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

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Basic information

Entry
Database: EMDB / ID: EMD-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)
Sample
  • Complex: NFS1-ISD11-ISCU-FXN
    • Protein or peptide: Cysteine desulfurase, mitochondrial
    • Protein or peptide: Frataxin, mitochondrial
    • Protein or peptide: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
Function / homology
Function and homology information


negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / regulation of ferrochelatase activity / Molybdenum cofactor biosynthesis / [4Fe-4S] cluster assembly / proprioception / iron incorporation into metallo-sulfur cluster / positive regulation of lyase activity / positive regulation of succinate dehydrogenase activity / L-cysteine desulfurase complex ...negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / regulation of ferrochelatase activity / Molybdenum cofactor biosynthesis / [4Fe-4S] cluster assembly / proprioception / 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 iron-sulfur cluster assembly complex / positive regulation of aconitate hydratase activity / positive regulation of mitochondrial electron transport, NADH to ubiquinone / iron chaperone activity / cysteine desulfurase / cysteine desulfurase activity / negative regulation of organ growth / iron-sulfur cluster assembly complex / Mo-molybdopterin cofactor biosynthetic process / Mitochondrial protein import / [2Fe-2S] cluster assembly / adult walking behavior / oxidative phosphorylation / response to iron ion / embryo development ending in birth or egg hatching / iron-sulfur cluster assembly / heme biosynthetic process / muscle cell cellular homeostasis / negative regulation of multicellular organism growth / organ growth / positive regulation of catalytic activity / ferroxidase / iron-sulfur cluster binding / negative regulation of release of cytochrome c from mitochondria / ferroxidase activity / protein autoprocessing / mitochondrion organization / ferric iron binding / 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 / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
ISC system FeS cluster assembly, IscU scaffold / Frataxin / Cysteine desulfurase IscS / Frataxin/CyaY / Frataxin conserved site / Frataxin-like domain / Frataxin family signature. / Frataxin family profile. / Frataxin-like domain / NIF system FeS cluster assembly, NifU, N-terminal ...ISC system FeS cluster assembly, IscU scaffold / Frataxin / Cysteine desulfurase IscS / Frataxin/CyaY / Frataxin conserved site / Frataxin-like domain / Frataxin family signature. / Frataxin family profile. / Frataxin-like domain / NIF system FeS cluster assembly, NifU, N-terminal / NifU-like N terminal domain / Frataxin/CyaY superfamily / Cysteine desulfurase / Aminotransferase class-V, pyridoxal-phosphate binding site / Aminotransferases class-V pyridoxal-phosphate attachment site. / Aminotransferase class V domain / Aminotransferase class-V / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
Frataxin, mitochondrial / Iron-sulfur cluster assembly enzyme ISCU / Cysteine desulfurase
Similarity search - Component
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
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)AG15709-19 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.
History
DepositionAug 9, 2016-
Header (metadata) releaseAug 31, 2016-
Map releaseAug 31, 2016-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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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

emd_8301.png

Downloads & links

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Map

FileDownload / File: emd_8301.map.gz / Format: CCP4 / Size: 11.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman mitochondrial iron-sulfur cluster assembly machinery: grouping of segments corresponding to one half of the full map (EMD-8293)
Projections & slices

Image control

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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum0 - 8.551927
Average (Standard dev.)0.31592557 (±0.81216276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin464646
Dimensions165113165
Spacing113165165
CellA: 124.3 Å / B: 181.5 Å / C: 181.5 Å
α=β=γ: 90.0 °

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
Components
  • Complex: NFS1-ISD11-ISCU-FXN
    • Protein or peptide: Cysteine desulfurase, mitochondrial
    • Protein or peptide: Frataxin, mitochondrial
    • Protein or peptide: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial

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Supramolecule #1: NFS1-ISD11-ISCU-FXN

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

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Macromolecule #1: Cysteine desulfurase, mitochondrial

MacromoleculeName: Cysteine desulfurase, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: cysteine desulfurase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 43.429648 KDa
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria)
SequenceString: TPLDPRVLDA MLPYLINYYG NPHSRTHAYG WESEAAMERA RQQVASLIGA DPREIIFTSG ATESNNIAIK GVARFYRSRK KHLITTQTE HKCVLDSCRS LEAEGFQVTY LPVQKSGIID LKELEAAIQP DTSLVSVMTV NNEIGVKQPI AEIGRICSSR K VYFHTDAA ...String:
TPLDPRVLDA MLPYLINYYG NPHSRTHAYG WESEAAMERA RQQVASLIGA DPREIIFTSG ATESNNIAIK GVARFYRSRK KHLITTQTE HKCVLDSCRS LEAEGFQVTY LPVQKSGIID LKELEAAIQP DTSLVSVMTV NNEIGVKQPI AEIGRICSSR K VYFHTDAA QAVGKIPLDV NDMKIDLMSI SGHKIYGPKG VGAIYIRRRP RVRVEALQSG GGQERGMRSG TVPTPLVVGL GA ACEVAQQ EMEYDHKRIS KLSERLIQNI MKSLPDVVMN GDPKHHYPGC INLSFAYVEG ESLLMALKDV ALSSGSACTS ASL EPSYVL RAIGTDEDLA HSSIRFGIGR FTTEEEVDYT VEKCIQHVKR LREMSPLWEM VQDGIDLKSI KWTQH

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Macromolecule #2: Frataxin, mitochondrial

MacromoleculeName: Frataxin, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.849025 KDa
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria)
SequenceString:
LRTDIDATCT PRRASSNQRG LNQIWNVKKQ SVYLMNLRKS GTLGHPGSLD ETTYERLAEE TLDSLAEFFE DLADKPYTFE DYDVSFGSG VLTVKLGGDL GTYVINKQTP NKQIWLSSPS SGPKRYDWTG KNWVYSHDGV SLHELLAAEL TKALKTKLDL S SLAYSGKD A

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Macromolecule #3: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial

MacromoleculeName: Iron-sulfur cluster assembly enzyme ISCU, mitochondrial
type: protein_or_peptide / ID: 3 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.52558 KDa
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria)
SequenceString:
GSLDKTSKNV GTGLVGAPAC GDVMKLQIQV DEKGKIVDAR FKTFGCGSAI ASSSLATEWV KGKTVEEALT IKNTDIAKEL CLPPVKLHC SMLAEDAIKA ALADYKLKQE PKKGEAEKK

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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
Output model

PDB-5kz5:
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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