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- PDB-5tre: Zinc and the Iron Donor Frataxin Regulate Oligomerization of the ... -

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Basic information

Entry
Database: PDB / ID: 5tre
TitleZinc and the Iron Donor Frataxin Regulate Oligomerization of the Scaffold Protein to Form New Fe-S Cluster Assembly Centers
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 / Maturation of TCA enzymes and regulation of TCA cycle / Mitochondrial protein import / Complex III assembly / mitochondrial electron transport, succinate to ubiquinone / iron chaperone activity / tRNA wobble uridine modification / iron-sulfur cluster assembly complex / heme biosynthetic process / iron-sulfur cluster assembly ...Mitochondrial iron-sulfur cluster biogenesis / Maturation of TCA enzymes and regulation of TCA cycle / Mitochondrial protein import / Complex III assembly / mitochondrial electron transport, succinate to ubiquinone / iron chaperone activity / tRNA wobble uridine modification / iron-sulfur cluster assembly complex / heme biosynthetic process / iron-sulfur cluster assembly / response to iron(II) ion / ferroxidase / ferroxidase activity / ATPase activator activity / glutathione metabolic process / ferric iron binding / iron ion transport / ferrous iron binding / mitochondrial intermembrane space / 2 iron, 2 sulfur cluster binding / response to oxidative stress / intracellular iron ion homeostasis / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 15.6 Å
AuthorsRanatunga, W. / Gakh, O. / Galeano, B.K. / Smith IV, D.Y. / Thompson, J.R. / Isaya, G.
Funding support United States, 1items
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, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 7, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_volume ..._citation.country / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.name
Revision 1.4Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Nov 6, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / struct_sheet
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _struct_sheet.number_strands

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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
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area179150 Å2
ΔGint-645 kcal/mol
Surface area237950 Å2

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Components

#1: Protein ...
Iron sulfur cluster assembly protein 1, mitochondrial / 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
Has protein modificationY

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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: Macromolecular complex 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 chlorideNaCl1
SpecimenConc.: 0.11 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 FIELD / 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: 475
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
12EMAN22.063D reconstruction
13Situs2.3model refinement
14NAMD2.1model refinement
15Coot0.8.1model refinement
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: 4218
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 15.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4218 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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