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- PDB-5kz5: Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assem... -
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Basic information
Entry | Database: PDB / ID: 5kz5 | ||||||
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Title | Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Complex Formed by the Iron Donor, the Sulfur Donor, and the Scaffold | ||||||
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![]() | TRANSFERASE/OXIDOREDUCTASE / frataxin / iron-sulfur protein / mitochondria / protein complex / TRANSFERASE-OXIDOREDUCTASE complex | ||||||
Function / homology | ![]() regulation of ferrochelatase activity / negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / Molybdenum cofactor biosynthesis / proprioception / [4Fe-4S] cluster assembly / iron incorporation into metallo-sulfur cluster / positive regulation of lyase activity / positive regulation of succinate dehydrogenase activity / L-cysteine desulfurase complex ...regulation of ferrochelatase activity / negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / Molybdenum cofactor biosynthesis / proprioception / [4Fe-4S] cluster assembly / iron incorporation into metallo-sulfur cluster / positive regulation of lyase activity / positive regulation of succinate dehydrogenase activity / L-cysteine desulfurase complex / Mitochondrial iron-sulfur cluster biogenesis / mitochondrial [2Fe-2S] assembly complex / positive regulation of aconitate hydratase activity / positive regulation of mitochondrial electron transport, NADH to ubiquinone / iron chaperone activity / Maturation of TCA enzymes and regulation of TCA cycle / cysteine desulfurase / cysteine desulfurase activity / negative regulation of organ growth / Mo-molybdopterin cofactor biosynthetic process / iron-sulfur cluster assembly complex / Mitochondrial protein import / [2Fe-2S] cluster assembly / oxidative phosphorylation / adult walking behavior / response to iron ion / embryo development ending in birth or egg hatching / iron-sulfur cluster assembly / heme biosynthetic process / negative regulation of multicellular organism growth / organ growth / positive regulation of catalytic activity / muscle cell cellular homeostasis / ferroxidase / negative regulation of release of cytochrome c from mitochondria / iron-sulfur cluster binding / protein autoprocessing / ferroxidase activity / ferric iron binding / mitochondrion organization / ferrous iron binding / 2 iron, 2 sulfur cluster binding / cellular response to hydrogen peroxide / pyridoxal phosphate binding / Maturation of replicase proteins / iron ion transport / positive regulation of cell growth / intracellular iron ion homeostasis / molecular adaptor activity / mitochondrial matrix / iron ion binding / centrosome / positive regulation of cell population proliferation / negative regulation of apoptotic process / protein homodimerization activity / mitochondrion / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 14.3 Å | ||||||
![]() | Gakh, O. / Ranatunga, W. / Smith, D.Y. / Ahlgren, E.C. / Al-Karadaghi, S. / Thompson, J.R. / Isaya, G. | ||||||
Funding support | ![]()
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![]() | ![]() 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. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 1.3 MB | Display | ![]() |
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PDB format | ![]() | 1.1 MB | Display | ![]() |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 8301MC ![]() 8293C M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 43429.648 Da / Num. of mol.: 12 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #2: Protein | Mass: 18849.025 Da / Num. of mol.: 12 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #3: Protein | Mass: 12525.580 Da / Num. of mol.: 12 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: NFS1-ISD11-ISCU-FXN / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Source (natural) | Organism: ![]() | |||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||
Buffer solution | pH: 8 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO / Details: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl | |||||||||||||||
EM staining | Type: NEGATIVE / Details: 5 and 30 seconds / Material: 1% uranyl acetate | |||||||||||||||
Specimen support | Details: DV-502A vacuum evaporator (Denton Vacuum Inc.) / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Carbon-coated copper grids, EMS |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai F30 / Image courtesy: FEI Company |
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Microscopy | Model: FEI TECNAI F30 |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 115000 X / Calibrated magnification: 115000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 210 nm / Calibrated defocus min: 210 nm / Calibrated defocus max: 3000 nm / Cs: 2 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: SIDE ENTRY, EUCENTRIC |
Image recording | Electron dose: 30 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 466 |
Image scans | Width: 4096 / Height: 4096 |
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Processing
EM software |
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Image processing | Details: 432 symmetry | ||||||||||||||||||||||||||||||||
CTF correction | Details: The ctf.auto function from EMAN2 was applied. / Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 4124 | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: O (octahedral) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 14.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4124 / Algorithm: FOURIER SPACE / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL |