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- EMDB-70990: Compact, ligand-free state of Manduca sexta soluble guanylate cyc... -

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Basic information

Entry
Database: EMDB / ID: EMD-70990
TitleCompact, ligand-free state of Manduca sexta soluble guanylate cyclase mutant beta C122S
Map dataComposite Map of ligand-free C122S Ms sGC in the compact conformation. A composite map of local refinements of the HNOX and catalytic domains.
Sample
  • Complex: Ligand-free Manduca sexta soluble guanylase cyclase variant
    • Protein or peptide: Soluble guanylyl cyclase alpha-1 subunit
    • Protein or peptide: Guanylate cyclase soluble subunit beta-1
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
KeywordsCyclase / NO / SIGNALING PROTEIN
Function / homology
Function and homology information


guanylate cyclase complex, soluble / guanylate cyclase / guanylate cyclase activity / response to oxygen levels / : / heme binding / GTP binding
Similarity search - Function
Haem NO binding associated / Haem NO binding associated domain superfamily / Heme NO binding associated / Heme NO-binding / H-NOX domain superfamily / Haem-NO-binding / Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / NO signalling/Golgi transport ligand-binding domain superfamily / Adenylyl- / guanylyl cyclase, catalytic domain ...Haem NO binding associated / Haem NO binding associated domain superfamily / Heme NO binding associated / Heme NO-binding / H-NOX domain superfamily / Haem-NO-binding / Adenylyl cyclase class-4/guanylyl cyclase, conserved site / Guanylate cyclase signature. / NO signalling/Golgi transport ligand-binding domain superfamily / Adenylyl- / guanylyl cyclase, catalytic domain / Adenylate and Guanylate cyclase catalytic domain / Adenylyl cyclase class-3/4/guanylyl cyclase / Guanylate cyclase domain profile. / Nucleotide cyclase
Similarity search - Domain/homology
guanylate cyclase / Guanylate cyclase soluble subunit beta-1
Similarity search - Component
Biological speciesManduca sexta (tobacco hornworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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)5F32GM149060-02 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 3, 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_70990.png

Downloads & links

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Map

FileDownload / File: emd_70990.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite Map of ligand-free C122S Ms sGC in the compact conformation. A composite map of local refinements of the HNOX and catalytic domains.
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: 0.783
Minimum - Maximum-4.1759667 - 7.6547575
Average (Standard dev.)0.000015657208 (±0.07952573)
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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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: Soluble guanylyl cyclase alpha-1 subunit
    • Protein or peptide: Guanylate cyclase soluble subunit beta-1
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE

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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: #1-#2
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: Soluble guanylyl cyclase alpha-1 subunit

MacromoleculeName: Soluble guanylyl cyclase alpha-1 subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Manduca sexta (tobacco hornworm)
Molecular weightTheoretical: 78.598727 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
SequenceString: MTCPFRRASS QHQFANGGSS APKKPEFRSR TSSVHLTGPE EEDGERNTLT LKHMSEALQL LTAPSNECLH AAVTSLTKNQ SDHYHKYNC LRRLPDDVKT CRNYAYLQEI YDAVRATDSV NTKDFMAKLG EYLILTAFSH NCRLERAFKC LGTNLTEFLT T LDSVHDVL ...String:
MTCPFRRASS QHQFANGGSS APKKPEFRSR TSSVHLTGPE EEDGERNTLT LKHMSEALQL LTAPSNECLH AAVTSLTKNQ SDHYHKYNC LRRLPDDVKT CRNYAYLQEI YDAVRATDSV NTKDFMAKLG EYLILTAFSH NCRLERAFKC LGTNLTEFLT T LDSVHDVL HDQDTPLKDE TMEYEANFVC TTSQEGKIQL HLTTESEPVA YLLVGSLKAI AKRLYDTQTD IRLRSYTNDP RR FRYEINA VPLHQKSKED SCELVNEAAS VATSTKVTDL KIGVASFCKA FPWHFITDKR LELVQLGAGF MRLFGTHLAT HGS SLGTYF RLLRPRGVPL DFREILKRVN TPFMFCLKMP GSTALAEGLE IKGQMVFCAE SDSLLFVGSP FLDGLEGLTG RGLF ISDIP LHDATRDVIL VGEQARAQDG LRRRMDKLKN SIEEASKAVD KEREKNVSLL HLIFPPHIAK RLWLGEKIEA KSHDD VTML FSDIVGFTSI CATATPMMVI AMLEDLYSVF DIFCEELDVY KVETIGDAYC VASGLHRKVE THAPQIAWMA LRMVET CAQ HLTHEGNPIK MRIGLHTGTV LAGVVGKTML KYCLFGHNVT LANKFESGSE PLKINVSPTT YEWLIKFPGF DMEPRDR SC LPNSFPKDIH GTCYFLHKYT HPGTDPGEPQ VKHIREALKD YGIGQANSTD VDTEEPT

UniProtKB: guanylate cyclase

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Macromolecule #2: Guanylate cyclase soluble subunit beta-1

MacromoleculeName: Guanylate cyclase soluble subunit beta-1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: guanylate cyclase
Source (natural)Organism: Manduca sexta (tobacco hornworm)
Molecular weightTheoretical: 68.16593 KDa
Recombinant expressionOrganism: Spodoptera aff. frugiperda 2 RZ-2014 (butterflies/moths)
SequenceString: MYGFVNYALE LLVMKTFDEE TWETIKKKAD VAMEGSFLVR QIYEDEITYN LITAAVEVLQ IPADAILELF GKTFFEFCQD SGYDKILQV LGATPRDFLQ NLDGLHDHLG TLYPGMRSPS FRSTERPEDG ALVLHYYSDR PGLEHIVIGI VKTVASKLHN T EVKVEILK ...String:
MYGFVNYALE LLVMKTFDEE TWETIKKKAD VAMEGSFLVR QIYEDEITYN LITAAVEVLQ IPADAILELF GKTFFEFCQD SGYDKILQV LGATPRDFLQ NLDGLHDHLG TLYPGMRSPS FRSTERPEDG ALVLHYYSDR PGLEHIVIGI VKTVASKLHN T EVKVEILK TKEECDHVQF LITETSTTGR VSAPEIAEIE TLSLEPKVSP ATFCRVFPFH LMFDRDLNIV QAGRTVSRLL PR VTRPGCK ITDVLDTVRP HLEMTFANVL AHINTVYVLK TKPEEMSVTD PHEEIASLRL KGQMLYIPET DVVVFQCYPS VTN LDDLTR RGLCIADIPL HDATRDLVLM SEQFEADYKL TQNLEVLTDK LQQTFRELEL EKQKTDRLLY SVLPISVATE LRHR RPVPA RRYDTVTLLF SGIVGFANYC ARNSDHKGAM KIVRMLNDLY TAFDVLTDPK RNPNVYKVET VGDKYMAVSG LPEYE VAHA KHISLLALDM MDLSQTVTVD GEPVGITIGI HSGEVVTGVI GHRMPRYCLF GNTVNLTSRC ETTGVPGTIN VSEDTY NYL MREDNHDEQF ELTYRGHVTM KGKAEPMQTW FLTRKIH

UniProtKB: Guanylate cyclase soluble subunit beta-1

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Macromolecule #3: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 3 / Number of copies: 1 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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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 / Material: COPPER / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
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 reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: During refinement it became clear that there was minor interdomain flexibility within the 264,227 particles. In particular, the catalytic domain was at lower resolution and had considerable ...Details: During refinement it became clear that there was minor interdomain flexibility within the 264,227 particles. In particular, the catalytic domain was at lower resolution and had considerable missing density. 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. A composite map was used for model building.
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.

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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
Output model

PDB-9oxn:
Compact, ligand-free state of Manduca sexta soluble guanylate cyclase mutant beta C122S

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