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- EMDB-32378: 2.12 angstrom cryo-EM map of the pump-like channelrhodopsin ChRmi... -

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Basic information

Entry
Database: EMDB / ID: EMD-32378
Title2.12 angstrom cryo-EM map of the pump-like channelrhodopsin ChRmine with Fab antibody fragment
Map data
Sample
  • Complex: ChRmine
    • Protein or peptide: ChRmine with Fab
Keywordschannelrhodopsin / ion channel / photoreceptor / cryo-EM / MEMBRANE PROTEIN
Biological speciesRhodomonas lens (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.12 Å
AuthorsKishi KE / Kim Y / Fukuda M / Yamashita K / Deisseroth K / Kato HE
Funding support Japan, 8 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21wm0525018 Japan
Japan Society for the Promotion of Science (JSPS)21H05142 Japan
Japan Science and TechnologyJPMJPR1782 Japan
Japan Science and TechnologyJPMJFR204S Japan
Japan Science and TechnologyJPMJCR21P3 Japan
Other private Japan
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R01MH075957 Japan
Other private Japan
CitationJournal: Cell / Year: 2022
Title: Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.
Authors: Koichiro E Kishi / Yoon Seok Kim / Masahiro Fukuda / Masatoshi Inoue / Tsukasa Kusakizako / Peter Y Wang / Charu Ramakrishnan / Eamon F X Byrne / Elina Thadhani / Joseph M Paggi / Toshiki E ...Authors: Koichiro E Kishi / Yoon Seok Kim / Masahiro Fukuda / Masatoshi Inoue / Tsukasa Kusakizako / Peter Y Wang / Charu Ramakrishnan / Eamon F X Byrne / Elina Thadhani / Joseph M Paggi / Toshiki E Matsui / Keitaro Yamashita / Takashi Nagata / Masae Konno / Sean Quirin / Maisie Lo / Tyler Benster / Tomoko Uemura / Kehong Liu / Mikihiro Shibata / Norimichi Nomura / So Iwata / Osamu Nureki / Ron O Dror / Keiichi Inoue / Karl Deisseroth / Hideaki E Kato /
Abstract: ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created ...ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
History
DepositionDec 11, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.021
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.021
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_32378.png

Downloads & links

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Map

FileDownload / File: emd_32378.map.gz / Format: CCP4 / Size: 18.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.94318 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.021
Minimum - Maximum-0.033065453 - 0.10009595
Average (Standard dev.)-0.00026260447 (±0.004992477)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions170170170
Spacing170170170
CellA=B=C: 160.34094 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.943182352941180.943182352941180.94318235294118
M x/y/z170170170
origin x/y/z0.0000.0000.000
length x/y/z160.341160.341160.341
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS170170170
D min/max/mean-0.0330.100-0.000

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Supplemental data

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Mask #1

Fileemd_32378_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Histogram (log scale)

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Half map: #1

Fileemd_32378_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32378_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : ChRmine

EntireName: ChRmine
Components
  • Complex: ChRmine
    • Protein or peptide: ChRmine with Fab

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Supramolecule #1: ChRmine

SupramoleculeName: ChRmine / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rhodomonas lens (eukaryote)

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Macromolecule #1: ChRmine with Fab

MacromoleculeName: ChRmine with Fab / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Rhodomonas lens (eukaryote)
SequenceString: GPMAHAPGTD QMFYVGTMDG WYLDTKLNSV AIGAHWSCFI VLTITTFYLG YESWTSRGPS KRTSFYAGYQ EEQNLALFVN FFAMLSYFGK IVADTLGHNF GDVGPFIIGF GNYRYADYML TCPMLVYDLL YQLRAPYRVS CSAIIFAILM SGVLAEFYAE GDPRLRNGAY ...String:
GPMAHAPGTD QMFYVGTMDG WYLDTKLNSV AIGAHWSCFI VLTITTFYLG YESWTSRGPS KRTSFYAGYQ EEQNLALFVN FFAMLSYFGK IVADTLGHNF GDVGPFIIGF GNYRYADYML TCPMLVYDLL YQLRAPYRVS CSAIIFAILM SGVLAEFYAE GDPRLRNGAY AWYGFGCFWF IFAYSIVMSI VAKQYSRLAQ LAQDTGAEHS LHVLKFAVFT FSMLWILFPL VWAICPRGFG WIDDNWTEVA HCVCDIVAKS CYGFALARFR KTYDEELFRL LEQLGHDEDE FQKLELDMRL SSNGERLEVL FQ

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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
GridModel: Quantifoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -0.0016 µm / Nominal defocus min: -0.0008 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 51.533 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.12 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 185895
FSC plot (resolution estimation)

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