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- PDB-6zgj: GLIC pentameric ligand-gated ion channel, pH 5 -

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Basic information

Entry
Database: PDB / ID: 6zgj
TitleGLIC pentameric ligand-gated ion channel, pH 5
ComponentsProton-gated ion channel
KeywordsMEMBRANE PROTEIN / GLIC / pentameric ligand-gated ion channel / cryoem
Function / homology
Function and homology information


sodium channel activity / transmembrane transporter complex / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRovsnik, U. / Zhuang, Y. / Forsberg, B.O. / Carroni, M. / Yvonnesdotter, L. / Howard, R.J. / Lindahl, E.
Funding support Sweden, 2items
OrganizationGrant numberCountry
Swedish Research Council Sweden
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Life Sci Alliance / Year: 2021
Title: Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations.
Authors: Urška Rovšnik / Yuxuan Zhuang / Björn O Forsberg / Marta Carroni / Linnea Yvonnesdotter / Rebecca J Howard / Erik Lindahl /
Abstract: Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high- ...Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electron microscopy (cryo-EM) structures of the proton-activated ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1-β2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.
History
DepositionJun 18, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 26, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 8, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / em_imaging
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_imaging.nominal_defocus_max / _em_imaging.nominal_defocus_min
Revision 1.2Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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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


Theoretical massNumber of molelcules
Total (without water)181,4595
Polymers181,4595
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20700 Å2
ΔGint-167 kcal/mol
Surface area66800 Å2
MethodPISA

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Components

#1: Protein
Proton-gated ion channel / GLIC / Ligand-gated ion channel / LGIC


Mass: 36291.750 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gloeobacter violaceus (strain ATCC 29082 / PCC 7421) (bacteria)
Strain: ATCC 29082 / PCC 7421 / Gene: glvI, glr4197 / Production host: Escherichia coli (E. coli) / References: UniProt: Q7NDN8

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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 ion channel / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: NO
Source (natural)Organism: Gloeobacter violaceus (strain ATCC 29082 / PCC 7421) (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: blot for 1.5 s force -1

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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 magnification: 165000 X / Nominal defocus max: 3800 nm / Nominal defocus min: 2000 nm / Calibrated defocus min: 0.9 nm / Calibrated defocus max: 2.8 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 80 K
Image recordingAverage exposure time: 6 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6000
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9PHENIX1.15model refinement
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 700000
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 300000 / Symmetry type: POINT
Atomic model buildingB value: 278 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 4NPQ

4npq


Pdb chain-ID: A / Accession code: 4NPQ / Pdb chain residue range: 5-315 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511875
ELECTRON MICROSCOPYf_angle_d0.58516330
ELECTRON MICROSCOPYf_dihedral_angle_d4.8621665
ELECTRON MICROSCOPYf_chiral_restr0.0431975
ELECTRON MICROSCOPYf_plane_restr0.0042040

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