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Yorodumi- PDB-8usp: Structural and biochemical investigations of a HEAT-repeat protei... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8usp | |||||||||||||||
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Title | Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway | |||||||||||||||
Components | DNA repair/transcription protein MET18/MMS19 | |||||||||||||||
Keywords | METAL TRANSPORT / IRON-SULFUR CLUSTER / ASSEMBLY / CIA PATHWAY / MET18 | |||||||||||||||
Function / homology | Function and homology information cytosolic [4Fe-4S] assembly targeting complex / protein maturation by iron-sulfur cluster transfer / iron-sulfur cluster assembly / DNA metabolic process / response to UV / : / DNA repair / nucleus / cytoplasm / cytosol Similarity search - Function | |||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||||||||
Authors | Vasquez, S. / Drennan, C.L. | |||||||||||||||
Funding support | United States, 4items
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Citation | Journal: Commun Biol / Year: 2023 Title: Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway. Authors: Sheena Vasquez / Melissa D Marquez / Edward J Brignole / Amanda Vo / Sunnie Kong / Christopher Park / Deborah L Perlstein / Catherine L Drennan / Abstract: Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the ...Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the cytosolic iron-sulfur cluster assembly (CIA) pathway is not well understood. Here we report cryo-EM structures of the HEAT-repeat protein Met18 from Saccharomyces cerevisiae, a key component of the CIA targeting complex (CTC) that identifies cytosolic and nuclear client proteins and delivers a mature iron-sulfur cluster. We find that in the absence of other CTC proteins, Met18 adopts tetrameric and hexameric states. Using mass photometry and negative stain EM, we show that upon the addition of Cia2, these higher order oligomeric states of Met18 disassemble. We also use pulldown assays to identify residues of critical importance for Cia2 binding and recognition of the Leu1 client, many of which are buried when Met18 oligomerizes. Our structures show conformations of Met18 that have not been previously observed in any Met18 homolog, lending support to the idea that a highly flexible Met18 may be key to how the CTC is able to deliver iron-sulfur clusters to client proteins of various sizes and shapes, i.e. Met18 conforms to the dimensions needed. | |||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8usp.cif.gz | 1 MB | Display | PDBx/mmCIF format |
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PDB format | pdb8usp.ent.gz | 865.1 KB | Display | PDB format |
PDBx/mmJSON format | 8usp.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8usp_validation.pdf.gz | 827.1 KB | Display | wwPDB validaton report |
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Full document | 8usp_full_validation.pdf.gz | 888.5 KB | Display | |
Data in XML | 8usp_validation.xml.gz | 138.9 KB | Display | |
Data in CIF | 8usp_validation.cif.gz | 211.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/us/8usp ftp://data.pdbj.org/pub/pdb/validation_reports/us/8usp | HTTPS FTP |
-Related structure data
Related structure data | 42510MC 8usqC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 118007.078 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Strain: ATCC 204508 / S288C / Gene: MET18 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: P40469 |
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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: QUATERNARY STRUCTURE OF THE MET18 HEXAMER COMPLEX. / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||||||
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Molecular weight | Value: 118 kDa/nm / Experimental value: YES | ||||||||||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288C | ||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: BL21 | ||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Details: -15 mA Electron Microscopy Science Quantifoil 1.2/1.3 Cu 300 mesh Grid material: COPPER / Grid mesh size: 300 divisions/in. | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 49.59 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 379779 | ||||||||||||||||||||||||||||
Symmetry | Point symmetry: D3 (2x3 fold dihedral) | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171255 / Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT | ||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6TC0 Accession code: 6TC0 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||
Refinement | Highest resolution: 3.3 Å |