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- PDB-8usp: Structural and biochemical investigations of a HEAT-repeat protei... -

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Basic information

Entry
Database: PDB / ID: 8usp
TitleStructural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway
ComponentsDNA repair/transcription protein MET18/MMS19
KeywordsMETAL 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 / response to organic substance / DNA repair / nucleus / cytosol / cytoplasm
Similarity search - Function
MMS19, C-terminal / MMS19, N-terminal / DNA repair/transcription protein MET18/MMS19 / RNAPII transcription regulator C-terminal / Dos2-interacting transcription regulator of RNA-Pol-II / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
DNA repair/transcription protein MET18/MMS19
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsVasquez, S. / Drennan, C.L.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM126982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM121673 United States
National Science Foundation (NSF, United States)DGE-1247312 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM007287 United States
CitationJournal: 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
DepositionOct 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2024Group: Source and taxonomy
Category: em_entity_assembly_naturalsource / em_entity_assembly_recombinant / entity_src_gen
Item: _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.strain ..._em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.strain / _em_entity_assembly_recombinant.ncbi_tax_id / _em_entity_assembly_recombinant.strain / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name
Revision 1.2May 1, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA repair/transcription protein MET18/MMS19
B: DNA repair/transcription protein MET18/MMS19
C: DNA repair/transcription protein MET18/MMS19
D: DNA repair/transcription protein MET18/MMS19
E: DNA repair/transcription protein MET18/MMS19
F: DNA repair/transcription protein MET18/MMS19


Theoretical massNumber of molelcules
Total (without water)708,0426
Polymers708,0426
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
DNA repair/transcription protein MET18/MMS19


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

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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: QUATERNARY STRUCTURE OF THE MET18 HEXAMER COMPLEX. / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 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 solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMTris-HClTris1
2100 mMNaClSodium chloride1
35 %Glycerol1
45 mMBetamercaptoethanol1
550 mMHEPES1
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: -15 mA Electron Microscopy Science Quantifoil 1.2/1.3 Cu 300 mesh
Grid material: COPPER / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 49.59 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
4CTFFIND4CTF correction
7PHENIXmodel fitting
11RELION3classification
12RELION33D reconstruction
13Warp3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 379779
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171255 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6TC0

6tc0


Accession code: 6TC0 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.3 Å

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