTA KASVEVICAE LLRLRDRFVS AAPEITPFAL TLRLLVIALK HNVILNSTW VDSGEGPQVH VHRGVHLGFG AATERGLLVP VVTDAQDKNT RELASRVAEL ITGAREGTLT PAELRGSTF TVSNFGALGV DDGVPVINHP ...String:
GSPDVRPVHG VHARMAEK M TLSHKEIPTA KASVEVICAE LLRLRDRFVS AAPEITPFAL TLRLLVIALK HNVILNSTW VDSGEGPQVH VHRGVHLGFG AATERGLLVP VVTDAQDKNT RELASRVAEL ITGAREGTLT PAELRGSTF TVSNFGALGV DDGVPVINHP EAAILGLGAI KPRPVVVGGE VVARPTMTLT C VFDHRVVD GAQVAQFMCE LRDLIESPET ALLDL

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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

Concentration1.6 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
50.0 mMNaClSodium chloridesodium Chloride
25.0 mMTris-HclTris
GridMaterial: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Details: Power 5W
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot time 4 seconds before plunging.

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 2 / Number real images: 5009 / Average exposure time: 1.0 sec. / Average electron dose: 42.0 e/Å2

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Image processing

Particle selectionNumber selected: 323584
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.0) / Number images used: 111891
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6zzn


Chain - Chain ID: A / Chain - Residue range: 165-393
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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