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- EMDB-38016: Structure of human phagocyte NADPH oxidase in the resting state i... -

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Basic information

Entry
Database: EMDB / ID: EMD-38016
TitleStructure of human phagocyte NADPH oxidase in the resting state in the presence of 2 mM NADPH
Map datacomposite map after focused refinement
Sample
  • Complex: resting NOX2-p22 complex
    • Protein or peptide: Cytochrome b-245 light chain
    • Protein or peptide: Cytochrome b-245 heavy chain
    • Protein or peptide: 7D5 Fab heavy chain
    • Protein or peptide: 7D5 Fab light chain
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsNOX2 / p22 / CYBA / CYBB / TP1170 / NOX / OXIDOREDUCTASE
Function / homology
Function and homology information


smooth muscle hypertrophy / cellular response to L-glutamine / positive regulation of toll-like receptor 2 signaling pathway / hypoxia-inducible factor-1alpha signaling pathway / superoxide-generating NAD(P)H oxidase activity / positive regulation of defense response to bacterium / mucus secretion / Cross-presentation of particulate exogenous antigens (phagosomes) / perinuclear endoplasmic reticulum / cytochrome complex assembly ...smooth muscle hypertrophy / cellular response to L-glutamine / positive regulation of toll-like receptor 2 signaling pathway / hypoxia-inducible factor-1alpha signaling pathway / superoxide-generating NAD(P)H oxidase activity / positive regulation of defense response to bacterium / mucus secretion / Cross-presentation of particulate exogenous antigens (phagosomes) / perinuclear endoplasmic reticulum / cytochrome complex assembly / NADPH oxidase complex / WNT5:FZD7-mediated leishmania damping / respiratory burst / regulation of release of sequestered calcium ion into cytosol / ROS and RNS production in phagocytes / cellular response to ethanol / Oxidoreductases / superoxide anion generation / response to angiotensin / hydrogen peroxide biosynthetic process / positive regulation of mucus secretion / positive regulation of reactive oxygen species biosynthetic process / monoatomic ion channel complex / response to aldosterone / superoxide metabolic process / Detoxification of Reactive Oxygen Species / tertiary granule membrane / RHO GTPases Activate NADPH Oxidases / RAC2 GTPase cycle / RAC3 GTPase cycle / specific granule membrane / positive regulation of phagocytosis / monoatomic ion transmembrane transport / RAC1 GTPase cycle / cellular response to cadmium ion / response to nutrient / secretory granule / establishment of localization in cell / defense response / VEGFA-VEGFR2 Pathway / SH3 domain binding / phagocytic vesicle membrane / positive regulation of angiogenesis / positive regulation of interleukin-6 production / positive regulation of tumor necrosis factor production / nuclear envelope / flavin adenine dinucleotide binding / oxidoreductase activity / electron transfer activity / endosome / response to xenobiotic stimulus / inflammatory response / protein heterodimerization activity / innate immune response / neuronal cell body / dendrite / heme binding / Neutrophil degranulation / endoplasmic reticulum membrane / membrane / metal ion binding / plasma membrane
Similarity search - Function
Cytochrome b245, heavy chain / Cytochrome b558 alpha-subunit / Cytochrome Cytochrome b558 alpha-subunit / Ferric reductase, NAD binding domain / Ferric reductase NAD binding domain / FAD-binding 8 / FAD-binding domain / Ferric reductase transmembrane component-like domain / Ferric reductase like transmembrane component / FAD-binding domain, ferredoxin reductase-type ...Cytochrome b245, heavy chain / Cytochrome b558 alpha-subunit / Cytochrome Cytochrome b558 alpha-subunit / Ferric reductase, NAD binding domain / Ferric reductase NAD binding domain / FAD-binding 8 / FAD-binding domain / Ferric reductase transmembrane component-like domain / Ferric reductase like transmembrane component / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel
Similarity search - Domain/homology
Cytochrome b-245 heavy chain / Cytochrome b-245 light chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsChen L / Liu X
Funding support China, 4 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)91957201 China
National Natural Science Foundation of China (NSFC)31821091 China
National Natural Science Foundation of China (NSFC)31870833 China
Ministry of Science and Technology (MoST, China)2022YFA1303000 China
CitationJournal: Nature / Year: 2024
Title: Structure of human phagocyte NADPH oxidase in the activated state.
Authors: Xiaoyu Liu / Yiting Shi / Rui Liu / Kangcheng Song / Lei Chen /
Abstract: Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen. This process ...Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen. This process generates superoxide anions that are vital for killing pathogens. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.
History
DepositionNov 9, 2023-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38016.png

Downloads & links

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Map

FileDownload / File: emd_38016.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcomposite map after focused refinement
Voxel sizeX=Y=Z: 1.028 Å
Density
Contour LevelBy AUTHOR: 0.55
Minimum - Maximum-2.5657609 - 5.2427754
Average (Standard dev.)0.011110045 (±0.06671211)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 328.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_38016_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of consensus refinement

Fileemd_38016_half_map_1.map
Annotationhalf map of consensus refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map of consensus refinement

Fileemd_38016_half_map_2.map
Annotationhalf map of consensus refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : resting NOX2-p22 complex

EntireName: resting NOX2-p22 complex
Components
  • Complex: resting NOX2-p22 complex
    • Protein or peptide: Cytochrome b-245 light chain
    • Protein or peptide: Cytochrome b-245 heavy chain
    • Protein or peptide: 7D5 Fab heavy chain
    • Protein or peptide: 7D5 Fab light chain
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: resting NOX2-p22 complex

SupramoleculeName: resting NOX2-p22 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Cytochrome b-245 light chain

MacromoleculeName: Cytochrome b-245 light chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.005398 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGQIEWAMWA NEQALASGLI LITGGIVATA GRFTQWYFGA YSIVAGVFVC LLEYPRGKRK KGSTMERWGQ KYMTAVVKLF GPFTRNYYV RAVLHLLLSV PAGFLLATIL GTACLAIASG IYLLAAVRGE QWTPIEPKPR ERPQIGGTIK QPPSNPPPRP P AEARKKPS ...String:
MGQIEWAMWA NEQALASGLI LITGGIVATA GRFTQWYFGA YSIVAGVFVC LLEYPRGKRK KGSTMERWGQ KYMTAVVKLF GPFTRNYYV RAVLHLLLSV PAGFLLATIL GTACLAIASG IYLLAAVRGE QWTPIEPKPR ERPQIGGTIK QPPSNPPPRP P AEARKKPS EEEAAAAAGG PPGGPQVNPI PVTDEVV

UniProtKB: Cytochrome b-245 light chain

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Macromolecule #2: Cytochrome b-245 heavy chain

MacromoleculeName: Cytochrome b-245 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.412727 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGNWAVNEGL SIFVILVWLG LNVFLFVWYY RVYDIPPKFF YTRKLLGSAL ALARAPAACL NFNCMLILLP VCRNLLSFLR GSSACCSTR VRRQLDRNLT FHKMVAWMIA LHSAIHTIAH LFNVEWCVNA RVNNSDPYSV ALSELGDRQN ESYLNFARKR I KNPEGGLY ...String:
MGNWAVNEGL SIFVILVWLG LNVFLFVWYY RVYDIPPKFF YTRKLLGSAL ALARAPAACL NFNCMLILLP VCRNLLSFLR GSSACCSTR VRRQLDRNLT FHKMVAWMIA LHSAIHTIAH LFNVEWCVNA RVNNSDPYSV ALSELGDRQN ESYLNFARKR I KNPEGGLY LAVTLLAGIT GVVITLCLIL IITSSTKTIR RSYFEVFWYT HHLFVIFFIG LAIHGAERIV RGQTAESLAV HN ITVCEQK ISEWGKIKEC PIPQFAGNPP MTWKWIVGPM FLYLCERLVR FWRSQQKVVI TKVVTHPFKT IELQMKKKGF KME VGQYIF VKCPKVSKLE WHPFTLTSAP EEDFFSIHIR IVGDWTEGLF NACGCDKQEF QDAWKLPKIA VDGPFGTASE DVFS YEVVM LVGAGIGVTP FASILKSVWY KYCNNATNLK LKKIYFYWLC RDTHAFEWFA DLLQLLESQM QERNNAGFLS YNIYL TGWD ESQANHFAVH HDEEKDVITG LKQKTLYGRP NWDNEFKTIA SQHPNTRIGV FLCGPEALAE TLSKQSISNS ESGPRG VHF IFNKENF

UniProtKB: Cytochrome b-245 heavy chain

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Macromolecule #3: 7D5 Fab heavy chain

MacromoleculeName: 7D5 Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 27.603949 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYADVQLQES GPGLVKPSQS LSLTCSVTGY SLTSGSFWSW IRQFPGNKLE WMGYITYDGT SHFNPSLKS RFSITRDTSK NQFFLKLNSV TTEDTATYYC TRDPYRYDAM DFWGQGTSVT VSSAKTTPPS VYPLAPGSAA Q TNSMVTLG ...String:
MKKNIAFLLA SMFVFSIATN AYADVQLQES GPGLVKPSQS LSLTCSVTGY SLTSGSFWSW IRQFPGNKLE WMGYITYDGT SHFNPSLKS RFSITRDTSK NQFFLKLNSV TTEDTATYYC TRDPYRYDAM DFWGQGTSVT VSSAKTTPPS VYPLAPGSAA Q TNSMVTLG CLVKGYFPEP VTVTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IV PRDLEHH HHHH

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Macromolecule #4: 7D5 Fab light chain

MacromoleculeName: 7D5 Fab light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.238188 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYADIQLTQT TSSLSASLGD RVTISCRASQ DISNYLNWYQ QKPDGTVKLL IYYTSKLHSG VPSRFTASG SGRDYSLAIS NLEQEDIATY FCQQVFSLPW TFGGGTKLEI KRADAAPTVS IFPPSSEQLT SGGASVVCFL N NFYPKDIN ...String:
MKKNIAFLLA SMFVFSIATN AYADIQLTQT TSSLSASLGD RVTISCRASQ DISNYLNWYQ QKPDGTVKLL IYYTSKLHSG VPSRFTASG SGRDYSLAIS NLEQEDIATY FCQQVFSLPW TFGGGTKLEI KRADAAPTVS IFPPSSEQLT SGGASVVCFL N NFYPKDIN VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNE

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Macromolecule #6: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 6 / Number of copies: 1 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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Macromolecule #7: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 7 / Number of copies: 2 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: water

MacromoleculeName: water / type: ligand / ID: 10 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 183394
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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