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- EMDB-16981: FMRFa-bound Malacoceros FaNaC1 in lipid nanodiscs -

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Basic information

Entry
Database: EMDB / ID: EMD-16981
TitleFMRFa-bound Malacoceros FaNaC1 in lipid nanodiscs
Map datadeepEMhancer postprocessed FMRFa-bound FaNaC1 map, used in model building and figure preparation
Sample
  • Complex: FMRFamide-gated sodium channel 1 (FaNaC1)
    • Protein or peptide: FMRFamide-gated sodium channel 1 (FaNaC1)
    • Protein or peptide: FMRFamide, neuropeptide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsNeuropeptide / ion channel / DEG/ENaC / MEMBRANE PROTEIN
Biological speciesMalacoceros fuliginosus (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsKalienkova V / Dandamudi M / Paulino C / Lynagh T
Funding support Netherlands, Norway, Switzerland, 3 items
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, 2023-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16981.png

Downloads & links

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Map

FileDownload / File: emd_16981.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationdeepEMhancer postprocessed FMRFa-bound FaNaC1 map, used in model building and figure preparation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 220 pix.
= 224.84 Å		1.02 Å/pix.
x 220 pix.
= 224.84 Å		1.02 Å/pix.
x 220 pix.
= 224.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.022 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.0017476154 - 1.4023684
Average (Standard dev.)0.0022033432 (±0.032754682)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 224.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16981_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

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Additional map: refined FMRFa-bound FaNaC1 map, used in model refinement

Fileemd_16981_additional_1.map
Annotationrefined FMRFa-bound FaNaC1 map, used in model refinement
Projections & Slices
AxesZYX

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Histogram

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Half map: FMRFa-bound FaNaC1 refined halfmap1

Fileemd_16981_half_map_1.map
AnnotationFMRFa-bound FaNaC1 refined halfmap1
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: FMRFa-bound FaNaC1 refined halfmap2

Fileemd_16981_half_map_2.map
AnnotationFMRFa-bound FaNaC1 refined halfmap2
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : FMRFamide-gated sodium channel 1 (FaNaC1)

EntireName: FMRFamide-gated sodium channel 1 (FaNaC1)
Components
  • Complex: FMRFamide-gated sodium channel 1 (FaNaC1)
    • Protein or peptide: FMRFamide-gated sodium channel 1 (FaNaC1)
    • Protein or peptide: FMRFamide, neuropeptide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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

SupramoleculeName: FMRFamide-gated sodium channel 1 (FaNaC1) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Malacoceros fuliginosus (invertebrata)
Molecular weightTheoretical: 205 KDa

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

MacromoleculeName: FMRFamide-gated sodium channel 1 (FaNaC1) / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Malacoceros fuliginosus (invertebrata)
Molecular weightTheoretical: 68.605836 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSAIRDVMTK FAEQTTMHGV PKVINAKSSM GRLFWSLVCL AAGAMFCLQM SEVLQRYFSY PKKVTVEVVP TPVPFPSISI CNMRNLDVH ILNTLNRMFI EDDRPFSNIN KSEHEFIRAY MKKVAKYAPL FWNYQDEYPE VFQEIFSRTT FSANIDPEVI A LAAVQLEG ...String:
MSAIRDVMTK FAEQTTMHGV PKVINAKSSM GRLFWSLVCL AAGAMFCLQM SEVLQRYFSY PKKVTVEVVP TPVPFPSISI CNMRNLDVH ILNTLNRMFI EDDRPFSNIN KSEHEFIRAY MKKVAKYAPL FWNYQDEYPE VFQEIFSRTT FSANIDPEVI A LAAVQLEG FVVNCHYAGH RCNKTRDFYR FFDPYYFNCF TYKAHEPTDI EDNLSEGIEN GWSSILLSGS GMLDKNDEIR ML PGLHEWR SAVSASEGVR VVIHPPSTTP YPFTEGYDVP PGFSASFGIH PRRNIRIGPP HGNCSDKNPF GDGTERYRLM ACQ KMCMQH YIVETCGCAD VGLPKLPLQA NISWCRDDDN FPDECMFTAS EECLQLLMQL HNRIKCARSI KSKITKNTTA MEAC NCFPP CDEVSYDVSY SLSKWPSAGY EGDAAYFDVF GIEKFNERFN KTGTQGKYEL FTKYFNVSNR EESMKDFARL NVYIA DSNV VKTQESEDYT RNQLVSDIGG QLGLWVGISL ITLAEVLELI IDLFRLFSKH TYRSVPVIRQ SIKYKDKRNG AEMNYD TRY SQSNGGPHAR YLHHGHSIPK HPPELPDTSL ALEVLFQ

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Macromolecule #2: FMRFamide, neuropeptide

MacromoleculeName: FMRFamide, neuropeptide / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Malacoceros fuliginosus (invertebrata)
Molecular weightTheoretical: 598.76 Da
SequenceString:
FMRF(NH2)

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 9 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration1.44 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPESHEPES
30.0 uMFMRFamide
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: at 5 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 288.15 K / Instrument: FEI VITROBOT MARK IV
Detailsnanodisc-reconstituted Malacoceros FaNaC1 bound to the full agonist (FMRFamide)

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 90.0 K / Max: 105.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 2 / Number real images: 4479 / Average exposure time: 9.0 sec. / Average electron dose: 50.11 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3235905
Startup modelType of model: NONE / Details: Ab Initio reconstruction
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 458934
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1.0)
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC (ver. 3)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL
Output model

PDB-8on7:
FMRFa-bound Malacoceros FaNaC1 in lipid nanodiscs

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