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- EMDB-16316: Alvinella pompejana nicotinic acetylcholine receptor Alpo4 in apo... -

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Basic information

Entry
Database: EMDB / ID: EMD-16316
TitleAlvinella pompejana nicotinic acetylcholine receptor Alpo4 in apo state (Alpo4_comb dataset 3)
Map data
Sample
  • Complex: Pentameric complex of Alpo4 in apo state
    • Protein or peptide: Acetylcholine receptor
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water
KeywordsCys-loop receptor / pentameric ligand-gated ion channel / Alpo / nAChR / MEMBRANE PROTEIN
Biological speciesAlvinella pompejana (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsDe Gieter S / Efremov RG / Ulens C
Funding support Belgium, 5 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0H5916N Belgium
Research Foundation - Flanders (FWO)G054617N Belgium
Research Foundation - Flanders (FWO)G087921N Belgium
KU LeuvenC3/19/023 Belgium
KU LeuvenC14/17/093 Belgium
CitationJournal: Elife / Year: 2023
Title: Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor.
Authors: Steven De Gieter / Casey I Gallagher / Eveline Wijckmans / Diletta Pasini / Chris Ulens / Rouslan G Efremov /
Abstract: Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high ...Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm . Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the cryo-EM structure of the lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators.
History
DepositionDec 8, 2022-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateJul 12, 2023-
Current statusJul 12, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16316.png

Downloads & links

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Map

FileDownload / File: emd_16316.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.7596 Å
Density
Contour LevelBy AUTHOR: 0.164
Minimum - Maximum-1.0130112 - 1.6530012
Average (Standard dev.)0.0018768663 (±0.046696283)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 267.37918 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16316_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16316_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_16316_half_map_2.map
Projections & Slices
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Sample components

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Entire : Pentameric complex of Alpo4 in apo state

EntireName: Pentameric complex of Alpo4 in apo state
Components
  • Complex: Pentameric complex of Alpo4 in apo state
    • Protein or peptide: Acetylcholine receptor
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: water

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Supramolecule #1: Pentameric complex of Alpo4 in apo state

SupramoleculeName: Pentameric complex of Alpo4 in apo state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Alvinella pompejana (invertebrata)
Molecular weightTheoretical: 270 KDa

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Macromolecule #1: Acetylcholine receptor

MacromoleculeName: Acetylcholine receptor / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Alvinella pompejana (invertebrata)
Molecular weightTheoretical: 54.154934 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAGSKVDKTL CRLGDITGLL IVLSVLVSRG LCQDCNNTNA SSMADEKRLL KCILHDYDTA IRPVQNVSDV VNVALEVTVV KVIDLDEKE HVLTTNGWIY HEWNDFQLKW NPSDYSGLKK IRIPVDRIWT PDIVLFNNAD ESYRYVVDKL AVVYYTGKVM W VPHARLRS ...String:
MAGSKVDKTL CRLGDITGLL IVLSVLVSRG LCQDCNNTNA SSMADEKRLL KCILHDYDTA IRPVQNVSDV VNVALEVTVV KVIDLDEKE HVLTTNGWIY HEWNDFQLKW NPSDYSGLKK IRIPVDRIWT PDIVLFNNAD ESYRYVVDKL AVVYYTGKVM W VPHARLRS FCVLDLSRFP FDSQMCTLVF GSWTHDVSSV NVTLRNQSKV QYMIDGKEWQ VTSVQPKRYQ WTYNSNENYA GI ITGIKLK RTSIYYQYVF IMPTVLLAFL TLLMPFIPPL GKERITYGIG LVLGCTLLLM MLSDRMPTEL GNVPVVAAYL AYV FVMVAI NLLFAIMAIN MSMRDPKFGK VPGWVRWIFL TKLSKLVCLP VEPYTAVPAD LAYEETAAAR ELLDMNNGTA TADQ RVSGS DTKPALDRTL EDIRRYLRLV ATRTVVTPQL SHRDLVVQEW QQLTRVIDRL LFGSFLVLTV VITISMYAHY

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

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 5 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 4 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMNaPiPhosphate
150.0 mMNaClSodium Chlorate
0.003 %LMNGLMNG
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Specialist opticsPhase plate: OTHER / Energy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 25669 / Average electron dose: 59.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: OTHER / Details: Ab initio model from cryoSPARC
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 131380
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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