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- PDB-8ona: FMRFa-bound Malacoceros FaNaC1 in lipid nanodiscs in presence of ... -

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Basic information

Entry
Database: PDB / ID: 8ona
TitleFMRFa-bound Malacoceros FaNaC1 in lipid nanodiscs in presence of diminazene
Components
  • FMRFamide, neuropeptide
  • FMRFamide-gated sodium channel 1 (FaNaC1)
KeywordsMEMBRANE PROTEIN / Neuropeptide / ion channel / DEG/ENaC
Biological speciesMalacoceros fuliginosus (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsKalienkova, V. / Dandamudi, M. / Paulino, C. / Lynagh, T.
Funding support Netherlands, Norway, Switzerland, 3items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Norwegian Research Council234817 Norway
Swiss National Science FoundationP500PB_203053 Switzerland
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis for excitatory neuropeptide signaling.
Authors: Valeria Kalienkova / Mowgli Dandamudi / Cristina Paulino / Timothy Lynagh /
Abstract: Rapid signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain. The ...Rapid signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily is diverse in terms of its gating stimuli, with some DEG/ENaCs gated by neuropeptides, and others gated by pH, mechanical force or enzymatic activity. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. Here we dissected the structural basis for neuropeptide-gated activity of a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus, using cryo-electron microscopy. Structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes of channel gating, which we verified with complementary mutagenesis experiments. Our results illuminate channel gating in DEG/ENaCs and offer a structural template for experimental dissection of channel pharmacology and ion conduction.
History
DepositionApr 1, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 14, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2May 1, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: FMRFamide-gated sodium channel 1 (FaNaC1)
D: FMRFamide, neuropeptide
B: FMRFamide-gated sodium channel 1 (FaNaC1)
C: FMRFamide-gated sodium channel 1 (FaNaC1)
E: FMRFamide, neuropeptide
F: FMRFamide, neuropeptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)210,93221
Polymers207,6146
Non-polymers3,31815
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area23410 Å2
ΔGint-46 kcal/mol
Surface area67480 Å2
MethodPISA

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Components

#1: Protein FMRFamide-gated sodium channel 1 (FaNaC1)


Mass: 68605.836 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Malacoceros fuliginosus (invertebrata) / Gene: ON156825.1 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / Tissue (production host): kidney, embryo
#2: Protein/peptide FMRFamide, neuropeptide


Mass: 598.760 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Malacoceros fuliginosus (invertebrata)
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1FMRFamide-gated sodium channel 1 (FaNaC1)COMPLEX#1-#20MULTIPLE SOURCES
2FMRFamide-gated sodium channel 1 (FaNaC1)COMPLEX#11RECOMBINANT
3FMRFamide, neuropeptideCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.205 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Malacoceros fuliginosus (invertebrata)271776
33Malacoceros fuliginosus (invertebrata)271776
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Homo sapiens (human)9606HEK293S GnTI-
33Synthetic construct (others)32630
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mMHEPESHEPES1
330 uMFMRFamide1
4100 uMdiminazene aceturate1
SpecimenConc.: 1.83 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: nanodisc-reconstituted Malacoceros FaNaC1 bound to the full agonist (FMRFamide) in presence of a pore blocker diminazene
Specimen supportDetails: at 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 49407 X / Nominal defocus max: 3000 nm / Nominal defocus min: 300 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 47.19 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7284
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7Coot0.9model fitting
8ISOLDE1.4model fitting
10PHENIX1.20.1-4487model refinement
11cryoSPARC3initial Euler assignment
12RELION3.1.0final Euler assignment
13RELION3.1.0classification
14RELION3.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3893627
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 137558 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 8ON7

8on7


Accession code: 8ON7 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE

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