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- PDB-5t0v: Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assem... -

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Basic information

Entry
Database: PDB / ID: 5t0v
TitleArchitecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Sub-Complex Formed by the Iron Donor, Yfh1, and the Scaffold, Isu1
Components
  • Frataxin homolog, mitochondrial
  • Iron sulfur cluster assembly protein 1, mitochondrial
KeywordsOXIDOREDUCTASE / Friedreich Ataxia / frataxin / iron-sulfur protein / mitochondria / protein complex
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 17.5 Å
AuthorsRanatunga, W. / Gakh, O. / Galeano, B.K. / Smith IV, D.Y. / Soderberg, C.A. / Al-Karadaghi, S. / Thompson, J.R. / Isaya, G.
Funding support United States, 1items
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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 31, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Assembly

Deposited unit
a: Iron sulfur cluster assembly protein 1, mitochondrial
A: Frataxin homolog, mitochondrial
b: Iron sulfur cluster assembly protein 1, mitochondrial
B: Frataxin homolog, mitochondrial
c: Iron sulfur cluster assembly protein 1, mitochondrial
C: Frataxin homolog, mitochondrial
d: Iron sulfur cluster assembly protein 1, mitochondrial
D: Frataxin homolog, mitochondrial
e: Iron sulfur cluster assembly protein 1, mitochondrial
E: Frataxin homolog, mitochondrial
f: Iron sulfur cluster assembly protein 1, mitochondrial
F: Frataxin homolog, mitochondrial
g: Iron sulfur cluster assembly protein 1, mitochondrial
G: Frataxin homolog, mitochondrial
h: Iron sulfur cluster assembly protein 1, mitochondrial
H: Frataxin homolog, mitochondrial
i: Iron sulfur cluster assembly protein 1, mitochondrial
I: Frataxin homolog, mitochondrial
j: Iron sulfur cluster assembly protein 1, mitochondrial
J: Frataxin homolog, mitochondrial
k: Iron sulfur cluster assembly protein 1, mitochondrial
K: Frataxin homolog, mitochondrial
l: Iron sulfur cluster assembly protein 1, mitochondrial
L: Frataxin homolog, mitochondrial
m: Iron sulfur cluster assembly protein 1, mitochondrial
M: Frataxin homolog, mitochondrial
n: Iron sulfur cluster assembly protein 1, mitochondrial
N: Frataxin homolog, mitochondrial
o: Iron sulfur cluster assembly protein 1, mitochondrial
O: Frataxin homolog, mitochondrial
p: Iron sulfur cluster assembly protein 1, mitochondrial
P: Frataxin homolog, mitochondrial
q: Iron sulfur cluster assembly protein 1, mitochondrial
Q: Frataxin homolog, mitochondrial
r: Iron sulfur cluster assembly protein 1, mitochondrial
R: Frataxin homolog, mitochondrial
s: Iron sulfur cluster assembly protein 1, mitochondrial
S: Frataxin homolog, mitochondrial
t: Iron sulfur cluster assembly protein 1, mitochondrial
T: Frataxin homolog, mitochondrial
u: Iron sulfur cluster assembly protein 1, mitochondrial
U: Frataxin homolog, mitochondrial
v: Iron sulfur cluster assembly protein 1, mitochondrial
V: Frataxin homolog, mitochondrial
w: Iron sulfur cluster assembly protein 1, mitochondrial
W: Frataxin homolog, mitochondrial
x: Iron sulfur cluster assembly protein 1, mitochondrial
X: Frataxin homolog, mitochondrial


Theoretical massNumber of molelcules
Total (without water)692,15648
Polymers692,15648
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area254290 Å2
ΔGint-904 kcal/mol
Surface area152560 Å2

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Components

#1: Protein ...
Iron sulfur cluster assembly protein 1, mitochondrial / Isu1 / Iron sulfur cluster scaffold protein 1


Mass: 15383.872 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ISU1, NUA1, YPL135W / Plasmid: pET28b / Production host: Escherichia coli #1/H766 (bacteria) / References: UniProt: Q03020
#2: Protein ...
Frataxin homolog, mitochondrial


Mass: 13455.976 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: YFH1, YDL120W / Plasmid: pET24a / Production host: Escherichia coli #1/H766 (bacteria) / References: UniProt: Q07540, ferroxidase

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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: Yfh1-Isu1 / Type: COMPLEX
Details: macromolecule comprising 24-mer of Yfh1 and 24-mer of Isu1
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.7 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli #1/H766 (bacteria) / Plasmid: pET24a, pET28b
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPES-KOHC8H19KN2O5S1
2100 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 0.12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
Details: The 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.
EM stainingType: NEGATIVE
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.
Material: uranyl acetate
Specimen supportDetails: DV-502A instrument, Denton Vacuum Inc. / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: carbon-coated, 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: 2800 nm / Nominal defocus min: 210 nm / Calibrated defocus min: 210 nm / Calibrated defocus max: 2800 nm / Cs: 2 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder 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: 559
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1EMAN22.06particle selection
2DigitalMicrograph1.6image acquisition
4EMAN22.06CTF correction
7UCSF Chimera1.1model fitting
9Situs2.3model refinement
10NAMD2.1model refinement
11Coot0.8.1model refinement
15EMAN22.063D reconstruction
Image processingDetails: 432 symmetry applied for reconstruction
CTF correctionDetails: The ctf.auto function from EMAN2 was applied. / Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 4153
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 17.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4153 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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