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- EMDB-71152: Catalytic domain local map of extended M. sexta soluble guanylate... -

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Basic information

Entry
Database: EMDB / ID: EMD-71152
TitleCatalytic domain local map of extended M. sexta soluble guanylate cyclase mutant beta C122S
Map dataLocal (focused) refinement map of catalytic domain of BC122S M. sexta sGC in the extended conformation with CYR715 bound.
Sample
  • Complex: Extended soluble guanylase cyclase variant bC122S with CYR715 bound
    • Protein or peptide: Soluble guanylate cyclase
KeywordsCyclase / NO / SIGNALING PROTEIN
Biological speciesManduca sexta (tobacco hornworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsThomas WC / Houghton KA
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5F32 GM149060-02 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129541-02 United States
CitationJournal: Biochemistry / Year: 2025
Title: Molecular Aspects of Soluble Guanylate Cyclase Activation and Stimulator Function.
Authors: Kimberly A Houghton / William C Thomas / Michael A Marletta /
Abstract: Soluble guanylate cyclases (sGCs) are heme-containing, gas-sensing proteins which catalyze the formation of cGMP from GTP. In humans, sGCs are highly selective sensors of nitric oxide (NO) and play a ...Soluble guanylate cyclases (sGCs) are heme-containing, gas-sensing proteins which catalyze the formation of cGMP from GTP. In humans, sGCs are highly selective sensors of nitric oxide (NO) and play a critical role in NO-based regulation of cardiovascular and pulmonary function. The physiological importance of sGC signaling has led to the development of drugs, known as stimulators and activators, which increase sGC catalytic function. Here we characterize a newly developed stimulator, CYR715, which is a particularly potent stimulator of () sGC catalytic function even in the absence of NO, increasing activity of the NO-free enzyme to 45% of full catalytic activity. CYR715 also increased the catalytic activity of sGC βC122A and βC122S variants, with a marked stimulation of the NO-free βC122S variant to 74% of maximum. High-resolution cryo-electron microscopy structures were solved for CYR715 bound to sGC βC122S revealing that CYR715 occupies the same binding site as the characterized sGC stimulators YC-1 and riociguat. Additionally, the core scaffold of CYR715 makes a binding interaction with βC78 while the flexible tail can interact with αR429 or βY7 and E361. Conformational extension of sGC following NO, YC-1, or CYR715 binding was characterized using small-angle X-ray scattering, revealing that while ligand binding results in sGC extension this extension does not directly correlate to observed activity. This suggests that not all conformational extensions of sGC result in increased catalytic activity, and that effective stimulators assist in converting extension into catalytic function.
History
DepositionJun 11, 2025-
Header (metadata) releaseNov 12, 2025-
Map releaseNov 12, 2025-
UpdateNov 12, 2025-
Current statusNov 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71152.png

Downloads & links

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Map

FileDownload / File: emd_71152.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal (focused) refinement map of catalytic domain of BC122S M. sexta sGC in the extended conformation with CYR715 bound.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 320 pix.
= 275.2 Å		0.86 Å/pix.
x 320 pix.
= 275.2 Å		0.86 Å/pix.
x 320 pix.
= 275.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.85
Minimum - Maximum-2.372563 - 3.778305
Average (Standard dev.)0.0006190469 (±0.047341485)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 275.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map A of local (focused) refinement map of...

Fileemd_71152_half_map_1.map
AnnotationHalf-map A of local (focused) refinement map of H-NOX domain of BC122S M. sexta sGC in the extended conformation with CYR715 bound.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B of local (focused) refinement map of...

Fileemd_71152_half_map_2.map
AnnotationHalf-map B of local (focused) refinement map of H-NOX domain of BC122S M. sexta sGC in the extended conformation with CYR715 bound.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Extended soluble guanylase cyclase variant bC122S with CYR715 bound

EntireName: Extended soluble guanylase cyclase variant bC122S with CYR715 bound
Components
  • Complex: Extended soluble guanylase cyclase variant bC122S with CYR715 bound
    • Protein or peptide: Soluble guanylate cyclase

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Supramolecule #1: Extended soluble guanylase cyclase variant bC122S with CYR715 bound

SupramoleculeName: Extended soluble guanylase cyclase variant bC122S with CYR715 bound
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Heterodimeric sGC molecule in the CYR715-bound, extended state. Beta-C122S mutant variant.
Source (natural)Organism: Manduca sexta (tobacco hornworm)
Molecular weightTheoretical: 147 KDa

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Macromolecule #1: Soluble guanylate cyclase

MacromoleculeName: Soluble guanylate cyclase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Manduca sexta (tobacco hornworm)
Recombinant expressionOrganism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
SequenceString: MTCPFRRASS QHQFANGGSS APKKPEFRSR TSSVHLTGPE EEDGERNTLT LKHMSEALQL LTAPSNECLH AAVTSLTKNQ SDHYHKYNCL RRLPDDVKTC RNYAYLQEIY DAVRATDSVN TKDFMAKLGE YLILTAFSHN CRLERAFKCL GTNLTEFLTT LDSVHDVLHD ...String:
MTCPFRRASS QHQFANGGSS APKKPEFRSR TSSVHLTGPE EEDGERNTLT LKHMSEALQL LTAPSNECLH AAVTSLTKNQ SDHYHKYNCL RRLPDDVKTC RNYAYLQEIY DAVRATDSVN TKDFMAKLGE YLILTAFSHN CRLERAFKCL GTNLTEFLTT LDSVHDVLHD QDTPLKDETM EYEANFVCTT SQEGKIQLHL TTESEPVAYL LVGSLKAIAK RLYDTQTDIR LRSYTNDPRR FRYEINAVPL HQKSKEDSCE LVNEAASVAT STKVTDLKIG VASFCKAFPW HFITDKRLEL VQLGAGFMRL FGTHLATHGS SLGTYFRLLR PRGVPLDFRE ILKRVNTPFM FCLKMPGSTA LAEGLEIKGQ MVFCAESDSL LFVGSPFLDG LEGLTGRGLF ISDIPLHDAT RDVILVGEQA RAQDGLRRRM DKLKNSIEEA SKAVDKEREK NVSLLHLIFP PHIAKRLWLG EKIEAKSHDD VTMLFSDIVG FTSICATATP MMVIAMLEDL YSVFDIFCEE LDVYKVETIG DAYCVASGLH RKVETHAPQI AWMALRMVET CAQHLTHEGN PIKMRIGLHT GTVLAGVVGK TMLKYCLFGH NVTLANKFES GSEPLKINVS PTTYEWLIKF PGFDMEPRDR SCLPNSFPKD IHGTCYFLHK YTHPGTDPGE PQVKHIREAL KDYGIGQANS TDVDTEEPT MYGFVNYALE LLVMKTFDEE TWETIKKKAD VAMEGSFLVR QIYEDEITYN LITAAVEVLQ IPADAILELF GKTFFEFCQD SGYDKILQVL GATPRDFLQN LDGLHDHLGT LYPGMRSPSF RSTERPEDGA LVLHYYSDRP GLEHIVIGIV KTVASKLHNT EVKVEILKTK EECDHVQFLI TETSTTGRVS APEIAEIETL SLEPKVSPAT FCRVFPFHLM FDRDLNIVQA GRTVSRLLPR VTRPGCKITD VLDTVRPHLE MTFANVLAHI NTVYVLKTKP EEMSVTDPHE EIASLRLKGQ MLYIPETDVV VFQCYPSVTN LDDLTRRGLC IADIPLHDAT RDLVLMSEQF EADYKLTQNL EVLTDKLQQT FRELELEKQK TDRLLYSVLP ISVATELRHR RPVPARRYDT VTLLFSGIVG FANYCARNSD HKGAMKIVRM LNDLYTAFDV LTDPKRNPNV YKVETVGDKY MAVSGLPEYE VAHAKHISLL ALDMMDLSQT VTVDGEPVGI TIGIHSGEVV TGVIGHRMPR YCLFGNTVNL TSRCETTGVP GTINVSEDTY NYLMREDNHD EQFELTYRGH VTMKGKAEPM QTWFLTRKIH

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC6H15NO3Triethanolamine (TEA)
25.0 mMNaClSodium chloride
5.0 mMC4H10O2S2Dithiothreitol (DTT)
5.0 mMMgCl2Magnesium chloride
0.5 mMC20H25F13O11Fluorinated Octyl Maltoside
GridModel: Quantifoil R1.2/1.3 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Cryo-EM samples were prepared by applying 3 ul to a glow-discharged Quantifoil R1.2/1.3 holey-carbon cryo-EM grid. The grid was blotted for 4 s with Whatman #1 filter paper and then plunge- ...Details: Cryo-EM samples were prepared by applying 3 ul to a glow-discharged Quantifoil R1.2/1.3 holey-carbon cryo-EM grid. The grid was blotted for 4 s with Whatman #1 filter paper and then plunge-frozen in liquid ethane with a Mark IV Vitrobot (ThermoFisher) at 4 C and 100% humidity..
DetailsSamples were prepared in a Coy anaerobic chamber at RT. Protein was thawed at 4 C, reduced with 10 mM Na2S2O4 for 15 minutes at RT, and desalted using a Zeba spin column equilibrated with Buffer, 0.22 um filtered. Protein samples were then diluted to 10 uM in equivalent buffer but with addition of 0.5 mM FOM. Additionally, 250 uM CYR715 (stimulator) and 1 mM GpCpp (non-hydrolyzable substrate-analog) were added to the sample before freezing.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8433 / Average exposure time: 0.0354 sec. / Average electron dose: 1.25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1226641
Details: Autopicking was used to produce a stack of 2,299,895 particles. Particles showing poor alignment or broken complexes were removed using a series of 2D classification steps, leaving a ...Details: Autopicking was used to produce a stack of 2,299,895 particles. Particles showing poor alignment or broken complexes were removed using a series of 2D classification steps, leaving a particle stack of 902,997 particles that resembled sGC dimers.
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: The initial model of Ms sGC BC122S was built using SwissModel and Alphafold predicted structures of BC122S based on homology with solved structures of human sGC in the extended state.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Local refinement was performed in CryoSPARC after creating a mask of the catalytic domain alone.
Number images used: 598571
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Avg.num./class: 200000 / Software - Name: cryoSPARC
Details: The stack was subjected to 2-class ab initio refinement followed by heterogeneous refinement in cryoSPARC. 598,571 particles sorted into a higher resolution 3D class.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: SwissModel / Chain - Initial model type: in silico model
Details: Structure best matched homology and shape with 8HBF.
DetailsRefinement was performed using iterative rounds of Phenix real space refinement and manual modeling in Coot. Phenix refinement was performed for separate domains of the model using the higher-resolution local maps of those domains.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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