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Yorodumi- PDB-5t0v: Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assem... -
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-Basic information
Entry | Database: PDB / ID: 5t0v | ||||||
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Title | Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Sub-Complex Formed by the Iron Donor, Yfh1, and the Scaffold, Isu1 | ||||||
Components |
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Keywords | OXIDOREDUCTASE / 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 / tRNA wobble uridine modification / iron chaperone activity / 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 / tRNA wobble uridine modification / iron chaperone activity / 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 / 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 | ||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 17.5 Å | ||||||
Authors | Ranatunga, 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
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Citation | Journal: 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 |
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-Structure visualization
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 5t0v.cif.gz | 998.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5t0v.ent.gz | 830.3 KB | Display | PDB format |
PDBx/mmJSON format | 5t0v.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5t0v_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 5t0v_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 5t0v_validation.xml.gz | 152 KB | Display | |
Data in CIF | 5t0v_validation.cif.gz | 239.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/t0/5t0v ftp://data.pdbj.org/pub/pdb/validation_reports/t0/5t0v | HTTPS FTP |
-Related structure data
Related structure data | 8341MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 | 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
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Yfh1-Isu1 / Type: COMPLEX Details: macromolecule comprising 24-mer of Yfh1 and 24-mer of Isu1 Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 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 solution | pH: 7.3 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 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 staining | Type: 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 support | Details: DV-502A instrument, Denton Vacuum Inc. / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: carbon-coated, EMS |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai F30 / Image courtesy: FEI Company |
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Microscopy | Model: FEI TECNAI F30 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: SIDE ENTRY, EUCENTRIC |
Image recording | Electron 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 scans | Width: 4096 / Height: 4096 |
-Processing
EM software |
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Image processing | Details: 432 symmetry applied for reconstruction | ||||||||||||||||||||||||||||||||||||
CTF correction | Details: The ctf.auto function from EMAN2 was applied. / Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 4153 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: O (octahedral) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 17.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4153 / Algorithm: FOURIER SPACE / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL |