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- EMDB-26383: Cryo-EM structure of the core human NADPH oxidase NOX2 -

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Basic information

Entry
Database: EMDB / ID: EMD-26383
TitleCryo-EM structure of the core human NADPH oxidase NOX2
Map dataSharpened EM map
Sample
  • Complex: NOX2 core - Fab 7G5 complex
    • Protein or peptide: EGFP, Cytochrome b-245 heavy chain chimera
    • Protein or peptide: Cytochrome b-245 light chain
    • Protein or peptide: 7G5 - heavy chain
    • Protein or peptide: 7G5 - light chain
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
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 / respiratory burst / WNT5:FZD7-mediated leishmania damping / 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 / bioluminescence / response to nutrient / secretory granule / generation of precursor metabolites and energy / 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 / Green fluorescent protein, GFP ...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 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / 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
EGFP / Cytochrome b-245 heavy chain / Cytochrome b-245 light chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsNoreng S / Ota N / Sun Y / Masureel M / Payandeh J / Yi T / Koerber JT
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Structure of the core human NADPH oxidase NOX2.
Authors: Sigrid Noreng / Naruhisa Ota / Yonglian Sun / Hoangdung Ho / Matthew Johnson / Christopher P Arthur / Kellen Schneider / Isabelle Lehoux / Christopher W Davies / Kyle Mortara / Kit Wong / ...Authors: Sigrid Noreng / Naruhisa Ota / Yonglian Sun / Hoangdung Ho / Matthew Johnson / Christopher P Arthur / Kellen Schneider / Isabelle Lehoux / Christopher W Davies / Kyle Mortara / Kit Wong / Dhaya Seshasayee / Matthieu Masureel / Jian Payandeh / Tangsheng Yi / James T Koerber /
Abstract: NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that ...NOX2 is the prototypical member of the NADPH oxidase NOX superfamily and produces superoxide (O), a key reactive oxygen species (ROS) that is essential in innate and adaptive immunity. Mutations that lead to deficiency in NOX2 activity correlate with increased susceptibility to bacterial and fungal infections, resulting in chronic granulomatous disease. The core of NOX2 is formed by a heterodimeric transmembrane complex composed of NOX2 (formerly gp91) and p22, but a detailed description of its structural architecture is lacking. Here, we present the structure of the human NOX2 core complex bound to a selective anti-NOX2 antibody fragment. The core complex reveals an intricate extracellular topology of NOX2, a four-transmembrane fold of the p22 subunit, and an extensive transmembrane interface which provides insights into NOX2 assembly and activation. Functional assays uncover an inhibitory activity of the 7G5 antibody mediated by internalization-dependent and internalization-independent mechanisms. Overall, our results provide insights into the NOX2 core complex architecture, disease-causing mutations, and potential avenues for selective NOX2 pharmacological modulation.
History
DepositionMar 8, 2022-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26383.png

Downloads & links

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Map

FileDownload / File: emd_26383.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened EM map
Voxel sizeX=Y=Z: 0.731 Å
Density
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.61075294 - 1.0850171
Average (Standard dev.)0.0005245311 (±0.016614323)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 280.704 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened EM map

Fileemd_26383_additional_1.map
AnnotationUnsharpened EM map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_26383_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_26383_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NOX2 core - Fab 7G5 complex

EntireName: NOX2 core - Fab 7G5 complex
Components
  • Complex: NOX2 core - Fab 7G5 complex
    • Protein or peptide: EGFP, Cytochrome b-245 heavy chain chimera
    • Protein or peptide: Cytochrome b-245 light chain
    • Protein or peptide: 7G5 - heavy chain
    • Protein or peptide: 7G5 - light chain
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE

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Supramolecule #1: NOX2 core - Fab 7G5 complex

SupramoleculeName: NOX2 core - Fab 7G5 complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: EGFP, Cytochrome b-245 heavy chain chimera

MacromoleculeName: EGFP, Cytochrome b-245 heavy chain chimera / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Oxidoreductases
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 97.669766 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KGSGMVSKGE ELFTGVVPIL VELDGDVNGH KFSVSGEGEG DATYGKLTLK FICTTGKLP VPWPTLVTTL TYGVQCFSRY PDHMKQHDFF KSAMPEGYVQ ERTIFFKDDG NYKTRAEVKF EGDTLVNRIE L KGIDFKED ...String:
MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KGSGMVSKGE ELFTGVVPIL VELDGDVNGH KFSVSGEGEG DATYGKLTLK FICTTGKLP VPWPTLVTTL TYGVQCFSRY PDHMKQHDFF KSAMPEGYVQ ERTIFFKDDG NYKTRAEVKF EGDTLVNRIE L KGIDFKED GNILGHKLEY NYNSHNVYIM ADKQKNGIKV NFKIRHNIED GSVQLADHYQ QNTPIGDGPV LLPDNHYLST QS ALSKDPN EKRDHMVLLE FVTAAGITLG MDELYKGSGE NLYFQGGSGL VPRGSGSGGN WAVNEGLSIF VILVWLGLNV FLF VWYYRV YDIPPKFFYT RKLLGSALAL ARAPAACLNF NCMLILLPVC RNLLSFLRGS SACCSTRVRR QLDRNLTFHK MVAW MIALH SAIHTIAHLF NVEWCVNARV NNSDPYSVAL SELGDRQNES YLNFARKRIK NPEGGLYLAV TLLAGITGVV ITLCL ILII TSSTKTIRRS YFEVFWYTHH LFVIFFIGLA IHGAERIVRG QTAESLAVHN ITVCEQKISE WGKIKECPIP QFAGNP PMT WKWIVGPMFL YLCERLVRFW RSQQKVVITK VVTHPFKTIE LQMKKKGFKM EVGQYIFVKC PKVSKLEWHP FTLTSAP EE DFFSIHIRIV GDWTEGLFNA CGCDKQEFQD AWKLPKIAVD GPFGTASEDV FSYEVVMLVG AGIGVTPFAS ILKSVWYK Y CNNATNLKLK KIYFYWLCRD THAFEWFADL LQLLESQMQE RNNAGFLSYN IYLTGWDESQ ANHFAVHHDE EKDVITGLK QKTLYGRPNW DNEFKTIASQ HPNTRIGVFL CGPEALAETL SKQSISNSES GPRGVHFIFN KENF

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Macromolecule #2: Cytochrome b-245 light chain

MacromoleculeName: Cytochrome b-245 light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 21.033451 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 EEEAAVAAGG PPGGPQVNPI PVTDEVV

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Macromolecule #3: 7G5 - heavy chain

MacromoleculeName: 7G5 - heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 23.599393 KDa
Recombinant expressionOrganism: Escherichia phage EcSzw-2 (virus)
SequenceString: QSLEESGGDL VKPGASLTLT CTASGIDFSG YHYMCWVRQA PGKGLEWIGC THSGDGTTYY ARWAKGRFTI SKTSSTTVTL QMTSLTAAD TATYFCARRY VFSGGYSGLD SWGPGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT ...String:
QSLEESGGDL VKPGASLTLT CTASGIDFSG YHYMCWVRQA PGKGLEWIGC THSGDGTTYY ARWAKGRFTI SKTSSTTVTL QMTSLTAAD TATYFCARRY VFSGGYSGLD SWGPGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCD

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Macromolecule #4: 7G5 - light chain

MacromoleculeName: 7G5 - light chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 23.521154 KDa
Recombinant expressionOrganism: Escherichia phage EcSzw-2 (virus)
SequenceString: ALVMTQTPSS VSAAVRGTVT IKCQASENIY SNLAWYQQKP GQPPKLLIYG ASKLASGVPS RFKGSGSGTD YTLTIRDLEA ADAATYYCQ QFYDSLNTDN AFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE ...String:
ALVMTQTPSS VSAAVRGTVT IKCQASENIY SNLAWYQQKP GQPPKLLIYG ASKLASGVPS RFKGSGSGTD YTLTIRDLEA ADAATYYCQ QFYDSLNTDN AFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS G NSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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Macromolecule #5: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 5 / Number of copies: 3 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 8 / Details: 0.03% DDM, 150 mM NaCl, 20 mM Tris
GridModel: Quantifoil / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Details: Solarus plasma cleaner (Gatan)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: OTHER / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Details: AlphaFold2 models. Uniprot P04839 - gp91 Uniprot P13498 - p22
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 70486
Initial angle assignmentType: OTHER / Details: Ab initio in cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD

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