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- EMDB-35161: Cryo-EM structure of nanodisc (asolectin) reconstituted GLIC at pH 7.5 -

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Basic information

Entry
Database: EMDB / ID: EMD-35161
TitleCryo-EM structure of nanodisc (asolectin) reconstituted GLIC at pH 7.5
Map datapostprocess masked of GLIC pH7.5 asolectin
Sample
  • Complex: pentameric ligand-gated ion channel
    • Protein or peptide: Proton-gated ion channel
  • Ligand: DIUNDECYL PHOSPHATIDYL CHOLINE
Keywordspentameric ligand-gated ion channels / cis-loop / cryo-EM / nanodisc / MEMBRANE PROTEIN
Function / homology
Function and homology information


sodium channel activity / potassium channel activity / extracellular ligand-gated monoatomic ion channel activity / transmembrane signaling receptor activity / identical protein binding / plasma membrane
Similarity search - Function
Gamma-aminobutyric acid A receptor/Glycine receptor alpha / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
Similarity search - Domain/homology
Proton-gated ion channel
Similarity search - Component
Biological speciesGloeobacter violaceus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsBharambe N / Li Z / Basak S
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Other private Singapore
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM structures of prokaryotic ligand-gated ion channel GLIC provide insights into gating in a lipid environment.
Authors: Nikhil Bharambe / Zhuowen Li / David Seiferth / Asha Manikkoth Balakrishna / Philip C Biggin / Sandip Basak /
Abstract: GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite ...GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite extensive studies conducted on GLIC, the molecular mechanism of channel gating in the lipid environment requires further investigation. Here, we present the cryo-EM structures of nanodisc-reconstituted GLIC at neutral and acidic pH in the resolution range of 2.6 - 3.4 Å. In our apo state at pH 7.5, the extracellular domain (ECD) displays conformational variations compared to the existing apo structures. At pH 4.0, three distinct conformational states (C1, C2 and O states) are identified. The protonated structures exhibit a compacted and counter-clockwise rotated ECD compared with our apo state. A gradual widening of the pore in the TMD is observed upon reducing the pH, with the widest pore in O state, accompanied by several layers of water pentagons. The pore radius and molecular dynamics (MD) simulations suggest that the O state represents an open conductive state. We also observe state-dependent interactions between several lipids and proteins that may be involved in the regulation of channel gating. Our results provide comprehensive insights into the importance of lipids impact on gating.
History
DepositionJan 18, 2023-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35161.png

Downloads & links

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Map

FileDownload / File: emd_35161.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocess masked of GLIC pH7.5 asolectin
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 272.384 Å		1.06 Å/pix.
x 256 pix.
= 272.384 Å		1.06 Å/pix.
x 256 pix.
= 272.384 Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0068
Minimum - Maximum-0.022104437 - 0.05043143
Average (Standard dev.)0.00012892694 (±0.0013418751)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 272.384 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35161_msk_1.map
Projections & Slices
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Additional map: postprocess unmasked of GLIC pH7.5 asolectin

Fileemd_35161_additional_1.map
Annotationpostprocess unmasked of GLIC pH7.5 asolectin
Projections & Slices
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Additional map: refinement of GLIC pH7.5 asolectin

Fileemd_35161_additional_2.map
Annotationrefinement of GLIC pH7.5 asolectin
Projections & Slices
AxesZYX

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Half map: half map 1 of GLIC pH 7.5 asolectin

Fileemd_35161_half_map_1.map
Annotationhalf map 1 of GLIC pH 7.5 asolectin
Projections & Slices
AxesZYX

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Histogram

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Half map: half map 2 of GLIC pH 7.5 asolectin

Fileemd_35161_half_map_2.map
Annotationhalf map 2 of GLIC pH 7.5 asolectin
Projections & Slices
AxesZYX

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Sample components

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Entire : pentameric ligand-gated ion channel

EntireName: pentameric ligand-gated ion channel
Components
  • Complex: pentameric ligand-gated ion channel
    • Protein or peptide: Proton-gated ion channel
  • Ligand: DIUNDECYL PHOSPHATIDYL CHOLINE

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Supramolecule #1: pentameric ligand-gated ion channel

SupramoleculeName: pentameric ligand-gated ion channel / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Gloeobacter violaceus (bacteria)

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Macromolecule #1: Proton-gated ion channel

MacromoleculeName: Proton-gated ion channel / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Gloeobacter violaceus (bacteria)
Molecular weightTheoretical: 35.846266 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: VSPPPPIADE PLTVNTGIYL IECYSLDDKA ETFKVNAFLS LSWKDRRLAF DPVRSGVRVK TYEPEAIWIP EIRFVNVENA RDADVVDIS VSPDGTVQYL ERFSARVLSP LDFRRYPFDS QTLHIYLIVR SVDTRNIVLA VDLEKVGKND DVFLTGWDIE S FTAVVKPA ...String:
VSPPPPIADE PLTVNTGIYL IECYSLDDKA ETFKVNAFLS LSWKDRRLAF DPVRSGVRVK TYEPEAIWIP EIRFVNVENA RDADVVDIS VSPDGTVQYL ERFSARVLSP LDFRRYPFDS QTLHIYLIVR SVDTRNIVLA VDLEKVGKND DVFLTGWDIE S FTAVVKPA NFALEDRLES KLDYQLRISR QYFSYIPNII LPMLFILFIS WTAFWSTSYE ANVTLVVSTL IAHIAFNILV ET NLPKTPY MTYTGAIIFM IYLFYFVAVI EVTVQHYLKV ESQPARAASI TRASRIAFPV VFLLANIILA FLFFGF

UniProtKB: Proton-gated ion channel

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Macromolecule #2: DIUNDECYL PHOSPHATIDYL CHOLINE

MacromoleculeName: DIUNDECYL PHOSPHATIDYL CHOLINE / type: ligand / ID: 2 / Number of copies: 25 / Formula: PLC
Molecular weightTheoretical: 622.834 Da
Chemical component information

ChemComp-PLC:
DIUNDECYL PHOSPHATIDYL CHOLINE / phospholipid*YM

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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

Concentration1.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES buffer
150.0 mMsodium chlorideNaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 635408
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4pnq

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 69953
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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