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
Citation
Journal: 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.
Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The protein was reconstituted in lipid nanodiscs.
Average exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16366
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Processing
EM software
ID
Name
Version
Category
1
cryoSPARC
3.3.2
particleselection
4
cryoSPARC
3.3.2
CTFcorrection
7
MOLREP
modelfitting
9
PHENIX
modelrefinement
10
Servalcat
modelrefinement
11
cryoSPARC
3.3.2
initialEulerassignment
12
cryoSPARC
3.3.2
finalEulerassignment
13
cryoSPARC
3.3.2
classification
14
cryoSPARC
3.3.2
3Dreconstruction
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 5403048
3D reconstruction
Resolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 581115 / Algorithm: FOURIER SPACE / Details: With C3 symmetry / Symmetry type: POINT
Atomic model building
Protocol: RIGID BODY FIT / Space: REAL
Atomic model building
Source name: AlphaFold / Type: in silico model
Refinement
Resolution: 2.71→2.71 Å / Cor.coef. Fo:Fc: 0.83 / SU B: 2.676 / SU ML: 0.085 / ESU R: 0.103 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS