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- EMDB-35713: Cryo-EM structure of the potassium-selective channelrhodopsin HcK... -
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Basic information
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Title | Cryo-EM structure of the potassium-selective channelrhodopsin HcKCR1 H225F mutant in lipid nanodisc | ||||||||||||||||||||||||
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![]() | Membrane Protein / Cryo-EM | ||||||||||||||||||||||||
Biological species | ![]() | ||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.66 Å | ||||||||||||||||||||||||
![]() | Tajima S / Kim Y / Nakamura S / Yamashita K / Fukuda M / Deisseroth K / Kato HE | ||||||||||||||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 17.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20.4 KB 20.4 KB | Display Display | ![]() |
Images | ![]() | 177.2 KB | ||
Masks | ![]() | 18.7 MB | ![]() | |
Filedesc metadata | ![]() | 6.3 KB | ||
Others | ![]() ![]() ![]() | 17.1 MB 17.4 MB 17.4 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8iu0MC ![]() 8h86C ![]() 8h87C M: atomic model generated by this map C: citing same article ( |
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Links
EMDB pages | ![]() ![]() |
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Map
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Voxel size | X=Y=Z: 0.83 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
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-Additional map: #1
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-Half map: #2
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-Half map: #1
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Sample components
-Entire : HcKCR1
Entire | Name: HcKCR1 |
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Components |
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-Supramolecule #1: HcKCR1
Supramolecule | Name: HcKCR1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: HcKCR1
Macromolecule | Name: HcKCR1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 31.408795 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: RETINAL
Macromolecule | Name: RETINAL / type: ligand / ID: 2 / Number of copies: 1 / Formula: RET |
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Molecular weight | Theoretical: 284.436 Da |
Chemical component information | ![]() ChemComp-RET: |
-Macromolecule #3: (7R,17E,20E)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)MET...
Macromolecule | Name: (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 |
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Molecular weight | Theoretical: 759.068 Da |
Chemical component information | ![]() ChemComp-PSC: |
-Macromolecule #4: PALMITIC ACID
Macromolecule | Name: PALMITIC ACID / type: ligand / ID: 4 / Number of copies: 4 / Formula: PLM |
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Molecular weight | Theoretical: 256.424 Da |
Chemical component information | ![]() ChemComp-PLM: |
-Macromolecule #5: water
Macromolecule | Name: water / type: ligand / ID: 5 / Number of copies: 26 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ![]() ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Sugar embedding | Material: Lipid / Details: nanodisc composed of MSP1D1E3 and soybean lipids |
Grid | Model: 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. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 48.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination 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 model | Type of model: NONE |
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Final reconstruction | Applied 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 assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |