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- PDB-8i42: Cryo-EM structure of nanodisc (PE:PS:PC) reconstituted GLIC at pH 7.5 -

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Basic information

Entry
Database: PDB / ID: 8i42
TitleCryo-EM structure of nanodisc (PE:PS:PC) reconstituted GLIC at pH 7.5
ComponentsProton-gated ion channel
KeywordsMEMBRANE PROTEIN / pentameric ligand-gated ion channels / cis-loop / cryo-EM / nanodisc
Function / homology
Function and homology information


sodium channel activity / potassium channel activity / transmembrane transporter complex / 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
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Proton-gated ion channel
Similarity search - Component
Biological speciesGloeobacter violaceus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsBharambe, N. / Li, Z. / Basak, S.
Funding support Singapore, 1items
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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proton-gated ion channel
B: Proton-gated ion channel
C: Proton-gated ion channel
D: Proton-gated ion channel
E: Proton-gated ion channel
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,25745
Polymers178,4955
Non-polymers29,76140
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Proton-gated ion channel


Mass: 35699.094 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gloeobacter violaceus (bacteria) / Gene: glvI / Production host: Escherichia coli (E. coli) / References: UniProt: Q7NDN8
#2: Chemical...
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 40 / Source method: obtained synthetically / Formula: C41H78NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: DOPE, phospholipid*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: pentameric ligand-gated ion channel / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Gloeobacter violaceus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES buffer1
2150 mMsodium chlorideNaCl1
31 mMEDTAC10H16N2O81
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1400 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 71 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 14001
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7Coot0.9.8.7model fitting
9PHENIX1.21model refinement
10RELION4.0.1initial Euler assignment
11RELION4.0.1final Euler assignment
12RELION4.0.1classification
13RELION4.0.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 788636
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152938 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314365
ELECTRON MICROSCOPYf_angle_d0.58919355
ELECTRON MICROSCOPYf_dihedral_angle_d13.3172535
ELECTRON MICROSCOPYf_chiral_restr0.0422125
ELECTRON MICROSCOPYf_plane_restr0.0052315

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