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- PDB-8scx: Cryo-EM structure of the core TIM23 complex from S. cerevisiae -

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Basic information

Entry
Database: PDB / ID: 8scx
TitleCryo-EM structure of the core TIM23 complex from S. cerevisiae
Components
  • (Antibody Fab fragment ...) x 2
  • (Mitochondrial import inner membrane translocase subunit ...) x 3
KeywordsTRANSLOCASE
Function / homology
Function and homology information


TIM23 mitochondrial import inner membrane translocase complex / protein import into mitochondrial matrix / protein transmembrane transporter activity / protein-folding chaperone binding / mitochondrial inner membrane
Similarity search - Function
Mitochondrial inner membrane translocase complex, subunit Tim17 / Mitochondrial inner membrane translocase complex, subunit Tim23 / Tim23-like / Mitochondrial import inner membrane translocase subunit Tim44 / Tim44-like / Tim44-like domain / Tim44-like domain / Tim44 / Tim17/Tim22/Tim23/Pmp24 family / NTF2-like domain superfamily
Similarity search - Domain/homology
CARDIOLIPIN / PHOSPHATIDYLETHANOLAMINE / Mitochondrial import inner membrane translocase subunit TIM17 / Mitochondrial import inner membrane translocase subunit TIM44 / Mitochondrial import inner membrane translocase subunit TIM23
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsSim, S.I. / Park, E.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nature / Year: 2023
Title: Structural basis of mitochondrial protein import by the TIM23 complex.
Authors: Sue Im Sim / Yuanyuan Chen / Diane L Lynch / James C Gumbart / Eunyong Park /
Abstract: Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical studies have shown that the ...Mitochondria import nearly all of their approximately 1,000-2,000 constituent proteins from the cytosol across their double-membrane envelope. Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel. However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17-Tim23-Tim44) from Saccharomyces cerevisiae. Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis.
History
DepositionApr 5, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 4, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Mitochondrial import inner membrane translocase subunit TIM17
B: Mitochondrial import inner membrane translocase subunit TIM23
C: Mitochondrial import inner membrane translocase subunit TIM44
L: Antibody Fab fragment light chain
H: Antibody Fab fragment heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,6687
Polymers140,4705
Non-polymers2,1982
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Mitochondrial import inner membrane translocase subunit ... , 3 types, 3 molecules ABC

#1: Protein Mitochondrial import inner membrane translocase subunit TIM17


Mass: 16602.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS3076, PACBIOSEQ_LOCUS3090, PACBIOSEQ_LOCUS3120, PACBIOSEQ_LOCUS3122, PACBIOSEQ_LOCUS3162, PACBIOSEQ_LOCUS3163, PACBIOSEQ_LOCUS3183, SCNYR20_0009006200, SCP684_0009006100
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5PVU8
#2: Protein Mitochondrial import inner membrane translocase subunit TIM23


Mass: 23266.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS5249, SCNYR20_0008030700, SCP684_0008030200
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q5E3
#3: Protein Mitochondrial import inner membrane translocase subunit TIM44


Mass: 48933.418 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PACBIOSEQ_LOCUS1611, PACBIOSEQ_LOCUS1621, PACBIOSEQ_LOCUS1657, SCNYR20_0013012900, SCP684_0013013400
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q2Y5

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Antibody , 2 types, 2 molecules LH

#4: Antibody Antibody Fab fragment light chain


Mass: 26173.201 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#5: Antibody Antibody Fab fragment heavy chain


Mass: 25494.990 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL


Mass: 1464.043 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C81H156O17P2 / Comment: phospholipid*YM
#7: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Comment: phospholipid*YM

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Core TIM23 complex (core+Pam16/18)COMPLEX#1-#50RECOMBINANT
2Mitochondrial import inner membrane translocase subunit TIM17, Mitochondrial import inner membrane translocase subunit TIM23, Mitochondrial import inner membrane translocase subunit TIM44COMPLEX#1-#31RECOMBINANT
3Antibody Fab fragment light chain, Antibody Fab fragment heavy chainCOMPLEX#4-#51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
23Mus musculus (house mouse)10090
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELD / Nominal defocus max: 2100 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.7image acquisition
12cryoSPARC2.15classification
13cryoSPARC2.153D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 159958 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045867
ELECTRON MICROSCOPYf_angle_d0.5147914
ELECTRON MICROSCOPYf_dihedral_angle_d8.782911
ELECTRON MICROSCOPYf_chiral_restr0.041857
ELECTRON MICROSCOPYf_plane_restr0.0031015

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