|Entry||Database: EMDB / ID: EMD-5876|
|Title||Single-particle reconstruction of conformation XVI of ligand-free sGC|
|Sample||Soluble Guanylate Cyclase, ligand-freeSoluble guanylyl cyclase|
|Keywords||soluble guanylate cyclase / conformational heterogeneity|
|Biological species||Rattus norvegicus (Norway rat)|
|Method||single particle reconstruction / negative staining / Resolution: 30 Å|
|Authors||Campbell MG / Underbakke ES / Potter CS / Carragher B / Marletta MA|
|Citation||Journal: 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.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_5876.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.06 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Soluble Guanylate Cyclase, ligand-free
|Entire||Name: Soluble Guanylate Cyclase, ligand-freeSoluble guanylyl cyclase|
Number of Components: 1 / Oligomeric State: Heterodimer
|Mass||Theoretical: 150 kDa / Experimental: 150 kDa|
-Component #1: protein, Soluble Guanylate Cyclase
|Protein||Name: Soluble Guanylate CyclaseSoluble guanylyl cyclase / a.k.a: sGC / Oligomeric Details: Heterodimer / Recombinant expression: Yes / Number of Copies: 1|
|Mass||Theoretical: 150 kDa / Experimental: 150 kDa|
|Source||Species: Rattus norvegicus (Norway rat)|
|Source (engineered)||Expression System: Spodoptera frugiperda (fall armyworm) / Vector: pFastBac1/sGCALPHA1 and pFastBac1/sGCBETA1 / Cell of expression system: Sf9|
|Specimen||Specimen State: Particle / Method: negative staining|
|Sample solution||Buffer solution: 50 mM TEA, 150 mM NaCl, 5 mM DTT / pH: 7.5|
|Support film||Glow discharged C-flat grid with 2-micron-diameter holes overlaid by thin 1.5 nm continuous carbon|
|Staining||3 microliters of sample were applied to grid. The specimen was stained twice with 2% uranyl formate, then allowed to air-dry.|
|Vitrification||Cryogen Name: NONE|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20 / Date: Jan 26, 2013|
|Electron gun||Electron Source: FIELD EMISSION GUN / Accelerating Voltage: 200 kV / Electron Dose: 35 e/Å2 / Illumination Mode: FLOOD BEAM|
|Lens||Magnification: 80000 X (nominal) / Cs: 2 mm / Imaging Mode: BRIGHT FIELD / Defocus: 1200 - 2200 nm|
|Specimen Holder||Model: SIDE ENTRY, EUCENTRIC / Tilt Angle: -55 - 0 ° / Temperature: 298|
|Camera||Detector: TVIPS TEMCAM-F416 (4k x 4k)|
|Image acquisition||Number of Digital Images: 2204|
|Processing||Method: single particle reconstruction / Number of Class Averages: 1 / Applied Symmetry: C1 (asymmetric) / Number of Projections: 280 / Details: See publication|
|3D reconstruction||Algorithm: RCT / Software: SPIDER / CTF correction: Each micrograph / Resolution: 30 Å / Resolution Method: FSC 0.5, semi-independent|
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