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- EMDB-8458: Zinc and the Iron Donor Frataxin Regulate Oligomerization of the ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8458
TitleZinc and the Iron Donor Frataxin Regulate Oligomerization of the Scaffold Protein to Form New Fe-S Cluster Assembly Centers
Map dataIron sulfur cluster assembly protein 1 / Frataxin complex (generated using EMAN2)
Sample
  • Complex: Yfh1-Isu1
    • Protein or peptide: Iron sulfur cluster assembly protein 1, mitochondrial
    • Protein or peptide: Frataxin homolog, mitochondrial
Function / homology
Function and homology information


Mitochondrial iron-sulfur cluster biogenesis / Mitochondrial protein import / mitochondrial electron transport, succinate to ubiquinone / iron chaperone activity / tRNA wobble uridine modification / iron-sulfur cluster assembly complex / response to iron(II) ion / iron-sulfur cluster assembly / heme biosynthetic process / ferroxidase ...Mitochondrial iron-sulfur cluster biogenesis / Mitochondrial protein import / mitochondrial electron transport, succinate to ubiquinone / iron chaperone activity / tRNA wobble uridine modification / iron-sulfur cluster assembly complex / response to iron(II) ion / iron-sulfur cluster assembly / heme biosynthetic process / ferroxidase / ATPase activator activity / ferroxidase activity / glutathione metabolic process / ferric iron binding / ferrous iron binding / mitochondrial intermembrane space / 2 iron, 2 sulfur cluster binding / iron ion transport / response to oxidative stress / mitochondrial inner membrane / intracellular iron ion homeostasis / mitochondrial matrix / iron ion binding / mitochondrion / zinc ion binding / identical protein binding / cytoplasm
Similarity search - Function
ISC system FeS cluster assembly, IscU scaffold / Frataxin / 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: 15.6 Å
AuthorsRanatunga W / Gakh O / Galeano BK / Smith IV DY / Thompson JR / Isaya G
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute on Aging (NIH/NIA)AG15709 United States
CitationJournal: Metallomics / Year: 2017
Title: Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers.
Authors: B K Galeano / W Ranatunga / O Gakh / D Y Smith / J R Thompson / G Isaya /
Abstract: Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast ...Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery.
History
DepositionOct 26, 2016-
Header (metadata) releaseNov 9, 2016-
Map releaseJun 7, 2017-
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
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  • Surface view colored by radius
  • Surface level: 0.8
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  • Surface view with fitted model
  • Atomic models: PDB-5tre
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5tre
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8458.png

Downloads & links

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Map

FileDownload / File: emd_8458.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIron sulfur cluster assembly protein 1 / Frataxin complex (generated using EMAN2)
Voxel sizeX=Y=Z: 1.034 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-5.767055 - 8.18915
Average (Standard dev.)0.023244726 (±0.5704755)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-5.7678.1890.023

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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: Macromolecular complex comprising 24-mer of Yfh1 and 24-mer of Isu1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli #1/H766 (bacteria) / Recombinant plasmid: pET24a, pET28b
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

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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

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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

Concentration0.11 mg/mL
BufferpH: 7.3
Component:
ConcentrationFormulaName
10.0 mMC8H19KN2O5SHEPES-KOH
100.0 mMNaClSodium chloridesodium 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 / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.8 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: 2.8 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: 475 / 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: 4218
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: 15.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.06) / Number images used: 4218
Details432 symmetry applied for reconstruction
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5tre:
Zinc and the Iron Donor Frataxin Regulate Oligomerization of the Scaffold Protein to Form New Fe-S Cluster Assembly Centers

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