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- EMDB-8293: Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assem... -

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Entry
Database: EMDB / ID: EMD-8293
TitleArchitecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Complex Formed by the Iron Donor, the Sulfur Donor, and the Scaffold
Map dataHuman Fe-S Assembly
Sample
  • Complex: NFS1-ISD11-ISCU-FXN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 14.3 Å
AuthorsGakh O / Ranatunga W / Smith DY / Ahlgren EC / Al-Karadaghi S / Thompson JR / Isaya G
CitationJournal: J Biol Chem / Year: 2016
Title: Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery.
Authors: Oleksandr Gakh / Wasantha Ranatunga / Douglas Y Smith / Eva-Christina Ahlgren / Salam Al-Karadaghi / James R Thompson / Grazia Isaya /
Abstract: Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human ...Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN]·[NFS1]·[ISD11]·[ISCU] complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN]·[ISCU] sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN trimer at each of its eight vertices. Binding of 12 [NFS1]·[ISD11] sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN to ISCU.
History
DepositionAug 5, 2016-
Header (metadata) releaseAug 31, 2016-
Map releaseAug 31, 2016-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8293.png

Downloads & links

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Map

FileDownload / File: emd_8293.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman Fe-S Assembly
Voxel sizeX=Y=Z: 1.034 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 1
Minimum - Maximum-8.422809000000001 - 9.254791000000001
Average (Standard dev.)0.023395902 (±0.63186723)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 264.704 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0341.0341.034
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z264.704264.704264.704
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-8.4239.2550.023

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Supplemental data

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Sample components

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Entire : NFS1-ISD11-ISCU-FXN

EntireName: NFS1-ISD11-ISCU-FXN
Components
  • Complex: NFS1-ISD11-ISCU-FXN

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Supramolecule #1: NFS1-ISD11-ISCU-FXN

SupramoleculeName: NFS1-ISD11-ISCU-FXN / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: E.coli (others) / Recombinant plasmid: pCDF, pET

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H12ClNO3Tris-HClTris
150.0 mMNaClSodium chlorideSodium chloride
StainingType: NEGATIVE / Material: 1% uranyl acetate / Details: 5 and 30 seconds
GridModel: Carbon-coated copper grids, EMS / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Details: DV-502A vacuum evaporator (Denton Vacuum Inc.)
Details50 mM Tris-HCl, pH 8.0, 150 mM NaCl

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 0.21 µm / Calibrated magnification: 115000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.21 µm / Nominal magnification: 115000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 466 / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4124
CTF correctionSoftware - Name: EMAN2 (ver. 2.06) / Details: The ctf.auto function from EMAN2 was applied.
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 14.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.06) / Number images used: 4124
Details432 symmetry
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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