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- EMDB-43438: Structure of the voltage-gated sodium channel NavPas from America... -

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Basic information

Entry
Database: EMDB / ID: EMD-43438
TitleStructure of the voltage-gated sodium channel NavPas from American Cockroach Periplaneta Americana in complex with scorpion alpha-toxin LqhaIT
Map dataSharpened Map
Sample
  • Complex: NavPas-LqhaIT
    • Protein or peptide: Sodium channel protein PaFPC1
    • Protein or peptide: Alpha-insect toxin LqhaIT
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsNavPas / scorpion toxin / ion channel / voltage-gated sodium channel / MEMBRANE PROTEIN
Function / homology
Function and homology information


membrane depolarization during action potential / voltage-gated sodium channel complex / sodium channel inhibitor activity / voltage-gated monoatomic cation channel activity / voltage-gated sodium channel activity / neuronal action potential / defense response / toxin activity / extracellular region
Similarity search - Function
Scorpion long chain toxin / LCN-type cysteine-stabilized alpha/beta (CS-alpha/beta) domain / LCN-type cysteine-stabilized alpha/beta (CS-alpha/beta) domain profile. / Scorpion long chain toxin/defensin / Scorpion toxin-like domain / Knottins / Knottin, scorpion toxin-like / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Voltage gated sodium channel, alpha subunit ...Scorpion long chain toxin / LCN-type cysteine-stabilized alpha/beta (CS-alpha/beta) domain / LCN-type cysteine-stabilized alpha/beta (CS-alpha/beta) domain profile. / Scorpion long chain toxin/defensin / Scorpion toxin-like domain / Knottins / Knottin, scorpion toxin-like / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Voltage gated sodium channel, alpha subunit / Knottin, scorpion toxin-like superfamily / Voltage-gated cation channel calcium and sodium / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Sodium channel protein PaFPC1 / Alpha-insect toxin LqhaIT
Similarity search - Component
Biological speciesPeriplaneta americana (American cockroach) / Leiurus quinquestriatus hebraeus (scorpion)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsPhulera S / Khoshouei M / Whicher J / Weihofen WA
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Structure / Year: 2024
Title: Scorpion α-toxin LqhαIT specifically interacts with a glycan at the pore domain of voltage-gated sodium channels.
Authors: Swastik Phulera / Callum J Dickson / Christopher J Schwalen / Maryam Khoshouei / Samantha J Cassell / Yishan Sun / Tara Condos / Jonathan Whicher / Wilhelm A Weihofen /
Abstract: Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on ...Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on Nav channels. To elucidate the mechanism of action of LqhαIT, we determined a 3.9 Å cryoelectron microscopy (cryo-EM) structure of LqhαIT in complex with the Nav channel from Periplaneta americana (NavPas). We found that LqhαIT binds to voltage sensor domain 4 and traps it in an "S4 down" conformation. The functionally essential C-terminal epitope of LqhαIT forms an extensive interface with the glycan scaffold linked to Asn330 of NavPas that augments a small protein-protein interface between NavPas and LqhαIT. A combination of molecular dynamics simulations, structural comparisons, and prior mutagenesis experiments demonstrates the functional importance of this toxin-glycan interaction. These findings establish a structural basis for the specificity achieved by scorpion α-toxins and reveal the conserved glycan as an essential component of the toxin-binding epitope.
History
DepositionJan 18, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43438.png

Downloads & links

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Map

FileDownload / File: emd_43438.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 480 pix.
= 316.8 Å		0.66 Å/pix.
x 480 pix.
= 316.8 Å		0.66 Å/pix.
x 480 pix.
= 316.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.63955677 - 0.9815459
Average (Standard dev.)-0.00045047057 (±0.019531872)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 316.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map

Fileemd_43438_half_map_1.map
AnnotationHalf Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map

Fileemd_43438_half_map_2.map
AnnotationHalf Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NavPas-LqhaIT

EntireName: NavPas-LqhaIT
Components
  • Complex: NavPas-LqhaIT
    • Protein or peptide: Sodium channel protein PaFPC1
    • Protein or peptide: Alpha-insect toxin LqhaIT
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: NavPas-LqhaIT

SupramoleculeName: NavPas-LqhaIT / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Voltage-gated sodium channel NavPas from American Cockroach Periplaneta Americana in complex with scorpion alpha-toxin LqhaIT
Source (natural)Organism: Periplaneta americana (American cockroach)

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Macromolecule #1: Sodium channel protein PaFPC1

MacromoleculeName: Sodium channel protein PaFPC1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Periplaneta americana (American cockroach)
Molecular weightTheoretical: 183.875422 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MASWSHPQFE KGGGARGGSG GGSWSHPQFE KGFDYKDDDD KGTMADNSPL IREERQRLFR PYTRAMLTAP SAQPAKENGK TEENKDNSR DKGRGANKDR DGSAHPDQAL EQGSRLPARM RNIFPAELAS TPLEDFDPFY KNKKTFVVVT KAGDIFRFSG E KSLWMLDP ...String:
MASWSHPQFE KGGGARGGSG GGSWSHPQFE KGFDYKDDDD KGTMADNSPL IREERQRLFR PYTRAMLTAP SAQPAKENGK TEENKDNSR DKGRGANKDR DGSAHPDQAL EQGSRLPARM RNIFPAELAS TPLEDFDPFY KNKKTFVVVT KAGDIFRFSG E KSLWMLDP FTPIRRVAIS TMVQPIFSYF IMITILIHCI FMIMPATQTT YILELVFLSI YTIEVVVKVL ARGFILHPFA YL RDPWNWL DFLVTLIGYI TLVVDLGHLY ALRAFRVLRS WRTVTIVPGW RTIVDALSLS ITSLKDLVLL LLFSLFVFAV LGL QIYMGV LTQKCVKHFP ADGSWGNFTD ERWFNYTSNS SHWYIPDDWI EYPLCGNSSG AGMCPPGYTC LQGYGGNPNY GYTS FDTFG WAFLSVFRLV TLDYWEDLYQ LALRSAGPWH ILFFIIVVFY GTFCFLNFIL AVVVMSYTHM VKRADEEKAA ERELK KEKK AASVANNTAN GQEQTTIEMN GDEAVVIDNN DQAARQQSDP ETPAPSVTQR LTDFLCVWDC CVPWQKLQGA IGAVVL SPF FELFIAVIIV LNITFMALDH HDMNIEFERI LRTGNYIFTS IYIVEAVLKI IALSPKFYFK DSWNVFDFII VVFAILE LG LEGVQGLSVF RSFRLLRVFR LAKFWPTLNN FMSVMTKSYG AFVNVMYVMF LLLFIFAIIG MQLFGMNYID NMERFPDG D LPRWNFTDFL HSFMIVFRAL CGEWIESMWD CMLVGDWSCI PFFVAVFFVG NLVILNLLIA LLLNNYGSFC TSPTSDEED SKDEDALAQI VRIFKRFKPN LNAVKLSPMK PDSEDIVESQ EIQGNNIADA EDVLAGEFPP DCCCNAFYKC FPSRPARDSS VQRMWSNIR RVCFLLAKNK YFQKFVTAVL VITSVLLALE DIYLPQRPVL VNITLYVDYV LTAFFVIEMI IMLFAVGFKK Y FTSKWYWL DFIVVVAYLL NFVLMCAGIE ALQTLRLLRV FRLFRPLSKV NGMQVVTSTL VEAVPHIFNV ILVGIFFWLV FA IMGVQLF AGKFYKCVDE NSTVLSHEIT MDRNDCLHEN YTWENSPMNF DHVGNAYLSL LQVATFKGWL QIMNDAIDSR EVH KQPIRE TNIYMYLYFI FFIVFGSFFI LKLFVCILID IFRQQRRKAE GLSATDSRTQ LIYRRAVMRT MSAKPVKRIP KPTC HPQSL MYDISVNRKF EYTMMILIIL NVAVMAIDHY GQSMEFSEVL DYLNLIFIII FFVECVIKVS GLRHHYFKDP WNIID FLYV VLAIAGLMLS DVIEKYFISP TLLRILRILR VGRLLRYFQS ARGMRLLLLA LRKALRTLFN VSFLLFVIMF VYAVFG MEF FMHIRDAGAI DDVYNFKTFG QSIILLFQLA TSAGWDGVYF AIANEEDCRA PDHELGYPGN CGSRALGIAY LVSYLII TC LVVINMYAAV ILDYVLEVYE DSKEGLTDDD YDMFFEVWQQ FDPEATQYIR YDQLSELLEA LQPPLQVQKP NKYKILSM N IPICKDDHIF YKDVLEALVK DVFSRRGSPV EAGDVQAPNV DEAEYKPVSS TLQRQREEYC VRLIQNAWRK HKQQN

UniProtKB: Sodium channel protein PaFPC1

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Macromolecule #2: Alpha-insect toxin LqhaIT

MacromoleculeName: Alpha-insect toxin LqhaIT / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Leiurus quinquestriatus hebraeus (scorpion)
Molecular weightTheoretical: 7.396455 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MVRDAYIAKN YNCVYECFRD AYCNELCTKN GASSGYCQWA GKYGNACWCY ALPDNVPIRV PGKCR

UniProtKB: Alpha-insect toxin LqhaIT

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 2 / 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

Concentration6.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mM(HOCH2)3CNH2Tris
50.0 mMNaClSodium Chloride
0.1 % w/vC56H92O29Digitonin

Details: 25 mM Tris pH 7.5, 50 mM NaCl, 0.1% (w/v) digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
Details: Electron microscopy grids (Quantifoil Au, 200-mesh copper R1.2/1.3) were glow-discharged for 90 s using PELCO easiGlow
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Sample was applied on the grid in Vitrobot Mark IV chamber (Thermo Fisher Scientific), blotted for 4 secs with a blot force of 25 and then plunged into ethane.
DetailsMonodisperse sample post SEC

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

6698

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.1) / Details: using Cryosparc / Number images used: 164000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5x0m


Chain - Chain ID: a / Chain - Residue range: 47-1521 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: maximum likelihood
Output model

PDB-8vqc:
Structure of the voltage-gated sodium channel NavPas from American Cockroach Periplaneta Americana in complex with scorpion alpha-toxin LqhaIT

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