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- EMDB-10099: Structure of ovine transhydrogenase in the apo state -

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Basic information

Entry
Database: EMDB / ID: EMD-10099
TitleStructure of ovine transhydrogenase in the apo state
Map dataDensity for apo-NNT. dI density from consensus full NNT refinement and dIIdIII density from focused refinement.
Sample
  • Complex: Nicotinamide nucleotide transhydrogenase in the apo state
    • Protein or peptide: Nicotinamide nucleotide transhydrogenase
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
Keywordsmitochondrial / proton-translocating / nicotinamide nucleotide transhydrogenase / MEMBRANE PROTEIN
Function / homology
Function and homology information


: / proton-translocating NAD(P)+ transhydrogenase / NADPH regeneration / proton transmembrane transport / NADP binding / mitochondrial inner membrane / identical protein binding
Similarity search - Function
NAD(P) transhydrogenase, alpha subunit / NAD(P) transhydrogenase, alpha subunit, C-terminal / 4TM region of pyridine nucleotide transhydrogenase, mitoch / NADP transhydrogenase beta-like domain / NAD(P) transhydrogenase beta subunit / Alanine dehydrogenase/NAD(P) transhydrogenase, conserved site-1 / Alanine dehydrogenase & pyridine nucleotide transhydrogenase signature 1. / Alanine dehydrogenase/pyridine nucleotide transhydrogenase, conserved site-2 / Alanine dehydrogenase & pyridine nucleotide transhydrogenase signature 2. / Alanine dehydrogenase/PNT, C-terminal domain ...NAD(P) transhydrogenase, alpha subunit / NAD(P) transhydrogenase, alpha subunit, C-terminal / 4TM region of pyridine nucleotide transhydrogenase, mitoch / NADP transhydrogenase beta-like domain / NAD(P) transhydrogenase beta subunit / Alanine dehydrogenase/NAD(P) transhydrogenase, conserved site-1 / Alanine dehydrogenase & pyridine nucleotide transhydrogenase signature 1. / Alanine dehydrogenase/pyridine nucleotide transhydrogenase, conserved site-2 / Alanine dehydrogenase & pyridine nucleotide transhydrogenase signature 2. / Alanine dehydrogenase/PNT, C-terminal domain / Alanine dehydrogenase/pyridine nucleotide transhydrogenase, N-terminal / Alanine dehydrogenase/PNT, N-terminal domain / Alanine dehydrogenase/PNT, C-terminal domain / Alanine dehydrogenase/PNT, N-terminal domain / Alanine dehydrogenase/pyridine nucleotide transhydrogenase, NAD(H)-binding domain / DHS-like NAD/FAD-binding domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
NAD(P) transhydrogenase, mitochondrial
Similarity search - Component
Biological speciesOvis aries (sheep)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsKampjut D / Sazanov LA
Funding support Austria, 1 items
OrganizationGrant numberCountry
European Union665385 Austria
CitationJournal: Nature / Year: 2019
Title: Structure and mechanism of mitochondrial proton-translocating transhydrogenase.
Authors: Domen Kampjut / Leonid A Sazanov /
Abstract: Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. ...Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyses the transfer of a hydride between NADH and NADP, coupled to the translocation of one proton across the membrane. Its main physiological function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those observed in type II diabetes. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resolution structures of intact NNT, the details of this process remain unclear. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the soluble domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochemical work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischaemia reperfusion injury, metabolic syndrome and some cancers.
History
DepositionJul 1, 2019-
Header (metadata) releaseJul 24, 2019-
Map releaseAug 28, 2019-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6s59
  • Surface level: 0.023
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10099.png

Downloads & links

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Map

FileDownload / File: emd_10099.map.gz / Format: CCP4 / Size: 14 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity for apo-NNT. dI density from consensus full NNT refinement and dIIdIII density from focused refinement.
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.023
Minimum - Maximum-0.04270431 - 0.08285316
Average (Standard dev.)0.0020778421 (±0.0064960094)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions149147167
Spacing167149147
CellA: 140.28 Å / B: 125.159996 Å / C: 123.479996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z167149147
origin x/y/z0.0000.0000.000
length x/y/z140.280125.160123.480
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ167149147
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS147149167
D min/max/mean-0.0430.0830.002

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Supplemental data

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Sample components

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Entire : Nicotinamide nucleotide transhydrogenase in the apo state

EntireName: Nicotinamide nucleotide transhydrogenase in the apo state
Components
  • Complex: Nicotinamide nucleotide transhydrogenase in the apo state
    • Protein or peptide: Nicotinamide nucleotide transhydrogenase
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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Supramolecule #1: Nicotinamide nucleotide transhydrogenase in the apo state

SupramoleculeName: Nicotinamide nucleotide transhydrogenase in the apo state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Ovis aries (sheep)
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Nicotinamide nucleotide transhydrogenase

MacromoleculeName: Nicotinamide nucleotide transhydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Ovis aries (sheep) / Organ: heart
Molecular weightTheoretical: 114.010305 KDa
SequenceString: MANLLKTVVT GCSCPFLSNL GSCKVLPGKK NFLRTFHTHR ILWCKAPVKP GIPYKQLTVG VPKEIFQNEK RVALSPAGVQ ALVKQGFNV VVESGAGEAS KFSDDHYRAA GAQIQGAKEV LASDLVVKVR APMLNPTLGI HEADLLKTSG TLISFIYPAQ N PDLLNKLS ...String:
MANLLKTVVT GCSCPFLSNL GSCKVLPGKK NFLRTFHTHR ILWCKAPVKP GIPYKQLTVG VPKEIFQNEK RVALSPAGVQ ALVKQGFNV VVESGAGEAS KFSDDHYRAA GAQIQGAKEV LASDLVVKVR APMLNPTLGI HEADLLKTSG TLISFIYPAQ N PDLLNKLS KRNTTVLAMD QVPRVTIAQG YDALSSMANI AGYKAVVLAA NHFGRFFTGQ ITAAGKVPPA KILIVGGGVA GL ASAGAAK SMGAIVRGFD TRAAALEQFK SLGAEPLEVD LKESGEGQGG YAKEMSKEFI EAEMKLFAQQ CKEVDILIST ALI PGKKAP ILFNKEMIES MKEGSVVVDL AAEAGGNFET TKPGELYVHK GITHIGYTDL PSRMATQAST LYSNNITKLL KAIS PDKDN FYFEVKDDFD FGTMGHVIRG TVVMKDGQVI FPAPTPKNIP QGAPVKQKTV AELEAEKAAT ITPFRKTMTS ASVYT AGLT GILGLGIAAP NLAFSQMVTT FGLAGIVGYH TVWGVTPALH SPLMSVTNAI SGLTAVGGLV LMGGHLYPST TSQGLA ALA TFISSVNIAG GFLVTQRMLD MFKRPTDPPE YNYLYLLPAG TFVGGYLASL YSGYNIEQIM YLGSGLCCVG ALAGLST QG TARLGNALGM IGVAGGLAAT LGGLKPCPEL LAQMSGAMAL GGTIGLTIAK RIQISDLPQL VAAFHSLVGL AAVLTCIA E YIIEYPHFAT DAAANLTKIV AYLGTYIGGV TFSGSLVAYG KLQGILKSAP LLLPGRHLLN AGLLAASVGG IIPFMMDPS FTTGITCLGS VSALSAVMGV TLTAAIGGAD MPVVITVLNS YSGWALCAEG FLLNNNLLTI VGALIGSSGA ILSYIMCVAM NRSLANVIL GGYGTTSTAG GKPMEISGTH TEINLDNAID MIREANSIII TPGYGLCAAK AQYPIADLVK MLSEQGKKVR F GIHPVAGR MPGQLNVLLA EAGVPYDIVL EMDEINHDFP DTDLVLVIGA NDTVNSAAQE DPNSIIAGMP VLEVWKSKQV IV MKRSLGV GYAAVDNPIF YKPNTAMLLG DAKKTCDALQ AKVRESYQK

UniProtKB: NAD(P) transhydrogenase, mitochondrial

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Macromolecule #2: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 2 / Number of copies: 8 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHEPES
50.0 mMSodium chlorideNaCl
1.0 mMEDTA
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 4-6 s blotting time.
DetailsSample was purified in LMNG (final conc. 0.06%) and contained 0.05% CHAPS as a secondary detergent.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 760 / Average exposure time: 3.25 sec. / Average electron dose: 90.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 500001
Startup modelType of model: OTHER / Details: Reconstruction from a previous dataset
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 67908
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6qti


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 137 / Target criteria: Correlation coefficient, EMRinger score
Output model

PDB-6s59:
Structure of ovine transhydrogenase in the apo state

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