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- EMDB-71036: Catalytic domain local map of ligand-free M. sexta soluble guanyl... -

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Basic information

Entry
Database: EMDB / ID: EMD-71036
TitleCatalytic domain local map of ligand-free M. sexta soluble guanylate cyclase mutant beta C122S
Map dataLocal (focused) map of catalytic domain of ligand-free, compact M. sexta sGC bC122S
Sample
  • Complex: Ligand-free Manduca sexta soluble guanylase cyclase variant
    • Protein or peptide: M. sexta soluble guanylate cyclase mutant variant beta C122S
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 4, 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_71036.png

Downloads & links

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Map

FileDownload / File: emd_71036.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal (focused) map of catalytic domain of ligand-free, compact M. sexta sGC bC122S
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 320 pix.
= 301.44 Å		0.94 Å/pix.
x 320 pix.
= 301.44 Å		0.94 Å/pix.
x 320 pix.
= 301.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.942 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.15
Minimum - Maximum-2.9840147 - 5.6267557
Average (Standard dev.)0.0000005669503 (±0.044901)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 301.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map A of local map of catalytic domain...

Fileemd_71036_half_map_1.map
AnnotationHalf-map A of local map of catalytic domain of ligand-free, compact M. sexta sGC bC122S
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B of local map of catalytic domain...

Fileemd_71036_half_map_2.map
AnnotationHalf-map B of local map of catalytic domain of ligand-free, compact M. sexta sGC bC122S
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ligand-free Manduca sexta soluble guanylase cyclase variant

EntireName: Ligand-free Manduca sexta soluble guanylase cyclase variant
Components
  • Complex: Ligand-free Manduca sexta soluble guanylase cyclase variant
    • Protein or peptide: M. sexta soluble guanylate cyclase mutant variant beta C122S

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Supramolecule #1: Ligand-free Manduca sexta soluble guanylase cyclase variant

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

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Macromolecule #1: M. sexta soluble guanylate cyclase mutant variant beta C122S

MacromoleculeName: M. sexta soluble guanylate cyclase mutant variant beta C122S
type: protein_or_peptide / ID: 1
Details: C-terminal domain focused refinement of a heterodimeric complex
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.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON I (4k x 4k) / Number grids imaged: 1 / Number real images: 11872 / Average exposure time: 0.2195 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 1,226,641 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 1,226,641 particles. Particles showing poor alignment or broken complexes were removed using a series of 2D classification steps, leaving a particle stack of 368,563 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 ModelAngelo in known sequence mode and with a sharpened global 3.3 A map as a starting point.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Separate masks were created for each domain, and local refinement as implemented in cryoSPARC was used to improve the resolution of the separate catalytic and H-NOX domains of the global map. ...Details: Separate masks were created for each domain, and local refinement as implemented in cryoSPARC was used to improve the resolution of the separate catalytic and H-NOX domains of the global map. The subsequent local refinement resulted in 3.0 A and 3.4 A maps for the H-NOX and catalytic domains respectively. Both have improved density for outer regions of the map and improved local resolution.
Number images used: 264227
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: After 2D classification for intact dimers, the particle stack was subjected to 2-class ab initio refinement followed by heterogeneous refinement in cryoSPARC. 264,227 particles sorted into a ...Details: After 2D classification for intact dimers, the particle stack was subjected to 2-class ab initio refinement followed by heterogeneous refinement in cryoSPARC. 264,227 particles sorted into a higher resolution 3D class, on which Non-Uniform (NU) refinement was performed. The resulting 3.3 A global map strongly resembled previous sGC structures in the ligand-free, contracted state, and the map was used for initial model building. Other refinement strategies were attempted, but did not lead to higher resolution global map densities.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model / Details: ModelAngelo and Alphafold
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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