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- EMDB-25135: Apo-ChRmine in MSP1E3D1 lipid nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-25135
TitleApo-ChRmine in MSP1E3D1 lipid nanodisc
Map dataApo-ChRmine in MSP1E3D1 lipid nanodisc
Sample
  • Complex: ChRmine trimer
    • Protein or peptide: ChRmine
KeywordsChannelrhodopsin / ion channel / MEMBRANE PROTEIN
Biological speciesTiarina fusa (eukaryote) / Rhodomonas lens (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsTucker K / Brohawn S
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structures of the channelrhodopsin ChRmine in lipid nanodiscs.
Authors: Kyle Tucker / Savitha Sridharan / Hillel Adesnik / Stephen G Brohawn /
Abstract: Microbial channelrhodopsins are light-gated ion channels widely used for optogenetic manipulation of neuronal activity. ChRmine is a bacteriorhodopsin-like cation channelrhodopsin (BCCR) more closely ...Microbial channelrhodopsins are light-gated ion channels widely used for optogenetic manipulation of neuronal activity. ChRmine is a bacteriorhodopsin-like cation channelrhodopsin (BCCR) more closely related to ion pump rhodopsins than other channelrhodopsins. ChRmine displays unique properties favorable for optogenetics including high light sensitivity, a broad, red-shifted activation spectrum, cation selectivity, and large photocurrents, while its slow closing kinetics impedes some applications. The structural basis for ChRmine function, or that of any other BCCR, is unknown. Here, we present cryo-EM structures of ChRmine in lipid nanodiscs in apo (opsin) and retinal-bound (rhodopsin) forms. The structures reveal an unprecedented trimeric architecture with a lipid filled central pore. Large electronegative cavities on either side of the membrane facilitate high conductance and selectivity for cations over protons. The retinal binding pocket structure suggests channel properties could be tuned with mutations and we identify ChRmine variants with ten-fold decreased and two-fold increased closing rates. A T119A mutant shows favorable properties relative to wild-type and previously reported ChRmine variants for optogenetics. These results provide insight into structural features that generate an ultra-potent microbial opsin and provide a platform for rational engineering of channelrhodopsins with improved properties that could expand the scale, depth, and precision of optogenetic experiments.
History
DepositionOct 11, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7shs
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25135.png

Downloads & links

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Map

FileDownload / File: emd_25135.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationApo-ChRmine in MSP1E3D1 lipid nanodisc
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 220 pix.
= 250.14 Å		1.14 Å/pix.
x 220 pix.
= 250.14 Å		1.14 Å/pix.
x 220 pix.
= 250.14 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.137 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.3
Minimum - Maximum-0.1824221 - 0.7685214
Average (Standard dev.)0.00074595265 (±0.03797524)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 250.14 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1371.1371.137
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z250.140250.140250.140
α/β/γ90.00090.00090.000
start NX/NY/NZ-140-140-140
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.1820.7690.001

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Supplemental data

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Sample components

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Entire : ChRmine trimer

EntireName: ChRmine trimer
Components
  • Complex: ChRmine trimer
    • Protein or peptide: ChRmine

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Supramolecule #1: ChRmine trimer

SupramoleculeName: ChRmine trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Tiarina fusa (eukaryote)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: ChRmine

MacromoleculeName: ChRmine / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Rhodomonas lens (eukaryote)
Molecular weightTheoretical: 36.471758 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: (AYA)HAPGTDQMF YVGTMDGWYL DTKLNSVAIG AHWSCFIVLT ITTFYLGYES WTSRGPSKRT SFYAGYQEEQ NLALFV NFF AMLSYFGKIV ADTLGHNFGD VGPFIIGFGN YRYADYMLTC PMLVYDLLYQ LRAPYRVSCS AIIFAILMSG VLAEFYA EG DPRLRNGAYA ...String:
(AYA)HAPGTDQMF YVGTMDGWYL DTKLNSVAIG AHWSCFIVLT ITTFYLGYES WTSRGPSKRT SFYAGYQEEQ NLALFV NFF AMLSYFGKIV ADTLGHNFGD VGPFIIGFGN YRYADYMLTC PMLVYDLLYQ LRAPYRVSCS AIIFAILMSG VLAEFYA EG DPRLRNGAYA WYGFGCFWFI FAYSIVMSIV AKQYSRLAQL AQDTGAEHSL HVLKFAVFTF SMLWILFPLV WAICPRGF G WIDDNWTEVA HCVCDIVAKS CYGFALARFR KTYDEELFRL LEQLGHDEDE FQKLELDMRL SSNGERLRRL SLNSLEVLF Q

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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

Concentration14.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3TRIS
150.0 mMNaClSodium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS TALOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Particle selectionNumber selected: 7054805
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 41053
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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