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- EMDB-2730: Conformational Snapshots of Inducible Nitric Oxide Synthase (iNOS) -

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Basic information

Entry
Database: EMDB / ID: EMD-2730
TitleConformational Snapshots of Inducible Nitric Oxide Synthase (iNOS)
Map dataSingle-particle reconstruction of iNOS: Group I, Conformation xiii
Sample
  • Sample: Murine Inducible Nitric Oxide Synthase
  • Protein or peptide: Inducible Nitric Oxide SynthaseNitric oxide synthase
Keywordsheterogeneity / random conical tilt / nitric oxide synthase / calmodulin / heme / electron transfer / flavin
Function / homology
Function and homology information


Nitric oxide stimulates guanylate cyclase / ROS and RNS production in phagocytes / G protein-coupled receptor signaling pathway coupled to cGMP nucleotide second messenger / Peroxisomal protein import / prostaglandin secretion / cAMP-dependent protein kinase regulator activity / positive regulation of killing of cells of another organism / tetrahydrobiopterin binding / arginine binding / cortical cytoskeleton ...Nitric oxide stimulates guanylate cyclase / ROS and RNS production in phagocytes / G protein-coupled receptor signaling pathway coupled to cGMP nucleotide second messenger / Peroxisomal protein import / prostaglandin secretion / cAMP-dependent protein kinase regulator activity / positive regulation of killing of cells of another organism / tetrahydrobiopterin binding / arginine binding / cortical cytoskeleton / superoxide metabolic process / cellular response to cytokine stimulus / cGMP-mediated signaling / nitric-oxide synthase binding / regulation of cytokine production involved in inflammatory response / peptidyl-cysteine S-nitrosylation / regulation of insulin secretion / blood vessel remodeling / regulation of heart contraction / nitric-oxide synthase (NADPH) / positive regulation of guanylate cyclase activity / response to tumor necrosis factor / nitric oxide mediated signal transduction / vesicle membrane / nitric-oxide synthase activity / cellular response to organic cyclic compound / arginine catabolic process / negative regulation of blood pressure / response to hormone / nitric oxide biosynthetic process / cellular response to interferon-gamma / response to bacterium / positive regulation of interleukin-8 production / positive regulation of interleukin-6 production / Hsp90 protein binding / peroxisome / circadian rhythm / cellular response to xenobiotic stimulus / negative regulation of protein catabolic process / regulation of blood pressure / beta-catenin binding / FMN binding / actin binding / regulation of cell population proliferation / flavin adenine dinucleotide binding / NADP binding / response to lipopolysaccharide / calmodulin binding / response to hypoxia / cellular response to lipopolysaccharide / cadherin binding / intracellular signal transduction / oxidoreductase activity / inflammatory response / iron ion binding / positive regulation of apoptotic process / defense response to bacterium / negative regulation of gene expression / heme binding / protein kinase binding / perinuclear region of cytoplasm / signal transduction / protein homodimerization activity / extracellular space / metal ion binding / identical protein binding / plasma membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
Ser/Thr protein kinase, TGFB receptor / Nitric-oxide synthase, eukaryote / Nitric oxide synthase, domain 3 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase (NOS) signature. / Nitric oxide synthase, oxygenase domain / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding ...Ser/Thr protein kinase, TGFB receptor / Nitric-oxide synthase, eukaryote / Nitric oxide synthase, domain 3 superfamily / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase (NOS) signature. / Nitric oxide synthase, oxygenase domain / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / FAD binding domain / NADPH-cytochrome p450 reductase, FAD-binding, alpha-helical domain superfamily / Flavodoxin-like / Flavoprotein pyridine nucleotide cytochrome reductase / Flavodoxin / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / Flavodoxin-like domain profile. / Flavodoxin/nitric oxide synthase / Ferredoxin reductase-type FAD binding domain profile. / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Riboflavin synthase-like beta-barrel / Flavoprotein-like superfamily
Similarity search - Domain/homology
Nitric oxide synthase, inducible
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 66.0 Å
AuthorsCampbell MG / Smith BC / Potter CS / Carragher B / Marletta MA
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Molecular architecture of mammalian nitric oxide synthases.
Authors: Melody G Campbell / Brian C Smith / Clinton S Potter / Bridget Carragher / Michael A Marletta /
Abstract: NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate ...NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoforms--inducible, endothelial, and neuronal NOS--that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.
History
DepositionJul 29, 2014-
Header (metadata) releaseSep 3, 2014-
Map releaseSep 10, 2014-
UpdateSep 17, 2014-
Current statusSep 17, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.818
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.818
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2730.map.gz / Format: CCP4 / Size: 162.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle reconstruction of iNOS: Group I, Conformation xiii
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 352 pix.
= 478.72 Å
1.36 Å/pix.
x 352 pix.
= 478.72 Å
1.36 Å/pix.
x 352 pix.
= 478.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.818 / Movie #1: 0.818
Minimum - Maximum-1.06709838 - 2.65295792
Average (Standard dev.)-0.00547693 (±0.1340338)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-176-176-176
Dimensions352352352
Spacing352352352
CellA=B=C: 478.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z478.720478.720478.720
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ442626
MAP C/R/S123
start NC/NR/NS-176-176-176
NC/NR/NS352352352
D min/max/mean-1.0672.653-0.005

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Supplemental data

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Sample components

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Entire : Murine Inducible Nitric Oxide Synthase

EntireName: Murine Inducible Nitric Oxide Synthase
Components
  • Sample: Murine Inducible Nitric Oxide Synthase
  • Protein or peptide: Inducible Nitric Oxide SynthaseNitric oxide synthase

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Supramolecule #1000: Murine Inducible Nitric Oxide Synthase

SupramoleculeName: Murine Inducible Nitric Oxide Synthase / type: sample / ID: 1000 / Details: Sample is highly flexible / Oligomeric state: Homodimer / Number unique components: 1
Molecular weightTheoretical: 260 KDa

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Macromolecule #1: Inducible Nitric Oxide Synthase

MacromoleculeName: Inducible Nitric Oxide Synthase / type: protein_or_peptide / ID: 1 / Name.synonym: iNOS / Number of copies: 1 / Oligomeric state: Homodimer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse / Cell: Macrophages
Molecular weightTheoretical: 260 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant cell: JM109 / Recombinant plasmid: pCWiNOS
SequenceUniProtKB: Nitric oxide synthase, inducible
GO: nitric oxide biosynthetic process, FMN binding, NADP binding, calmodulin binding, flavin adenine dinucleotide binding, heme binding, iron ion binding, nitric-oxide synthase activity
InterPro: Ser/Thr protein kinase, TGFB receptor

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 50 mM TEA pH 7.5, 150 mM NaCl, and 5 mM DTT
StainingType: NEGATIVE
Details: 3 microliters of sample were applied to grid. The specimen was stained twice with 2% uranyl formate, then allowed to air-dry.
GridDetails: Glow discharged C-flat grid with 2-micron-diameter holes overlaid by thin 1.5 nm continuous carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 114705 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55
DateMar 6, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 2226 / Average electron dose: 37 e/Å2
Details: Each area is imaged twice: once at 55 degrees and again with no tilt.
Tilt angle min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each Image
Final two d classificationNumber classes: 1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 66.0 Å / Resolution method: OTHER / Software - Name: Appion, Spider / Details: See publication. / Number images used: 226
DetailsSee publication.

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