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- PDB-8gi9: Cation channelrhodopsin from Hyphochytrium catenoides (HcCCR) emb... -

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Basic information

Entry
Database: PDB / ID: 8gi9
TitleCation channelrhodopsin from Hyphochytrium catenoides (HcCCR) embedded in peptidisc
ComponentsCation Channelrhodopsin
KeywordsTRANSPORT PROTEIN / Retinal Protein / Channelrhodopsin / Cation channel / Peptidisc / Optogenetics
Function / homologyCHOLESTEROL / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / RETINAL
Function and homology information
Biological speciesHyphochytrium catenoides (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsMorizumi, T. / Kim, K. / Li, H. / Spudich, J.L. / Ernst, O.P.
Funding support Canada, 3items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-04397 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2017-06862 Canada
Canadian Institutes of Health Research (CIHR)PJT-159464 Canada
CitationJournal: Nat Commun / Year: 2023
Title: Structures of channelrhodopsin paralogs in peptidiscs explain their contrasting K and Na selectivities.
Authors: Takefumi Morizumi / Kyumhyuk Kim / Hai Li / Elena G Govorunova / Oleg A Sineshchekov / Yumei Wang / Lei Zheng / Éva Bertalan / Ana-Nicoleta Bondar / Azam Askari / Leonid S Brown / John L ...Authors: Takefumi Morizumi / Kyumhyuk Kim / Hai Li / Elena G Govorunova / Oleg A Sineshchekov / Yumei Wang / Lei Zheng / Éva Bertalan / Ana-Nicoleta Bondar / Azam Askari / Leonid S Brown / John L Spudich / Oliver P Ernst /
Abstract: Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K over Na in the absence of the canonical ...Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K over Na in the absence of the canonical tetrameric K selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a highly homologous cation channelrhodopsin (HcCCR), a Na channel with >100-fold larger Na to K permeability ratio. Here, we use cryo-electron microscopy to determine atomic structures of these two channels embedded in peptidiscs to elucidate structural foundations of their dramatically different cation selectivity. Together with structure-guided mutagenesis, we show that K versus Na selectivity is determined at two distinct sites on the putative ion conduction pathway: in a patch of critical residues in the intracellular segment (Leu69/Phe69, Ile73/Ser73 and Asp116) and within a cluster of aromatic residues in the extracellular segment (primarily, Trp102 and Tyr222). The two filters are on the opposite sides of the photoactive site involved in channel gating.
History
DepositionMar 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 26, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting / em_3d_fitting_list
Item: _em_3d_fitting_list.3d_fitting_id
Revision 1.2May 1, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cation Channelrhodopsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,77214
Polymers30,2241
Non-polymers4,54813
Water27015
1
A: Cation Channelrhodopsin
hetero molecules

A: Cation Channelrhodopsin
hetero molecules

A: Cation Channelrhodopsin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,31742
Polymers90,6733
Non-polymers13,64439
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation2
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C3 (3 fold cyclic))

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Cation Channelrhodopsin


Mass: 30224.213 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hyphochytrium catenoides (eukaryote) / Plasmid: HcCCR_pPICZalpha-A / Production host: Komagataella pastoris (fungus) / Strain (production host): SMD1168

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Non-polymers , 5 types, 28 molecules

#2: Chemical ChemComp-RET / RETINAL / Retinal


Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na
#4: Chemical
ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C27H46O
#5: Chemical ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy


Mass: 744.034 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: Cation channelrhodopsin trimer reconstituted in peptidisc
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.030197 MDa / Experimental value: NO
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
220 mMHEPESC8H18N2O4S1
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was kept in the dark prior to freezing.
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: OTHER / Num. of grids imaged: 1 / Num. of real images: 5902 / Details: Falcon 4i detector
Image scansWidth: 4096 / Height: 4096

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1cryoSPARC4.1particle selection
2EPUimage acquisition
4cryoSPARC4.1CTF correction
7PHENIX1.2model fitting
9cryoSPARC4.1initial Euler assignment
10cryoSPARC4.1final Euler assignment
11cryoSPARC4.1classification
12cryoSPARC4.13D reconstruction
13PHENIX1.2model refinement
Image processingDetails: Falcon 4i detector was used for collection. Images were reference corrected.
CTF correctionDetails: CTF estimation done in cryoSPARC v4.1 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2674318
Details: Blob picking was performed on a subset of micrographs for generating templates for template picking.
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 298957 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 151.1 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross correlation coefficent / Details: Initial fitting done in Phenix
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 59.26 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00282362
ELECTRON MICROSCOPYf_angle_d0.44633235
ELECTRON MICROSCOPYf_chiral_restr0.0323352
ELECTRON MICROSCOPYf_plane_restr0.0032350
ELECTRON MICROSCOPYf_dihedral_angle_d15.423799

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