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- EMDB-11209: GLIC pentameric ligand-gated ion channel, pH 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-11209
TitleGLIC pentameric ligand-gated ion channel, pH 3
Map dataGLIC, pentameric ligand-gated ion channel, pH 3, sharpened map
Sample
  • Complex: Pentameric ion channel
    • Protein or peptide: Proton-gated ion channel
Function / homology
Function and homology information


sodium channel activity / extracellular ligand-gated monoatomic ion channel activity / potassium 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 (strain ATCC 29082 / PCC 7421) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsRovsnik U / Zhuang Y / Forsberg BO / Carroni M / Yvonnesdotter L / Howard RJ / Lindahl E
Funding support Sweden, 2 items
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, 2020-
Header (metadata) releaseMay 26, 2021-
Map releaseMay 26, 2021-
UpdateDec 8, 2021-
Current statusDec 8, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zgk
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11209.png

Downloads & links

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Map

FileDownload / File: emd_11209.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGLIC, pentameric ligand-gated ion channel, pH 3, sharpened map
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.14370015 - 0.25243294
Average (Standard dev.)0.00042262045 (±0.0066132187)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 209.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z209.920209.920209.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1440.2520.000

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Supplemental data

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Mask #1

Fileemd_11209_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: GLIC, pentameric ligand-gated ion channel, pH 3, unsharpened map

Fileemd_11209_additional_1.map
AnnotationGLIC, pentameric ligand-gated ion channel, pH 3, unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: GLIC, pentameric ligand-gated ion channel, pH 3, half map 1

Fileemd_11209_half_map_1.map
AnnotationGLIC, pentameric ligand-gated ion channel, pH 3, half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: GLIC, pentameric ligand-gated ion channel, pH 3, half map 2

Fileemd_11209_half_map_2.map
AnnotationGLIC, pentameric ligand-gated ion channel, pH 3, half map 2
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 ion channel

EntireName: Pentameric ion channel
Components
  • Complex: Pentameric ion channel
    • Protein or peptide: Proton-gated ion channel

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Supramolecule #1: Pentameric ion channel

SupramoleculeName: Pentameric ion channel / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Gloeobacter violaceus (strain ATCC 29082 / PCC 7421) (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 180 KDa

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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 (strain ATCC 29082 / PCC 7421) (bacteria)
Strain: ATCC 29082 / PCC 7421
Molecular weightTheoretical: 36.29175 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AQDMVSPPPP IADEPLTVNT GIYLIECYSL DDKAETFKVN AFLSLSWKDR RLAFDPVRSG VRVKTYEPEA IWIPEIRFVN VENARDADV VDISVSPDGT VQYLERFSAR VLSPLDFRRY PFDSQTLHIY LIVRSVDTRN IVLAVDLEKV GKNDDVFLTG W DIESFTAV ...String:
AQDMVSPPPP IADEPLTVNT GIYLIECYSL DDKAETFKVN AFLSLSWKDR RLAFDPVRSG VRVKTYEPEA IWIPEIRFVN VENARDADV VDISVSPDGT VQYLERFSAR VLSPLDFRRY PFDSQTLHIY LIVRSVDTRN IVLAVDLEKV GKNDDVFLTG W DIESFTAV VKPANFALED RLESKLDYQL RISRQYFSYI PNIILPMLFI LFISWTAFWS TSYEANVTLV VSTLIAHIAF NI LVETNLP KTPYMTYTGA IIFMIYLFYF VAVIEVTVQH YLKVESQPAR AASITRASRI AFPVVFLLAN IILAFLFFGF

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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

Concentration3 mg/mL
BufferpH: 3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 1.5 s force -1.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 0.0028 µm / Calibrated defocus min: 0.0009000000000000001 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.8000000000000003 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 6000 / Average exposure time: 6.0 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 900000
CTF correctionSoftware - Name: RELION
Startup modelType of model: OTHER / Details: SGD ab initio - Relion
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.1) / Details: 10%, 8%, 82% of the final map
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 177000
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4npq


Chain - Chain ID: A / Chain - Residue range: 5-315
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 278
Output model

PDB-6zgk:
GLIC pentameric ligand-gated ion channel, pH 3

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