|Entry||Database: EMDB / ID: EMD-2744|
|Title||Conformational Snapshots of Inducible Nitric Oxide Synthase (iNOS)|
|Map data||Single-particle reconstruction of iNOS: Group II, Conformation xii|
|Keywords||heterogeneity / 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 / cAMP-dependent protein kinase regulator activity / prostaglandin secretion / 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 / cAMP-dependent protein kinase regulator activity / prostaglandin secretion / 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 / regulation of cytokine production involved in inflammatory response / nitric-oxide synthase binding / regulation of insulin secretion / peptidyl-cysteine S-nitrosylation / blood vessel remodeling / regulation of heart contraction / response to tumor necrosis factor / nitric-oxide synthase (NADPH) / positive regulation of guanylate cyclase activity / nitric oxide mediated signal transduction / vesicle membrane / cellular response to organic cyclic compound / nitric-oxide synthase activity / arginine catabolic process / negative regulation of blood pressure / response to hormone / nitric oxide biosynthetic process / cellular response to type II interferon / response to bacterium / Hsp90 protein binding / positive regulation of interleukin-8 production / positive regulation of interleukin-6 production / peroxisome / circadian rhythm / negative regulation of protein catabolic process / regulation of blood pressure / beta-catenin binding / cellular response to xenobiotic stimulus / FMN binding / regulation of cell population proliferation / actin binding / flavin adenine dinucleotide binding / NADP binding / response to lipopolysaccharide / calmodulin binding / response to hypoxia / cellular response to lipopolysaccharide / cadherin binding / intracellular signal transduction / inflammatory response / iron ion binding / oxidoreductase activity / 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 1 superfamily / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, domain 3 superfamily / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase, oxygenase domain / Nitric oxide synthase (NOS) signature. / FAD binding domain ...Ser/Thr protein kinase, TGFB receptor / Nitric-oxide synthase, eukaryote / Nitric oxide synthase, domain 1 superfamily / Nitric oxide synthase, domain 2 superfamily / Nitric oxide synthase, domain 3 superfamily / Nitric oxide synthase, N-terminal / Nitric oxide synthase, N-terminal domain superfamily / Nitric oxide synthase, oxygenase domain / Nitric oxide synthase (NOS) signature. / FAD binding domain / Sulfite reductase [NADPH] flavoprotein alpha-component-like, FAD-binding / 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-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / FAD-binding domain, ferredoxin reductase-type / Riboflavin synthase-like beta-barrel / Flavoprotein-like superfamily
Similarity search - Domain/homology
Nitric oxide synthase, inducible
Similarity search - Component
|Biological species||Mus musculus (house mouse)|
|Method||single particle reconstruction / negative staining / Resolution: 77.0 Å|
|Authors||Campbell MG / Smith BC / Potter CS / Carragher B / Marletta MA|
|Citation||Journal: 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.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_2744.map.gz / Format: CCP4 / Size: 162.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Annotation||Single-particle reconstruction of iNOS: Group II, Conformation xii|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.36 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire : Murine Inducible Nitric Oxide Synthase
|Entire||Name: Murine Inducible Nitric Oxide Synthase|
-Supramolecule #1000: Murine Inducible Nitric Oxide Synthase
|Supramolecule||Name: Murine Inducible Nitric Oxide Synthase / type: sample / ID: 1000 / Details: Sample is highly flexible / Oligomeric state: Homodimer / Number unique components: 1|
|Molecular weight||Theoretical: 260 KDa|
-Macromolecule #1: Inducible Nitric Oxide Synthase
|Macromolecule||Name: 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 weight||Theoretical: 260 KDa|
|Recombinant expression||Organism: Escherichia coli (E. coli) / Recombinant cell: JM109 / Recombinant plasmid: pCWiNOS|
|Sequence||UniProtKB: 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
|Processing||single particle reconstruction|
|Buffer||pH: 7.5 / Details: 50 mM TEA pH 7.5, 150 mM NaCl, and 5 mM DTT|
Details: 3 microliters of sample were applied to grid. The specimen was stained twice with 2% uranyl formate, then allowed to air-dry.
|Grid||Details: Glow discharged C-flat grid with 2-micron-diameter holes overlaid by thin 1.5 nm continuous carbon|
|Vitrification||Cryogen name: NONE / Instrument: OTHER|
|Microscope||FEI TECNAI F20|
|Electron beam||Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN|
|Electron optics||Calibrated 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 stage||Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55|
|Date||Mar 6, 2013|
|Image recording||Category: 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 min||0|
Model: Tecnai F20 / Image courtesy: FEI Company
|CTF correction||Details: Each Image|
|Final two d classification||Number classes: 1|
|Final reconstruction||Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 77.0 Å / Resolution method: OTHER / Software - Name: Appion, Spider / Details: See publication. / Number images used: 180|
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