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- EMDB-36339: Cryo-EM structure of nanodisc (PE:PS:PC) reconstituted GLIC at pH 2.5 -

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Basic information

Entry
Database: EMDB / ID: EMD-36339
TitleCryo-EM structure of nanodisc (PE:PS:PC) reconstituted GLIC at pH 2.5
Map dataPostprocess masked map, pH 2.5, GLIC.
Sample
  • Complex: pentameric ligand-gated ion channel
    • Protein or peptide: Proton-gated ion channel
  • Ligand: CHLORIDE ION
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water
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: 2.6476 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
DepositionMay 29, 2023-
Header (metadata) releaseApr 17, 2024-
Map releaseApr 17, 2024-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36339.png

Downloads & links

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Map

FileDownload / File: emd_36339.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocess masked map, pH 2.5, GLIC.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 256 pix.
= 219.75 Å		0.86 Å/pix.
x 256 pix.
= 219.75 Å		0.86 Å/pix.
x 256 pix.
= 219.75 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8584 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0033
Minimum - Maximum-0.02142092 - 0.043091826
Average (Standard dev.)0.00009376981 (±0.0014945527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 219.7504 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36339_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Refinement map, pH 2.5, GLIC.

Fileemd_36339_additional_1.map
AnnotationRefinement map, pH 2.5, GLIC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Postprocess unmasked map, pH 2.5, GLIC.

Fileemd_36339_additional_2.map
AnnotationPostprocess unmasked map, pH 2.5, GLIC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refinement half map 1, pH 2.5, GLIC.

Fileemd_36339_half_map_1.map
AnnotationRefinement half map 1, pH 2.5, GLIC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refinement half map 2, pH 2.5, GLIC.

Fileemd_36339_half_map_2.map
AnnotationRefinement half map 2, pH 2.5, GLIC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: CHLORIDE ION
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water

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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: 35.699094 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 FLFFG

UniProtKB: Proton-gated ion channel

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Macromolecule #2: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 2 / Number of copies: 5 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #3: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 3 / Number of copies: 45 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

ChemComp-PEE:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE, phospholipid*YM

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 265 / 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

Concentration0.3 mg/mL
BufferpH: 2.5
Component:
ConcentrationNameFormula
10.0 mMsodium citrate buffer
150.0 mMsodium chlorideNaCl
1.0 mMEDTA
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 6449 / Average exposure time: 6.0 sec. / Average electron dose: 74.0 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: 0.6 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: 329848
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8i48

Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.6476 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0.1) / Number images used: 98776
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0.1)
Final 3D classificationSoftware - Name: RELION (ver. 4.0.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8i48


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8jj3:
Cryo-EM structure of nanodisc (PE:PS:PC) reconstituted GLIC at pH 2.5

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