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- EMDB-4635: Structure of ovine transhydrogenase in the presence of NADP+ in a... -

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Basic information

Entry
Database: EMDB / ID: EMD-4635
TitleStructure of ovine transhydrogenase in the presence of NADP+ in a "double face-down" conformation
Map dataCombined map from focused refinement of two domains separately: (dII plus dIII) and (dI).
Sample
  • Complex: Nicotinamide nucleotide transhydrogenase in the presence of NADP+
    • Protein or peptide: Nicotinamide nucleotide transhydrogenase
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: DODECANE
Function / homology
Function and homology information


NAD(P)+ transhydrogenase activity / proton-translocating NAD(P)+ transhydrogenase / proton transmembrane transport / 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) / Sheep (sheep)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 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
DepositionFeb 25, 2019-
Header (metadata) releaseJul 24, 2019-
Map releaseAug 28, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qti
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4635.png

Downloads & links

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Map

FileDownload / File: emd_4635.map.gz / Format: CCP4 / Size: 6.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCombined map from focused refinement of two domains separately: (dII plus dIII) and (dI).
Voxel sizeX=Y=Z: 1.065 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.096151374 - 0.19715394
Average (Standard dev.)0.0024106095 (±0.014039831)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions119117128
Spacing128119117
CellA: 136.32 Å / B: 126.73501 Å / C: 124.605 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0651.0651.065
M x/y/z128119117
origin x/y/z0.0000.0000.000
length x/y/z136.320126.735124.605
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128119117
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS117119128
D min/max/mean-0.0960.1970.002

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Supplemental data

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Sample components

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Entire : Nicotinamide nucleotide transhydrogenase in the presence of NADP+

EntireName: Nicotinamide nucleotide transhydrogenase in the presence of NADP+
Components
  • Complex: Nicotinamide nucleotide transhydrogenase in the presence of NADP+
    • Protein or peptide: Nicotinamide nucleotide transhydrogenase
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: NICOTINAMIDE-ADENINE-DINUCLEOTIDE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: DODECANE

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Supramolecule #1: Nicotinamide nucleotide transhydrogenase in the presence of NADP+

SupramoleculeName: Nicotinamide nucleotide transhydrogenase in the presence of NADP+
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: Sheep (sheep)
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

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Macromolecule #2: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 2 / Number of copies: 2 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate

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Macromolecule #3: NICOTINAMIDE-ADENINE-DINUCLEOTIDE

MacromoleculeName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 2 / Formula: NAD
Molecular weightTheoretical: 663.425 Da
Chemical component information

ChemComp-NAD:
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD*YM / Nicotinamide adenine dinucleotide

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Macromolecule #4: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 4 / Number of copies: 12 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

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

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Macromolecule #5: DODECANE

MacromoleculeName: DODECANE / type: ligand / ID: 5 / Number of copies: 3 / Formula: D12
Molecular weightTheoretical: 170.335 Da
Chemical component information

ChemComp-D12:
DODECANE / Dodecane

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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 chlorideNaClSodium chloride
1.0 mMEDTAEthylenediaminetetraacetic acid
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0007 kPa
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 as well as 5 mM NADP+.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 2160 / Average exposure time: 10.0 sec. / Average electron dose: 72.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1076677
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.5)
Startup modelType of model: OTHER / Details: Reconstruction from a previous dataset
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 98294
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1d4o

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 75.7 / Target criteria: Correlation coefficient, EMRinger score
Output model

PDB-6qti:
Structure of ovine transhydrogenase in the presence of NADP+ in a "double face-down" conformation

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