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- EMDB-39198: Cryo-EM structure of the channelrhodopsin GtCCR2 -

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Basic information

Entry
Database: EMDB / ID: EMD-39198
TitleCryo-EM structure of the channelrhodopsin GtCCR2
Map data
Sample
  • Complex: GtCCR2
    • Protein or peptide: GtCCR2
  • 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 / plasma membrane / Uncharacterized protein
Function and homology information
Biological speciesGuillardia theta (eukaryote) / Guillardia theta CCMP2712 (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.73 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-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39198.png

Downloads & links

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Map

FileDownload / File: emd_39198.map.gz / Format: CCP4 / Size: 10.9 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 110 pix.
= 104.343 Å		0.95 Å/pix.
x 160 pix.
= 151.771 Å		0.95 Å/pix.
x 162 pix.
= 153.669 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: 2.3
Minimum - Maximum-8.611862 - 12.572191999999999
Average (Standard dev.)0.0020130458 (±0.65845066)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin615990
Dimensions160162110
Spacing162160110
CellA: 153.66858 Å / B: 151.77142 Å / C: 104.34286 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : GtCCR2

EntireName: GtCCR2
Components
  • Complex: GtCCR2
    • Protein or peptide: GtCCR2
  • Ligand: RETINAL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: water

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Supramolecule #1: GtCCR2

SupramoleculeName: GtCCR2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Guillardia theta (eukaryote)

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Macromolecule #1: GtCCR2

MacromoleculeName: GtCCR2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Guillardia theta CCMP2712 (eukaryote) / Strain: CCMP2712
Molecular weightTheoretical: 52.825227 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKTIIALSYI FCLVFADYKD DDDAMGVASS AVITANWISF LAISASFIIL LVISLRYKGP GGTESFYNGF KEQNMLTVFI NLWCALAYF AKVLQSHSND NGFAPLTVIP YVDYCTTCPL LTLDLLWCLD APYKISSAVL VFTCLVIAVA CSLAVAPFSY C WFAMGMVL ...String:
MKTIIALSYI FCLVFADYKD DDDAMGVASS AVITANWISF LAISASFIIL LVISLRYKGP GGTESFYNGF KEQNMLTVFI NLWCALAYF AKVLQSHSND NGFAPLTVIP YVDYCTTCPL LTLDLLWCLD APYKISSAVL VFTCLVIAVA CSLAVAPFSY C WFAMGMVL FTFTYVFILS IVRQRLDFFT LCARDSNAKQ SLKHLKTAVF IYFGIWLLFP LLWLLSYRAA NVISNDINHI FH CILDVIA KSVYGFALLY FKMYFDKKLI ESGIDEDDFA KFSKVVTTHR SEEKQKRKPA VSQSPKMYYD EAAYQDGEVE SNL QSKIRK SLSRKDKTPG SPSRSPMTPR TAYSTRKSPG MHLNDHPAWG SLQASAHSWE NEREGPVGDH DDEEFSEFCQ SLPK KAIPP LNSQPHMYNS EDEDDGAYLE RAKMAYQKAK ERHDNANGQR ERRDRSGSRE SDFENLYFQ

UniProtKB: Uncharacterized protein

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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: 2 / 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: 6 / 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

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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: 9775 / 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: DARK 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: 5791254
Startup modelType of model: NONE
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Details: With C3 symmetry / Number images used: 457536
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: 3 / Avg.num./class: 407614 / Software - Name: RELION (ver. 3.1)

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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-8yek:
Cryo-EM structure of the channelrhodopsin GtCCR2

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