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- EMDB-5863: Single-particle reconstruction of conformation III of ligand-free sGC -

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Basic information

Entry
Database: EMDB / ID: EMD-5863
TitleSingle-particle reconstruction of conformation III of ligand-free sGC
Map dataSingle-particle reconstruction of conformation III of ligand-free sGC
Sample
  • Sample: Soluble Guanylate Cyclase, ligand-free
  • Protein or peptide: Soluble Guanylate Cyclase
Keywordssoluble guanylate cyclase / conformational heterogeneity
Biological speciesRattus norvegicus (Norway rat)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsCampbell MG / Underbakke ES / Potter CS / Carragher B / Marletta MA
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase.
Authors: Melody G Campbell / Eric S Underbakke / Clinton S Potter / Bridget Carragher / Michael A Marletta /
Abstract: Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. The NO-signaling pathways mediate diverse physiological ...Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. The NO-signaling pathways mediate diverse physiological processes, including vasodilation, neurotransmission, and myocardial functions. sGC is a heterodimer assembled from two homologous subunits, each comprised of four domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length sGC. We used single-particle electron microscopy to obtain the structure of the complete sGC heterodimer and determine its higher-order domain architecture. Overall, the protein is formed of two rigid modules: the catalytic dimer and the clustered Per/Art/Sim and heme-NO/O2-binding domains, connected by a parallel coiled coil at two hinge points. The quaternary assembly demonstrates a very high degree of flexibility. We captured hundreds of individual conformational snapshots of free sGC, NO-bound sGC, and guanosine-5'-[(α,β)-methylene]triphosphate-bound sGC. The molecular architecture and pronounced flexibility observed provides a significant step forward in understanding the mechanism of NO signaling.
History
DepositionJan 17, 2014-
Header (metadata) releaseMar 19, 2014-
Map releaseMar 19, 2014-
UpdateMar 19, 2014-
Current statusMar 19, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.802
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.802
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5863.jpg

Downloads & links

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Map

FileDownload / File: emd_5863.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle reconstruction of conformation III of ligand-free sGC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 224 pix.
= 237.44 Å		1.06 Å/pix.
x 224 pix.
= 237.44 Å		1.06 Å/pix.
x 224 pix.
= 237.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.802 / Movie #1: 0.802
Minimum - Maximum-1.26284504 - 3.24775505
Average (Standard dev.)0.00721366 (±0.26069891)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-112-112-112
Dimensions224224224
Spacing224224224
CellA=B=C: 237.43999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z237.440237.440237.440
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-112-112-112
NC/NR/NS224224224
D min/max/mean-1.2633.2480.007

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Supplemental data

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Sample components

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Entire : Soluble Guanylate Cyclase, ligand-free

EntireName: Soluble Guanylate Cyclase, ligand-free
Components
  • Sample: Soluble Guanylate Cyclase, ligand-free
  • Protein or peptide: Soluble Guanylate Cyclase

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Supramolecule #1000: Soluble Guanylate Cyclase, ligand-free

SupramoleculeName: Soluble Guanylate Cyclase, ligand-free / type: sample / ID: 1000 / Oligomeric state: Heterodimer / Number unique components: 1
Molecular weightExperimental: 150 KDa / Theoretical: 150 KDa

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Macromolecule #1: Soluble Guanylate Cyclase

MacromoleculeName: Soluble Guanylate Cyclase / type: protein_or_peptide / ID: 1 / Name.synonym: sGC / Number of copies: 1 / Oligomeric state: Heterodimer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Norwegian rat
Molecular weightExperimental: 150 KDa / Theoretical: 150 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9
Recombinant plasmid: pFastBac1/sGCALPHA1 and pFastBac1/sGCBETA1

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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, 150 mM NaCl, 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
TemperatureAverage: 298 K
DateJan 26, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 2204 / Average electron dose: 35 e/Å2
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -55
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsSee publication
CTF correctionDetails: Each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: SPIDER / Number images used: 288
Final two d classificationNumber classes: 1

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