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- PDB-9j9b: Substrate-engaged TIM23 complex from yeast -

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Basic information

Entry
Database: PDB / ID: 9j9b
TitleSubstrate-engaged TIM23 complex from yeast
Components
  • (Mitochondrial import inner membrane translocase subunit ...) x 3
  • (anti-Tim44-Fab ...) x 2
  • Protein MGR2
  • Substrate
KeywordsPROTEIN TRANSPORT / Mitochondrial protein import / TOM / TIM23 protein translocation
Function / homology
Function and homology information


PAM complex, Tim23 associated import motor / L-lactate dehydrogenase (cytochrome) / L-lactate dehydrogenase (cytochrome) activity / extrinsic component of mitochondrial inner membrane / Mitochondrial protein import / TIM23 mitochondrial import inner membrane translocase complex / mitochondrion targeting sequence binding / protein insertion into mitochondrial inner membrane / lactate metabolic process / protein import into mitochondrial matrix ...PAM complex, Tim23 associated import motor / L-lactate dehydrogenase (cytochrome) / L-lactate dehydrogenase (cytochrome) activity / extrinsic component of mitochondrial inner membrane / Mitochondrial protein import / TIM23 mitochondrial import inner membrane translocase complex / mitochondrion targeting sequence binding / protein insertion into mitochondrial inner membrane / lactate metabolic process / protein import into mitochondrial matrix / transmembrane protein transporter activity / intracellular protein transport / mitochondrial intermembrane space / protein-folding chaperone binding / protein-macromolecule adaptor activity / mitochondrial inner membrane / heme binding / mitochondrion / ATP binding / metal ion binding / nucleus / cytosol
Similarity search - Function
Romo1/Mgr2 / Reactive mitochondrial oxygen species modulator 1 / Reactive mitochondrial oxygen species modulator 1 / 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 / L-mandelate/L-lactate dehydrogenase, FMN-binding domain / Cytochrome b5, heme-binding site ...Romo1/Mgr2 / Reactive mitochondrial oxygen species modulator 1 / Reactive mitochondrial oxygen species modulator 1 / 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 / L-mandelate/L-lactate dehydrogenase, FMN-binding domain / Cytochrome b5, heme-binding site / Cytochrome b5 family, heme-binding domain signature. / FMN-dependent alpha-hydroxy acid dehydrogenase, active site / FMN hydroxy acid dehydrogenase domain / FMN-dependent alpha-hydroxy acid dehydrogenases active site. / FMN-dependent alpha-hydroxy acid dehydrogenase domain profile. / Cytochrome b5 family, heme-binding domain profile. / Cytochrome b5-like heme/steroid binding domain / Cytochrome b5-like heme/steroid binding domain superfamily / Cytochrome b5-like Heme/Steroid binding domain / Cytochrome b5-like Heme/Steroid binding domain / FMN-dependent dehydrogenase / FMN-dependent dehydrogenase / Tim44-like domain / Tim44-like domain / Tim44 / Tim17/Tim22/Tim23/Pmp24 family / NTF2-like domain superfamily / Aldolase-type TIM barrel
Similarity search - Domain/homology
CARDIOLIPIN / L-lactate dehydrogenase (cytochrome) / Mitochondrial import inner membrane translocase subunit TIM23 / Mitochondrial import inner membrane translocase subunit TIM17 / Mitochondrial import inner membrane translocase subunit TIM44 / Protein MGR2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.83 Å
AuthorsYang, Y.Q. / Wang, G.P.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32271269 China
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Dynamic TOM-TIM23 supercomplex directs mitochondrial protein translocation and sorting.
Authors: Yuqi Yang / Shanshan Wang / Guopeng Wang / Yuke Lian / Lingfeng Xue / Wenhong Jiang / Qiang Guo / Chen Song / Long Li /
Abstract: The mitochondrial translocase of the outer membrane (TOM) and translocase of the inner membrane 23 (TIM23) complexes are coupled to control protein import across the outer and inner membranes, ...The mitochondrial translocase of the outer membrane (TOM) and translocase of the inner membrane 23 (TIM23) complexes are coupled to control protein import across the outer and inner membranes, respectively. However, the mechanisms of protein recognition and sorting in the TOM-TIM23 pathway remain unclear. Here we report cryo-electron microscopy structures of a translocating polypeptide substrate captured in the active TOM-TIM23 supercomplex from Saccharomyces cerevisiae. In the TOM complex, the polypeptide substrate adopts multiple conformations stabilized by hydrophilic residues from distinct regions of the Tom40 channel. In the TIM23 complex, the Tim17 and Mgr2 subunits create the translocation pathway, with a central restriction formed by four highly conserved hydrophobic residues. The substrate primarily interacts with hydrophobic residues along the Tim17-Mgr2 pathway. Substrate hydrophobicity modulates the association of Mgr2 with Tim17, enabling dynamic regulation of protein sorting toward either the matrix or membrane. These findings reveal a sophisticated translocation mechanism of the TOM-TIM23 supercomplex that ensures the efficient import of diverse mitochondrial proteins.
History
DepositionAug 22, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 10, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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-lactate dehydrogenase (cytochrome),sfGFP
D: Protein MGR2
H: anti-Tim44-Fab H chain
L: anti-Tim44-Fab L chain
E: Substrate
hetero molecules


Theoretical massNumber of molelcules
Total (without water)191,1198
Polymers189,6557
Non-polymers1,4641
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
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 / Mitochondrial inner membrane protein MIM17 / Mitochondrial protein import protein 2


Mass: 16602.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: TIM17, MIM17, MPI2, SMS1, YJL143W, J0648 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P39515
#2: Protein Mitochondrial import inner membrane translocase subunit TIM23 / Membrane import machinery protein MIM23 / Mitochondrial protein import protein 3 / Mitochondrial ...Membrane import machinery protein MIM23 / Mitochondrial protein import protein 3 / Mitochondrial protein import protein MAS6


Mass: 23266.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: TIM23, MAS6, MIM23, MPI3, YNR017W, N3180 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32897
#3: Protein Mitochondrial import inner membrane translocase subunit TIM44,L-lactate dehydrogenase (cytochrome),sfGFP / Inner membrane import site protein 45 / ISP45 / Membrane import machinery protein MIM44 / ...Inner membrane import site protein 45 / ISP45 / Membrane import machinery protein MIM44 / Mitochondrial protein import protein 1 / Cytochrome b2 / Flavocytochrome b2 / FCB2 / L-lactate ferricytochrome c oxidoreductase / L-LCR


Mass: 84451.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast), (gene. exp.) synthetic construct (others)
Gene: TIM44, ISP45, MIM44, MPI1, YIL022W, CYB2, YML054C, YM9958.08C
Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: Q01852, UniProt: P00175, L-lactate dehydrogenase (cytochrome)

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Antibody , 2 types, 2 molecules HL

#5: Antibody anti-Tim44-Fab H chain


Mass: 25635.932 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Saccharomyces cerevisiae (brewer's yeast)
#6: Antibody anti-Tim44-Fab L chain


Mass: 26140.150 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Saccharomyces cerevisiae (brewer's yeast)

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Protein / Protein/peptide / Non-polymers , 3 types, 3 molecules DE

#4: Protein Protein MGR2 / Mitochondrial genome-required protein 2


Mass: 12264.158 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: MGR2, YPL098C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02889
#7: Protein/peptide Substrate


Mass: 1294.587 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Production host: Saccharomyces cerevisiae (brewer's yeast)
#8: 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 / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM

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Details

Has ligand of interestY
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: Substrate-engaged TIM23 complex from yeast / Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 1200 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPU3 5 1.6034image acquisition
8PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 160770 / Symmetry type: POINT
RefinementHighest resolution: 3.83 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046166
ELECTRON MICROSCOPYf_angle_d0.7238310
ELECTRON MICROSCOPYf_dihedral_angle_d14.2062254
ELECTRON MICROSCOPYf_chiral_restr0.048900
ELECTRON MICROSCOPYf_plane_restr0.0061065

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