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- EMDB-8301: Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assem... -

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Entry
Database: EMDB / ID: 8301
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 mitochondrial iron-sulfur cluster assembly machinery: grouping of segments corresponding to one half of the full map (EMD-8293)
SampleNFS1-ISD11-ISCU-FXN
  • Cysteine desulfurase, mitochondrial
  • Frataxin, mitochondrial
  • Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
Function / homologyPyridoxal phosphate-dependent transferase domain 1 / NIF system FeS cluster assembly, NifU, N-terminal / Pyridoxal phosphate-dependent transferase, major domain / ISC system FeS cluster assembly, IscU scaffold / Cysteine desulfurase IscS / Pyridoxal phosphate-dependent transferase / Cysteine desulfurase / Frataxin / Aminotransferase class-V, pyridoxal-phosphate binding site / Frataxin/CyaY ...Pyridoxal phosphate-dependent transferase domain 1 / NIF system FeS cluster assembly, NifU, N-terminal / Pyridoxal phosphate-dependent transferase, major domain / ISC system FeS cluster assembly, IscU scaffold / Cysteine desulfurase IscS / Pyridoxal phosphate-dependent transferase / Cysteine desulfurase / Frataxin / Aminotransferase class-V, pyridoxal-phosphate binding site / Frataxin/CyaY / Frataxin conserved site / Frataxin/CyaY superfamily / Aminotransferase class-V / Aminotransferase class V domain / Frataxin-like domain / NifU-like N terminal domain / Aminotransferases class-V pyridoxal-phosphate attachment site. / Frataxin family signature. / Frataxin family profile. / Mitochondrial protein import / Mitochondrial iron-sulfur cluster biogenesis / Molybdenum cofactor biosynthesis / regulation of ferrochelatase activity / L-cysteine desulfurase complex / positive regulation of succinate dehydrogenase activity / positive regulation of aconitate hydratase activity / positive regulation of lyase activity / proprioception / small molecule metabolic process / iron incorporation into metallo-sulfur cluster / sulfur amino acid metabolic process / [2Fe-2S] cluster assembly / cysteine desulfurase activity / cysteine desulfurase / iron chaperone activity / negative regulation of multicellular organism growth / molybdopterin cofactor biosynthetic process / negative regulation of organ growth / oxidative phosphorylation / adult walking behavior / iron-sulfur cluster assembly / Mo-molybdopterin cofactor biosynthetic process / embryo development ending in birth or egg hatching / iron-sulfur cluster binding / heme biosynthetic process / response to iron ion / protein autoprocessing / ferroxidase / ferroxidase activity / go:0032947: / negative regulation of release of cytochrome c from mitochondria / aerobic respiration / positive regulation of catalytic activity / ion transport / ferric iron binding / mitochondrion organization / ferrous iron binding / 2 iron, 2 sulfur cluster binding / cellular iron ion homeostasis / cellular response to hydrogen peroxide / 4 iron, 4 sulfur cluster binding / protein-containing complex assembly / pyridoxal phosphate binding / positive regulation of cell growth / mitochondrial matrix / iron ion binding / positive regulation of cell population proliferation / negative regulation of apoptotic process / protein homodimerization activity / mitochondrion / nucleoplasm / metal ion binding / nucleus / cytosol / cytoplasm / Frataxin, mitochondrial / Iron-sulfur cluster assembly enzyme ISCU, mitochondrial / Cysteine desulfurase, mitochondrial
Function and homology information
SourceHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / 14.3 Å resolution
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.
Validation ReportPDB-ID: 5kz5

SummaryFull reportAbout validation report
DateDeposition: Aug 9, 2016 / Header (metadata) release: Aug 31, 2016 / Map release: Aug 31, 2016 / Last update: Jul 18, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5kz5
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8301.map.gz (map file in CCP4 format, 12306 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
165 pix
1.1 Å/pix.
= 181.5 Å
165 pix
1.1 Å/pix.
= 181.5 Å
113 pix
1.1 Å/pix.
= 124.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour Level:0.8 (by author), 0.8 (movie #1):
Minimum - Maximum0. - 8.551926999999999
Average (Standard dev.)0.31592557 (0.81216276)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions165113165
Origin46.46.46.
Limit210.158.210.
Spacing113165165
CellA: 124.3 Å / B: 181.5 Å / C: 181.5 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z113165165
origin x/y/z0.0000.0000.000
length x/y/z124.300181.500181.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS464646
NC/NR/NS113165165
D min/max/mean0.0008.5520.316

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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 / Number of components: 4

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Component #1: protein, NFS1-ISD11-ISCU-FXN

ProteinName: NFS1-ISD11-ISCU-FXN / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pCDF, pET

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Component #2: protein, Cysteine desulfurase, mitochondrial

ProteinName: Cysteine desulfurase, mitochondrial / Number of Copies: 12 / Recombinant expression: No
MassTheoretical: 43.429648 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli #1/H766 (bacteria)

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Component #3: protein, Frataxin, mitochondrial

ProteinName: Frataxin, mitochondrial / Number of Copies: 12 / Recombinant expression: No
MassTheoretical: 18.849025 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli #1/H766 (bacteria)

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Component #4: protein, Iron-sulfur cluster assembly enzyme ISCU, mitochondrial

ProteinName: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
Number of Copies: 12 / Recombinant expression: No
MassTheoretical: 12.52558 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli #1/H766 (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining
Sample solutionSpecimen conc.: 0.3 mg/ml / pH: 8
Support filmDV-502A vacuum evaporator (Denton Vacuum Inc.)
Staining5 and 30 seconds
VitrificationCryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F30
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 3 e/Å2 / Illumination mode: OTHER
LensMagnification: 115000. X (nominal), 115000. X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 210.0 - 3000.0 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 466

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: O (octahedral) / Number of projections: 4124 / Details: 432 symmetry
3D reconstructionAlgorithm: FOURIER SPACE
CTF correction: The ctf.auto function from EMAN2 was applied.
Resolution: 14.3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Output model

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