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- PDB-9giw: Structure of the human mitochondrial pyruvate carrier inhibited b... -

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Basic information

Entry
Database: PDB / ID: 9giw
TitleStructure of the human mitochondrial pyruvate carrier inhibited by zaprinast
Components
  • Mitochondrial pyruvate carrier 1-like protein
  • Mitochondrial pyruvate carrier 2
  • Nanobody,Maltose/maltodextrin-binding periplasmic protein
KeywordsMEMBRANE PROTEIN / transporter SLC54 pyruvate UK5099-derivative
Function / homology
Function and homology information


pyruvate import into mitochondria / inner mitochondrial membrane protein complex / pyruvate transmembrane transporter activity / pyruvate decarboxylation to acetyl-CoA / Pyruvate metabolism / positive regulation of insulin secretion involved in cellular response to glucose stimulus / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport ...pyruvate import into mitochondria / inner mitochondrial membrane protein complex / pyruvate transmembrane transporter activity / pyruvate decarboxylation to acetyl-CoA / Pyruvate metabolism / positive regulation of insulin secretion involved in cellular response to glucose stimulus / carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / outer membrane-bounded periplasmic space / mitochondrial inner membrane / mitochondrion / identical protein binding / nucleus
Similarity search - Function
Mitochondrial pyruvate carrier / Mitochondrial pyruvate carriers / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
: / Mitochondrial pyruvate carrier 2 / Maltose/maltodextrin-binding periplasmic protein / Mitochondrial pyruvate carrier 1-like protein
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.92 Å
AuthorsSichrovsky, M. / Lacabanne, D. / Ruprecht, J.J. / Rana, J.J. / Stanik, K. / Dionysopoulou, M. / Sowton, A.P. / King, M.S. / Jones, S. / Cooper, L. ...Sichrovsky, M. / Lacabanne, D. / Ruprecht, J.J. / Rana, J.J. / Stanik, K. / Dionysopoulou, M. / Sowton, A.P. / King, M.S. / Jones, S. / Cooper, L. / Hardwick, S.W. / Paris, G. / Chirgadze, D.Y. / Ding, S. / Fearnley, I.M. / Palmer, S. / Pardon, E. / Steyaert, J. / Leone, V. / Forrest, L.R. / Tavoulari, S. / Kunji, E.R.S.
Funding support United Kingdom, European Union, Belgium, United States, 4items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UU_00028/2 United Kingdom
European Union (EU)Instruct-ERIC part of the European Strategy Forum on Research infrastructures (ESFRI) (PID: 22183)European Union
Research Foundation - Flanders (FWO) Belgium
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS003139 United States
CitationJournal: Sci Adv / Year: 2025
Title: Molecular basis of pyruvate transport and inhibition of the human mitochondrial pyruvate carrier.
Authors: Maximilian Sichrovsky / Denis Lacabanne / Jonathan J Ruprecht / Jessica J Rana / Klaudia Stanik / Mariangela Dionysopoulou / Alice P Sowton / Martin S King / Scott A Jones / Lee Cooper / ...Authors: Maximilian Sichrovsky / Denis Lacabanne / Jonathan J Ruprecht / Jessica J Rana / Klaudia Stanik / Mariangela Dionysopoulou / Alice P Sowton / Martin S King / Scott A Jones / Lee Cooper / Steven W Hardwick / Giulia Paris / Dimitri Y Chirgadze / Shujing Ding / Ian M Fearnley / Shane M Palmer / Els Pardon / Jan Steyaert / Vanessa Leone / Lucy R Forrest / Sotiria Tavoulari / Edmund R S Kunji /
Abstract: The mitochondrial pyruvate carrier transports pyruvate, produced by glycolysis from sugar molecules, into the mitochondrial matrix, as a crucial transport step in eukaryotic energy metabolism. The ...The mitochondrial pyruvate carrier transports pyruvate, produced by glycolysis from sugar molecules, into the mitochondrial matrix, as a crucial transport step in eukaryotic energy metabolism. The carrier is a drug target for the treatment of cancers, diabetes mellitus, neurodegeneration, and metabolic dysfunction-associated steatotic liver disease. We have solved the structure of the human MPC1L/MPC2 heterodimer in the inward- and outward-open states by cryo-electron microscopy, revealing its alternating access rocker-switch mechanism. The carrier has a central binding site for pyruvate, which contains an essential lysine and histidine residue, important for its ΔpH-dependent transport mechanism. We have also determined the binding poses of three chemically distinct inhibitor classes, which exploit the same binding site in the outward-open state by mimicking pyruvate interactions and by using aromatic stacking interactions.
History
DepositionAug 19, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 7, 2025Provider: repository / Type: Initial release
Revision 1.0May 7, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 7, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 7, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 7, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 7, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 7, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 7, 2025Data content type: Primary map / Data content type: Primary map / Provider: 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 pyruvate carrier 1-like protein
B: Mitochondrial pyruvate carrier 2
C: Nanobody,Maltose/maltodextrin-binding periplasmic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,8284
Polymers86,5563
Non-polymers2711
Water00
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 Mitochondrial pyruvate carrier 1-like protein


Mass: 15155.544 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MPC1L / Plasmid: pBEVY-GU / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): W303-1B / References: UniProt: P0DKB6
#2: Protein Mitochondrial pyruvate carrier 2 / Brain protein 44


Mass: 15093.728 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: MPC2 expressed with a TEV-cleavable His tag. Sequence is of purified protein post-TEV cleavage
Source: (gene. exp.) Homo sapiens (human) / Gene: MPC2, BRP44 / Plasmid: pBEVY-GU / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): W303-1B / References: UniProt: O95563
#3: Antibody Nanobody,Maltose/maltodextrin-binding periplasmic protein / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP


Mass: 56307.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Pro-macrobody is a MBP-nanobody-MBP fusion protein with a 3C protease-cleavable tag between the N-terminal MBP and nanobody. Sequence shows final purified protein post cleavage.,Pro- ...Details: Pro-macrobody is a MBP-nanobody-MBP fusion protein with a 3C protease-cleavable tag between the N-terminal MBP and nanobody. Sequence shows final purified protein post cleavage.,Pro-macrobody is a MBP-nanobody-MBP fusion protein with a 3C protease-cleavable tag between the N-terminal MBP and nanobody. Sequence shows final purified protein post cleavage.
Source: (gene. exp.) synthetic construct (others) / Gene: malE, Z5632, ECs5017 / Plasmid: pBXNPHM3 / Production host: Escherichia coli MC1061 (bacteria) / References: UniProt: P0AEY0
#4: Chemical ChemComp-A1IL3 / 5-(2-propoxyphenyl)-3,4-dihydro-[1,2,3]triazolo[4,5-d]pyrimidin-7-one


Mass: 271.275 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C13H13N5O2 / Feature type: SUBJECT OF INVESTIGATION
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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of human mitochondrial pyruvate carrier (MPC1L/MPC2) inhibited by zaprinast with a pro-macrobodyCOMPLEX#1-#30MULTIPLE SOURCES
2Mitochondrial pyruvate carrier (MPC1L/MPC2)COMPLEX#1-#21RECOMBINANT
3Pro-macrobody (fusion of a nanobody with Maltose-binding protein)COMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
22Saccharomyces cerevisiae (brewer's yeast)4932W303-1BpBEVY-GU
33Escherichia coli MC1061 (bacteria)1211845
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMBis-Tris pH7.01
2150 mMSodium chlorideNaCl1
30.1 mg/mlTetraoleoyl cardiolipin1
40.1 %LMNG detergent1
52 mMD-maltose1
60.035 %Fluorinated octyl maltoside1
7250 uMZaprinast1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 10 mA / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / 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 magnification: 165000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.39 sec. / Electron dose: 53.44 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
2EPUimage acquisition
4WarpCTF correction
7Coot0.9.8.92model fitting
8ISOLDEmodel fitting
9UCSF ChimeraX1.7.1model fitting
11cryoSPARC3.3.2initial Euler assignment
12cryoSPARC3.3.2final Euler assignment
13cryoSPARC3.3.2classification
14cryoSPARC3.3.23D reconstruction
15REFMAC5.8.0419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1739679
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 295345 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingDetails: PDB deposition D_1292140991 / Source name: Other / Type: other
RefinementResolution: 3.92→3.92 Å / Cor.coef. Fo:Fc: 0.961 / SU B: 38.635 / SU ML: 0.518 / ESU R: 0.694
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.35289 --
obs0.35289 21107 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 145.575 Å2
Refinement stepCycle: 1 / Total: 2661
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.0122738
ELECTRON MICROSCOPYr_bond_other_d00.0162549
ELECTRON MICROSCOPYr_angle_refined_deg1.381.8133714
ELECTRON MICROSCOPYr_angle_other_deg0.5051.7365829
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.2775330
ELECTRON MICROSCOPYr_dihedral_angle_2_deg4.512521
ELECTRON MICROSCOPYr_dihedral_angle_3_deg11.0610426
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0740.2389
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.023321
ELECTRON MICROSCOPYr_gen_planes_other0.0010.02714
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it3.70214.4611329
ELECTRON MICROSCOPYr_mcbond_other3.69914.4611329
ELECTRON MICROSCOPYr_mcangle_it6.81126.0961656
ELECTRON MICROSCOPYr_mcangle_other6.80926.0921657
ELECTRON MICROSCOPYr_scbond_it2.54614.8691409
ELECTRON MICROSCOPYr_scbond_other2.54514.8671410
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other5.14227.2212059
ELECTRON MICROSCOPYr_long_range_B_refined17.264169.1210414
ELECTRON MICROSCOPYr_long_range_B_other17.265169.1210412
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.9→4.001 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.312 1519 -
obs--100 %

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