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- EMDB-8341: Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assem... -

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Basic information

Entry
Database: EMDB / ID: EMD-8341
TitleArchitecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Sub-Complex Formed by the Iron Donor, Yfh1, and the Scaffold, Isu1
Map dataYfh1_Isu1 sub-complex
Sample
  • Complex: Yfh1-Isu1
    • Protein or peptide: Iron sulfur cluster assembly protein 1, mitochondrial
    • Protein or peptide: Frataxin homolog, mitochondrial
KeywordsFriedreich Ataxia / frataxin / iron-sulfur protein / mitochondria / protein complex / OXIDOREDUCTASE
Function / homology
Function and homology information


Maturation of TCA enzymes and regulation of TCA cycle / Mitochondrial iron-sulfur cluster biogenesis / Mitochondrial protein import / Complex III assembly / mitochondrial electron transport, succinate to ubiquinone / iron chaperone activity / tRNA wobble uridine modification / iron-sulfur cluster assembly complex / response to iron(II) ion / heme biosynthetic process ...Maturation of TCA enzymes and regulation of TCA cycle / Mitochondrial iron-sulfur cluster biogenesis / Mitochondrial protein import / Complex III assembly / mitochondrial electron transport, succinate to ubiquinone / iron chaperone activity / tRNA wobble uridine modification / iron-sulfur cluster assembly complex / response to iron(II) ion / heme biosynthetic process / iron-sulfur cluster assembly / ferroxidase / ferroxidase activity / ATPase activator activity / glutathione metabolic process / ferric iron binding / ferrous iron binding / mitochondrial intermembrane space / 2 iron, 2 sulfur cluster binding / iron ion transport / intracellular iron ion homeostasis / response to oxidative stress / mitochondrial inner membrane / mitochondrial matrix / iron ion binding / mitochondrion / zinc ion binding / identical protein binding / cytoplasm
Similarity search - Function
Frataxin / ISC system FeS cluster assembly, IscU scaffold / 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
Similarity search - Domain/homology
Iron sulfur cluster assembly protein 1, mitochondrial / Frataxin homolog, mitochondrial
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 17.5 Å
AuthorsRanatunga W / Gakh O
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 Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.
Authors: Wasantha Ranatunga / Oleksandr Gakh / Belinda K Galeano / Douglas Y Smith / Christopher A G Söderberg / Salam Al-Karadaghi / James R Thompson / Grazia Isaya /
Abstract: The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We ...The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly.
History
DepositionAug 16, 2016-
Header (metadata) releaseAug 31, 2016-
Map releaseAug 31, 2016-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing 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
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  • Surface view with fitted model
  • Atomic models: PDB-5t0v
  • Surface level: 1
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8341.png

Downloads & links

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Map

FileDownload / File: emd_8341.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYfh1_Isu1 sub-complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 288 pix.
= 297.792 Å		1.03 Å/pix.
x 288 pix.
= 297.792 Å		1.03 Å/pix.
x 288 pix.
= 297.792 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.034 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.8 / Movie #1: 1
Minimum - Maximum-8.725704 - 12.439627
Average (Standard dev.)0.042547934 (±0.8003871)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 297.79202 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0341.0341.034
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z297.792297.792297.792
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/NS288288288
D min/max/mean-8.72612.4400.043

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Supplemental data

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Sample components

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Entire : Yfh1-Isu1

EntireName: Yfh1-Isu1
Components
  • Complex: Yfh1-Isu1
    • Protein or peptide: Iron sulfur cluster assembly protein 1, mitochondrial
    • Protein or peptide: Frataxin homolog, mitochondrial

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Supramolecule #1: Yfh1-Isu1

SupramoleculeName: Yfh1-Isu1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: macromolecule comprising 24-mer of Yfh1 and 24-mer of Isu1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: Iron sulfur cluster assembly protein 1, mitochondrial

MacromoleculeName: Iron sulfur cluster assembly protein 1, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 15.383872 KDa
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria)
SequenceString:
GSHMSSITKR LYHPKVIEHY THPRNVGSLD KKLPNVGTGL VGAPACGDVM RLQIKVNDST GVIEDVKFKT FGCGSAIASS SYMTELVQG MTLDDAAKIK NTEIAKELSL PPVKLHCSML AEDAIKAAIK DYKSKRNTPT MLS

UniProtKB: Iron sulfur cluster assembly protein 1, mitochondrial

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Macromolecule #2: Frataxin homolog, mitochondrial

MacromoleculeName: Frataxin homolog, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 24 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 13.455976 KDa
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria)
SequenceString:
VESSTDGQVV PQEVLNLPLE KAHEEADDYL DHLLDSLEEL SEAHPDCIPD VELSHGVMTL EIPAFGTYVI NKQPPNKQIW LASPLSGPN RFDLLNGEWV SLRNGTKLTD ILTEEVEKAI SK

UniProtKB: Frataxin homolog, mitochondrial

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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

Concentration0.12 mg/mL
BufferpH: 7.3
Component:
ConcentrationFormulaName
10.0 mMC8H19KN2O5SHEPES-KOH
100.0 mMNaClsodium chloride
StainingType: NEGATIVE / Material: uranyl acetate
Details: Pre-incubated in HN100 buffer, the grid was placed on an 11-microliter drop of protein sample for 1 minute. Excess protein sample was blotted and washed for 3 seconds by placing the grid on ...Details: Pre-incubated in HN100 buffer, the grid was placed on an 11-microliter drop of protein sample for 1 minute. Excess protein sample was blotted and washed for 3 seconds by placing the grid on a drop of sterile water. After excess water was blotted, the grid was stained with 1% w/v uranyl acetate for 1 second and 30 seconds by successively placing it on two separate drops of uranyl acetate, with excess stain drawn off after each step.
GridModel: carbon-coated, EMS / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: DV-502A instrument, Denton Vacuum Inc.
DetailsThe protein complex was prepared by incubating Yfh1 and Isu1 (1:1.5 molar ratio) in HN100 buffer (10 mM HEPES-KOH, pH 7.3, 100 mM NaCl) and purified using Sephacryl S300 gel filtration chromatography.

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Electron microscopy

MicroscopeFEI TECNAI F30
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: 559 / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.8000000000000003 µm / Calibrated defocus min: 0.21 µm / Calibrated magnification: 115000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.21 µm / Nominal magnification: 115000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Details432 symmetry applied for reconstruction
Particle selectionNumber selected: 4153
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 17.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.06) / Number images used: 4153
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5t0v:
Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Sub-Complex Formed by the Iron Donor, Yfh1, and the Scaffold, Isu1

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