2025-11-26 REL RCSB RCSB RCSB 2025-06-11 2025-11-12 2025-11-12 2025-11-26 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 Consensus map of extended state M. sexta soluble guanylate cyclase mutant beta C122S with CYR715 Thomas WC Houghton KA Cyclase, NO, SIGNALING PROTEIN Houghton KA Thomas WC Marletta MA Molecular Aspects of Soluble Guanylate Cyclase Activation and Stimulator Function. Biochemistry US 64 4529 4541 2025 41146038 doi:10.1021/acs.biochem.5c00424 0006-2960 0033 BICHAW EMDB EMD-71150 associated EM volume Extended state Manduca sexta soluble guanylase cyclase variant C122S Extended state Manduca sexta soluble guanylase cyclase variant C122S 0 1
Heterodimeric sGC molecule in the extended, CYR715-bound state. Beta-C122S mutant variant.
Manduca sexta 0.147 M. sexta soluble guanylate cyclase variant Manduca sexta Spodoptera aff. frugiperda 2 RZ-2014 LEVO MTCPFRRASSQHQFANGGSSAPKKPEFRSRTSSVHLTGPEEEDGERNTLTLKHMSEALQLLTAPSNECLHAAVTSLTKNQSDHYHKYNCLRRLPDDVKTCRNYAYLQEIYDAVRATDSVNTKDFMAKLGEYLILTAFSHNCRLERAFKCLGTNLTEFLTTLDSVHDVLHDQDTPLKDETMEYEANFVCTTSQEGKIQLHLTTESEPVAYLLVGSLKAIAKRLYDTQTDIRLRSYTNDPRRFRYEINAVPLHQKSKEDSCELVNEAASVATSTKVTDLKIGVASFCKAFPWHFITDKRLELVQLGAGFMRLFGTHLATHGSSLGTYFRLLRPRGVPLDFREILKRVNTPFMFCLKMPGSTALAEGLEIKGQMVFCAESDSLLFVGSPFLDGLEGLTGRGLFISDIPLHDATRDVILVGEQARAQDGLRRRMDKLKNSIEEASKAVDKEREKNVSLLHLIFPPHIAKRLWLGEKIEAKSHDDVTMLFSDIVGFTSICATATPMMVIAMLEDLYSVFDIFCEELDVYKVETIGDAYCVASGLHRKVETHAPQIAWMALRMVETCAQHLTHEGNPIKMRIGLHTGTVLAGVVGKTMLKYCLFGHNVTLANKFESGSEPLKINVSPTTYEWLIKFPGFDMEPRDRSCLPNSFPKDIHGTCYFLHKYTHPGTDPGEPQVKHIREALKDYGIGQANSTDVDTEEPT MYGFVNYALELLVMKTFDEETWETIKKKADVAMEGSFLVRQIYEDEITYNLITAAVEVLQIPADAILELFGKTFFEFCQDSGYDKILQVLGATPRDFLQNLDGLHDHLGTLYPGMRSPSFRSTERPEDGALVLHYYSDRPGLEHIVIGIVKTVASKLHNTEVKVEILKTKEECDHVQFLITETSTTGRVSAPEIAEIETLSLEPKVSPATFCRVFPFHLMFDRDLNIVQAGRTVSRLLPRVTRPGCKITDVLDTVRPHLEMTFANVLAHINTVYVLKTKPEEMSVTDPHEEIASLRLKGQMLYIPETDVVVFQCYPSVTNLDDLTRRGLCIADIPLHDATRDLVLMSEQFEADYKLTQNLEVLTDKLQQTFRELELEKQKTDRLLYSVLPISVATELRHRRPVPARRYDTVTLLFSGIVGFANYCARNSDHKGAMKIVRMLNDLYTAFDVLTDPKRNPNVYKVETVGDKYMAVSGLPEYEVAHAKHISLLALDMMDLSQTVTVDGEPVGITIGIHSGEVVTGVIGHRMPRYCLFGNTVNLTSRCETTGVPGTINVSEDTYNYLMREDNHDEQFELTYRGHVTMKGKAEPMQTWFLTRKIH singleParticle particle 1.5 7.5 25.0 C6H15NO3 Triethanolamine (TEA) 25.0 NaCl Sodium chloride 5.0 C4H10O2S2 Dithiothreitol (DTT) 5.0 MgCl2 Magnesium chloride 0.5 C20H25F13O11 Fluorinated Octyl Maltoside Quantifoil R1.2/1.3 CARBON ETHANE 100 277 FEI VITROBOT MARK IV
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..
Samples 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.
 TFS KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 100.0 2.7 0.5 1.5 FEI TITAN KRIOS AUTOGRID HOLDER GATAN K3 (6k x 4k) 1 8433 0.0354 1.25 1 1226641
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.
 cryoSPARC PHASE FLIPPING AND AMPLITUDE CORRECTION 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. 3.7 FSC 0.143 CUT-OFF cryoSPARC
Non-Uniform (NU) refinement
598571 MAXIMUM LIKELIHOOD cryoSPARC MAXIMUM LIKELIHOOD cryoSPARC 2 200000.0 cryoSPARC
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.
 emd_71150.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 320 320 320 0 0 0 320 320 320 275.2 275.2 275.2 90.0 90.0 90.0 X Y Z -2.1677566 3.415273 0.00063671166 0.048876386 0.86 0.86 0.86 0.452 AUTHOR Consensus map of BC122S M. sexta sGC in the extended conformation with CYR715 bound. SwissModel in silico model
Structure best matched homology and shape with 8HBF.
 FLEXIBLE FIT
Refinement 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.
REAL emd_71150_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 320 320 320 0 0 0 320 320 320 275.2 275.2 275.2 90.0 90.0 90.0 X Y Z -0.23362552 0.73932403 0.00071680755 0.023428353 0.86 0.86 0.86 AUTHOR Half-map A of consensus map of BC122S M. sexta sGC in the extended conformation with CYR715 bound. emd_71150_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 320 320 320 0 0 0 320 320 320 275.2 275.2 275.2 90.0 90.0 90.0 X Y Z -0.19493619 0.7509187 0.00072494196 0.023463266 0.86 0.86 0.86 AUTHOR Half-map A of consensus map of BC122S M. sexta sGC in the extended conformation with CYR715 bound.