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- EMDB-35713: Cryo-EM structure of the potassium-selective channelrhodopsin HcK... -

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Basic information

Entry
Database: EMDB / ID: EMD-35713
TitleCryo-EM structure of the potassium-selective channelrhodopsin HcKCR1 H225F mutant in lipid nanodisc
Map data
Sample
  • Complex: HcKCR1
    • Protein or peptide: HcKCR1
  • Ligand: RETINAL
  • Ligand: (7R,17E,20E)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSA-17,20-DIEN-1-AMINIUM 4-OXIDE
  • Ligand: PALMITIC ACID
  • Ligand: water
KeywordsMembrane Protein / Cryo-EM
Biological speciesHyphochytrium catenoides (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsTajima S / Kim Y / Nakamura S / Yamashita K / Fukuda M / Deisseroth K / Kato HE
Funding support Japan, 7 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21wm0525018 Japan
Japan Society for the Promotion of Science (JSPS)22H04742 Japan
Japan Society for the Promotion of Science (JSPS)JP20K21383 Japan
Japan Society for the Promotion of Science (JSPS)JP21H01875 Japan
Japan Society for the Promotion of Science (JSPS)21H05142 Japan
Japan Society for the Promotion of Science (JSPS)22H00400 Japan
Japan Society for the Promotion of Science (JSPS)22K19265 Japan
CitationJournal: Cell / Year: 2023
Title: Structural basis for ion selectivity in potassium-selective channelrhodopsins.
Authors: Seiya Tajima / Yoon Seok Kim / Masahiro Fukuda / YoungJu Jo / Peter Y Wang / Joseph M Paggi / Masatoshi Inoue / Eamon F X Byrne / Koichiro E Kishi / Seiwa Nakamura / Charu Ramakrishnan / ...Authors: Seiya Tajima / Yoon Seok Kim / Masahiro Fukuda / YoungJu Jo / Peter Y Wang / Joseph M Paggi / Masatoshi Inoue / Eamon F X Byrne / Koichiro E Kishi / Seiwa Nakamura / Charu Ramakrishnan / Shunki Takaramoto / Takashi Nagata / Masae Konno / Masahiro Sugiura / Kota Katayama / Toshiki E Matsui / Keitaro Yamashita / Suhyang Kim / Hisako Ikeda / Jaeah Kim / Hideki Kandori / Ron O Dror / Keiichi Inoue / Karl Deisseroth / Hideaki E Kato /
Abstract: KCR channelrhodopsins (K-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K ...KCR channelrhodopsins (K-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K selectivity; rather than forming the symmetrical filter of canonical K channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K selectivity also provides a framework for next-generation optogenetics.
History
DepositionMar 23, 2023-
Header (metadata) releaseSep 6, 2023-
Map releaseSep 6, 2023-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35713.png

Downloads & links

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Map

FileDownload / File: emd_35713.map.gz / Format: CCP4 / Size: 18.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 170 pix.
= 141.1 Å		0.83 Å/pix.
x 170 pix.
= 141.1 Å		0.83 Å/pix.
x 170 pix.
= 141.1 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.613
Minimum - Maximum-1.6720041 - 2.4328141
Average (Standard dev.)0.0036998587 (±0.124590516)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions170170170
Spacing170170170
CellA=B=C: 141.09999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35713_msk_1.map
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Additional map: #1

Fileemd_35713_additional_1.map
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Half map: #2

Fileemd_35713_half_map_1.map
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Half map: #1

Fileemd_35713_half_map_2.map
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Sample components

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Entire : HcKCR1

EntireName: HcKCR1
Components
  • Complex: HcKCR1
    • Protein or peptide: HcKCR1
  • Ligand: RETINAL
  • Ligand: (7R,17E,20E)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSA-17,20-DIEN-1-AMINIUM 4-OXIDE
  • Ligand: PALMITIC ACID
  • Ligand: water

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Supramolecule #1: HcKCR1

SupramoleculeName: HcKCR1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)

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Macromolecule #1: HcKCR1

MacromoleculeName: HcKCR1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)
Molecular weightTheoretical: 31.408795 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GPMPFYDSRP PEGWPKGSIN DMDYPLLGSI CAVCCVFVAG SGIWMLYRLD LGMGYSCKPY KSGRAPEVNS LSGIICLLCG TMYAAKSFD FFDGGGTPFS LNWYWYLDYV FTCPLLILDF AFTLDLPHKI RYFFAVFLTL WCGVAAFVTP SAYRFAYYAL G CCWFTPFA ...String:
GPMPFYDSRP PEGWPKGSIN DMDYPLLGSI CAVCCVFVAG SGIWMLYRLD LGMGYSCKPY KSGRAPEVNS LSGIICLLCG TMYAAKSFD FFDGGGTPFS LNWYWYLDYV FTCPLLILDF AFTLDLPHKI RYFFAVFLTL WCGVAAFVTP SAYRFAYYAL G CCWFTPFA LSLMRHVKER YLVYPPKCQR WLFWACVIFF GFWPMFPILF IFSWLGTGHI SQQAFYIIFA FLDLTCKSIF GI LMTVFRL ELEEHTEVQG LPLNEPETLS LEVLFQ

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Macromolecule #2: RETINAL

MacromoleculeName: RETINAL / type: ligand / ID: 2 / Number of copies: 1 / Formula: RET
Molecular weightTheoretical: 284.436 Da
Chemical component information

ChemComp-RET:
RETINAL

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Macromolecule #3: (7R,17E,20E)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)MET...

MacromoleculeName: (7R,17E,20E)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSA-17,20-DIEN-1-AMINIUM 4-OXIDE
type: ligand / ID: 3 / Number of copies: 10 / Formula: PSC
Molecular weightTheoretical: 759.068 Da
Chemical component information

ChemComp-PSC:
(7R,17E,20E)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSA-17,20-DIEN-1-AMINIUM 4-OXIDE / phospholipid*YM

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Macromolecule #4: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 4 / Number of copies: 4 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 26 / 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

BufferpH: 7.5
Sugar embeddingMaterial: Lipid / Details: nanodisc composed of MSP1D1E3 and soybean lipids
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.0 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: 1.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 180294
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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