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- EMDB-39199: Cryo-EM structure of the channelrhodopsin GtCCR4 -

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Basic information

Entry
Database: EMDB / ID: EMD-39199
TitleCryo-EM structure of the channelrhodopsin GtCCR4
Map data
Sample
  • Complex: GtCCR4
    • Protein or peptide: Cation channel rhodopsin 4
  • Ligand: RETINAL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water
KeywordsMicrobial rhodopsin / MEMBRANE PROTEIN
Function / homologyBacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein / membrane / Cation channel rhodopsin 4
Function and homology information
Biological speciesGuillardia theta (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsTanaka T / Iida W / Sano FK / Oda K / Shihoya W / Nureki O
Funding support Japan, 8 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22KJ0577 Japan
Japan Society for the Promotion of Science (JSPS)19H05777 Japan
Japan Society for the Promotion of Science (JSPS)22K19371 Japan
Japan Society for the Promotion of Science (JSPS)22H02751 Japan
Japan Society for the Promotion of Science (JSPS)21H05037 Japan
Japan Agency for Medical Research and Development (AMED)JP233fa627001 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121002 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121012 Japan
CitationJournal: Mol Cell / Year: 2024
Title: The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4.
Authors: Tatsuki Tanaka / Shoko Hososhima / Yo Yamashita / Teppei Sugimoto / Toshiki Nakamura / Shunta Shigemura / Wataru Iida / Fumiya K Sano / Kazumasa Oda / Takayuki Uchihashi / Kota Katayama / ...Authors: Tatsuki Tanaka / Shoko Hososhima / Yo Yamashita / Teppei Sugimoto / Toshiki Nakamura / Shunta Shigemura / Wataru Iida / Fumiya K Sano / Kazumasa Oda / Takayuki Uchihashi / Kota Katayama / Yuji Furutani / Satoshi P Tsunoda / Wataru Shihoya / Hideki Kandori / Osamu Nureki /
Abstract: Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), ...Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.
History
DepositionFeb 22, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39199.png

Downloads & links

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Map

FileDownload / File: emd_39199.map.gz / Format: CCP4 / Size: 4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 98 pix.
= 92.96 Å		0.95 Å/pix.
x 104 pix.
= 98.651 Å		0.95 Å/pix.
x 104 pix.
= 98.651 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.94857 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.58
Minimum - Maximum-5.538939 - 7.9420033
Average (Standard dev.)-0.00652942 (±0.61596775)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin9187104
Dimensions10410498
Spacing10410498
CellA: 98.65143 Å / B: 98.65143 Å / C: 92.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : GtCCR4

EntireName: GtCCR4
Components
  • Complex: GtCCR4
    • Protein or peptide: Cation channel rhodopsin 4
  • Ligand: RETINAL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water

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Supramolecule #1: GtCCR4

SupramoleculeName: GtCCR4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Guillardia theta (eukaryote)

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Macromolecule #1: Cation channel rhodopsin 4

MacromoleculeName: Cation channel rhodopsin 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Guillardia theta (eukaryote)
Molecular weightTheoretical: 45.933734 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAMGTTSA PSLSDPNWQY GMGGWNNPRL PNFNLHDPTV IGVDWLGFLC LLGASLALMY KLMSFKGPD GDQEFFVGYR EEKCLSIYVN LIAAITYWGR ICAHFNNDMG LSLSVNYFKY LDYIFTCPIL TLDLLWSLNL P YKITYSLF ...String:
MKTIIALSYI FCLVFADYKD DDDAMGTTSA PSLSDPNWQY GMGGWNNPRL PNFNLHDPTV IGVDWLGFLC LLGASLALMY KLMSFKGPD GDQEFFVGYR EEKCLSIYVN LIAAITYWGR ICAHFNNDMG LSLSVNYFKY LDYIFTCPIL TLDLLWSLNL P YKITYSLF VGLTIACGVF CNAFEPPARY LWFMFGCFIF AFTWISIIRL VYARFQQFLN EDAKKIRAPL KLSLTLYFSI WC GYPALWL LTEFGAISQL AAHVTTVIMD VAAKSVYGFA LLKFQLGVDK RDVWLDELKS VRYRDVVPQI RPSKTREGRM EYS EDGDFM RPSKGKRAEG DYMNPRWDHH DDGRRLPDSR EMDEQVHEKD QEISSTMKQI ADLNKQLSAM QESEAVENLY FQ

UniProtKB: Cation channel rhodopsin 4

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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: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 3 / Number of copies: 3 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 9 / 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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris(hydroxymethyl)aminomethane
150.0 mMNaClSodium chloride
2.0 mMC4H10O2S2Dithiothreitol
0.01 %C56H92O29Digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThe protein was reconstituted in lipid nanodiscs.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 16366 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5403048
Startup modelType of model: NONE
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Details: With C3 symmetry / Number images used: 581115
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 2 / Avg.num./class: 301522 / Software - Name: cryoSPARC (ver. 3.3.2)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8yel:
Cryo-EM structure of the channelrhodopsin GtCCR4

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