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- EMDB-5940: 3D single particle reconstruction of the mammalian neuronal nitri... -

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Basic information

Entry
Database: EMDB / ID: EMD-5940
Title3D single particle reconstruction of the mammalian neuronal nitric oxide synthase bound to calmodulin
Map dataNegative-stain EM reconstruction of neuronal nitric oxide synthase bound to calmodulin
Sample
  • Sample: Rat neuronal nitric oxide synthase dimer bound to calmodulin
  • Protein or peptide: neuronal nitric oxide synthaseNitric oxide synthase
  • Protein or peptide: calmodulin
KeywordsNitric oxide synthase / calmodulin / electron transfer
Biological speciesRattus norvegicus (Norway rat) / unidentified (others)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsYokom AL / Morishima Y / Lau M / Su M / Glukhova A / Osawa Y / Southworth DR
CitationJournal: J Biol Chem / Year: 2014
Title: Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain.
Authors: Adam L Yokom / Yoshihiro Morishima / Miranda Lau / Min Su / Alisa Glukhova / Yoichi Osawa / Daniel R Southworth /
Abstract: Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and ...Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca(2+)-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis.
History
DepositionMar 31, 2014-
Header (metadata) releaseMay 28, 2014-
Map releaseMay 28, 2014-
UpdateJul 16, 2014-
Current statusJul 16, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.149
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.149
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5940.png

Downloads & links

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Map

FileDownload / File: emd_5940.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative-stain EM reconstruction of neuronal nitric oxide synthase bound to calmodulin
Voxel sizeX=Y=Z: 2.16 Å
Density
Contour LevelBy AUTHOR: 0.149 / Movie #1: 0.149
Minimum - Maximum-0.03602264 - 0.2222518
Average (Standard dev.)0.0095154 (±0.03729618)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.0360.2220.010

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Supplemental data

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Sample components

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Entire : Rat neuronal nitric oxide synthase dimer bound to calmodulin

EntireName: Rat neuronal nitric oxide synthase dimer bound to calmodulin
Components
  • Sample: Rat neuronal nitric oxide synthase dimer bound to calmodulin
  • Protein or peptide: neuronal nitric oxide synthaseNitric oxide synthase
  • Protein or peptide: calmodulin

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Supramolecule #1000: Rat neuronal nitric oxide synthase dimer bound to calmodulin

SupramoleculeName: Rat neuronal nitric oxide synthase dimer bound to calmodulin
type: sample / ID: 1000
Details: The sample was crosslinked with 0.01% glutaraldehyde prior to imaging using negative staining.
Oligomeric state: Homodimer of nNOS bound to two CaM proteins
Number unique components: 2
Molecular weightExperimental: 360 KDa / Theoretical: 360 KDa / Method: Multi-angle light scattering

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Macromolecule #1: neuronal nitric oxide synthase

MacromoleculeName: neuronal nitric oxide synthase / type: protein_or_peptide / ID: 1 / Name.synonym: nNOS / Number of copies: 2 / Oligomeric state: Homodimer bound to calmodulin / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: rat
Molecular weightExperimental: 360 KDa / Theoretical: 360 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9

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Macromolecule #2: calmodulin

MacromoleculeName: calmodulin / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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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

Concentration0.02 mg/mL
StainingType: NEGATIVE / Details: Protein was stained with 0.75% uranyl formate.
GridDetails: 400 mesh copper grids with a thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateJul 10, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 112 / Average electron dose: 30 e/Å2

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: EMAN2, RELION, Spider / Number images used: 12323
DetailsParticles were selected manually and 3D refinement was performed using RELION.

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Atomic model buiding 1

Initial modelPDB ID:

1rs9

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

1tll

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

3hr4

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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