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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
TitleArchitecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Complex Formed by the Iron Donor, the Sulfur Donor, and the Scaffold
Components
  • Cysteine desulfurase, mitochondrial
  • Frataxin, mitochondrial
  • Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
KeywordsTRANSFERASE/OXIDOREDUCTASE / frataxin / iron-sulfur protein / mitochondria / protein complex / TRANSFERASE-OXIDOREDUCTASE complex
Function / homology
Function and homology information


negative regulation of iron ion import across plasma membrane / regulation of ferrochelatase activity / L-cysteine desulfurase complex / positive regulation of succinate dehydrogenase activity / positive regulation of lyase activity / positive regulation of aconitate hydratase activity / proprioception / small molecule metabolic process / iron incorporation into metallo-sulfur cluster / [2Fe-2S] cluster assembly ...negative regulation of iron ion import across plasma membrane / regulation of ferrochelatase activity / L-cysteine desulfurase complex / positive regulation of succinate dehydrogenase activity / positive regulation of lyase activity / positive regulation of aconitate hydratase activity / proprioception / small molecule metabolic process / iron incorporation into metallo-sulfur cluster / [2Fe-2S] cluster assembly / cysteine desulfurase / positive regulation of mitochondrial electron transport, NADH to ubiquinone / cysteine desulfurase activity / molybdopterin cofactor biosynthetic process / iron chaperone activity / negative regulation of organ growth / negative regulation of multicellular organism growth / oxidative phosphorylation / adult walking behavior / Mo-molybdopterin cofactor biosynthetic process / iron-sulfur cluster assembly / embryo development ending in birth or egg hatching / heme biosynthetic process / response to iron ion / iron-sulfur cluster binding / ferroxidase / protein autoprocessing / molecular adaptor activity / negative regulation of release of cytochrome c from mitochondria / ferroxidase activity / positive regulation of catalytic activity / aerobic respiration / ion transport / mitochondrion organization / ferric iron binding / ferrous iron binding / 2 iron, 2 sulfur cluster binding / cellular iron ion homeostasis / cellular response to hydrogen peroxide / 4 iron, 4 sulfur cluster binding / pyridoxal phosphate binding / positive regulation of cell growth / mitochondrial matrix / iron ion binding / positive regulation of cell population proliferation / negative regulation of apoptotic process / mitochondrion / protein homodimerization activity / cell / nucleoplasm / metal ion binding / nucleus / cytosol / cytoplasm
Frataxin-like domain / NIF system FeS cluster assembly, NifU, N-terminal / Frataxin/CyaY superfamily / Frataxin conserved site / Aminotransferase class-V, pyridoxal-phosphate binding site / Frataxin / Cysteine desulfurase / Pyridoxal phosphate-dependent transferase / Pyridoxal phosphate-dependent transferase domain 1 / Pyridoxal phosphate-dependent transferase, major domain ...Frataxin-like domain / NIF system FeS cluster assembly, NifU, N-terminal / Frataxin/CyaY superfamily / Frataxin conserved site / Aminotransferase class-V, pyridoxal-phosphate binding site / Frataxin / Cysteine desulfurase / Pyridoxal phosphate-dependent transferase / Pyridoxal phosphate-dependent transferase domain 1 / Pyridoxal phosphate-dependent transferase, major domain / ISC system FeS cluster assembly, IscU scaffold / Aminotransferase class V domain / Cysteine desulfurase IscS / Frataxin/CyaY
Frataxin, mitochondrial / Iron-sulfur cluster assembly enzyme ISCU, mitochondrial / Cysteine desulfurase, mitochondrial
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 14.3 Å
AuthorsGakh, O. / Ranatunga, W. / Smith, D.Y. / Ahlgren, E.C. / Al-Karadaghi, S. / Thompson, J.R. / Isaya, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on AgingAG15709-19 United States
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 Report
SummaryFull reportAbout validation report
History
DepositionJul 22, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 31, 2016Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2016Group: Database references
Revision 1.2May 3, 2017Group: Derived calculations
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
1: Cysteine desulfurase, mitochondrial
2: Cysteine desulfurase, mitochondrial
3: Cysteine desulfurase, mitochondrial
4: Cysteine desulfurase, mitochondrial
M: Cysteine desulfurase, mitochondrial
N: Cysteine desulfurase, mitochondrial
O: Cysteine desulfurase, mitochondrial
P: Cysteine desulfurase, mitochondrial
Q: Cysteine desulfurase, mitochondrial
R: Cysteine desulfurase, mitochondrial
S: Cysteine desulfurase, mitochondrial
T: Cysteine desulfurase, mitochondrial
A: Frataxin, mitochondrial
a: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
B: Frataxin, mitochondrial
b: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
C: Frataxin, mitochondrial
c: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
D: Frataxin, mitochondrial
d: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
E: Frataxin, mitochondrial
e: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
F: Frataxin, mitochondrial
f: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
G: Frataxin, mitochondrial
g: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
H: Frataxin, mitochondrial
h: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
I: Frataxin, mitochondrial
i: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
J: Frataxin, mitochondrial
j: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
K: Frataxin, mitochondrial
k: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
L: Frataxin, mitochondrial
l: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial


Theoretical massNumber of molelcules
Total (without water)897,65136
Polymers897,65136
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area295710 Å2
ΔGint-1219 kcal/mol
Surface area202200 Å2

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Components

#1: Protein/peptide
Cysteine desulfurase, mitochondrial / / NFS1


Mass: 43429.648 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NFS1, NIFS, HUSSY-08 / Production host: Escherichia coli #1/H766 (bacteria) / References: UniProt: Q9Y697, cysteine desulfurase
#2: Protein/peptide
Frataxin, mitochondrial / / Friedreich ataxia protein / Fxn


Mass: 18849.025 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FXN, FRDA, X25 / Production host: Escherichia coli #1/H766 (bacteria) / References: UniProt: Q16595, ferroxidase
#3: Protein/peptide
Iron-sulfur cluster assembly enzyme ISCU, mitochondrial / NifU-like N-terminal domain-containing protein / NifU-like protein / iron sulfur cluster scaffold protein


Mass: 12525.580 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ISCU, NIFUN / Production host: Escherichia coli #1/H766 (bacteria) / References: UniProt: Q9H1K1

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: NFS1-ISD11-ISCU-FXN / Type: COMPLEX / Entity ID: 1,2,3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pCDF, pET
Buffer solutionpH: 8
Buffer component

Buffer-ID: 1

IDConc.NameFormula
150 mMTris-HClTrisC4H12ClNO3
2150 mMsodium chlorideNaClSodium chloride
SpecimenConc.: 0.3 mg/ml / Details: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Details: 5 and 30 seconds / Material: 1% uranyl acetate
Specimen supportDetails: 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
MicroscopyModel: FEI TECNAI F30
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / 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 holderCryogen: NITROGEN / Model: SIDE ENTRY, EUCENTRIC
Image recordingElectron 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 scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1DigitalMicrograph1.6image acquisition
3EMAN22.06CTF correction
6UCSF Chimera1.1model fitting
8Situs2.3model refinement
9NAMD2.1model refinement
10Coot0.8.1model refinement
14EMAN22.063D reconstruction
Image processingDetails: 432 symmetry
CTF correctionDetails: The ctf.auto function from EMAN2 was applied. / Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 4124
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 14.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4124 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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