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- EMDB-23002: Cryo-EM structure of the CRAC channel Orai in an open conformatio... -

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-23002
TitleCryo-EM structure of the CRAC channel Orai in an open conformation; H206A gain-of-function mutation in complex with an antibody
Map datacryo-EM map
Sample
  • Complex: Orai channel, H206A mutant, in complex with three Fab fragments
    • Complex: Orai channel, H206A mutant
      • Protein or peptide: Calcium release-activated calcium channel protein 1
    • Complex: Fab fragments
      • Protein or peptide: 19B5 Fab heavy chain
      • Protein or peptide: 19B5 Fab light chain
  • Ligand: CALCIUM IONCalcium
Function / homology
Function and homology information


store-operated calcium entry / store-operated calcium channel activity / positive regulation of calcineurin-NFAT signaling cascade / positive regulation of calcium ion transport / calcium channel regulator activity / calcium channel complex / calcium ion transmembrane transport / calcium channel activity / nervous system development / membrane ...store-operated calcium entry / store-operated calcium channel activity / positive regulation of calcineurin-NFAT signaling cascade / positive regulation of calcium ion transport / calcium channel regulator activity / calcium channel complex / calcium ion transmembrane transport / calcium channel activity / nervous system development / membrane / identical protein binding / plasma membrane
Similarity search - Function
Calcium release-activated calcium channel protein / Orai superfamily / Mediator of CRAC channel activity
Similarity search - Domain/homology
Calcium release-activated calcium channel protein 1
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLong SB / Hou X / Outhwaite IR
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131921 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM094273 United States
CitationJournal: Elife / Year: 2020
Title: Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation.
Authors: Xiaowei Hou / Ian R Outhwaite / Leanne Pedi / Stephen Barstow Long /
Abstract: The calcium release-activated calcium channel Orai regulates Ca entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca release from ...The calcium release-activated calcium channel Orai regulates Ca entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Orai in an open conformation (Hou et al., 2018). Here we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-electron microscopy. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance.
History
DepositionNov 18, 2020-
Header (metadata) releaseDec 9, 2020-
Map releaseDec 9, 2020-
UpdateDec 9, 2020-
Current statusDec 9, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kr5
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23002.png

Downloads & links

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Map

FileDownload / File: emd_23002.map.gz / Format: CCP4 / Size: 72.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM map
Voxel sizeX=Y=Z: 0.87078 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-3.6395504 - 5.2895193
Average (Standard dev.)0.0014398491 (±0.13753884)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin107107101
Dimensions267267267
Spacing267267267
CellA=B=C: 232.4972 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870775280898880.870775280898880.87077528089888
M x/y/z267267267
origin x/y/z0.0000.0000.000
length x/y/z232.497232.497232.497
α/β/γ90.00090.00090.000
start NX/NY/NZ107107101
NX/NY/NZ267267267
MAP C/R/S213
start NC/NR/NS107107101
NC/NR/NS267267267
D min/max/mean-3.6405.2900.001

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Supplemental data

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Sample components

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Entire : Orai channel, H206A mutant, in complex with three Fab fragments

EntireName: Orai channel, H206A mutant, in complex with three Fab fragments
Components
  • Complex: Orai channel, H206A mutant, in complex with three Fab fragments
    • Complex: Orai channel, H206A mutant
      • Protein or peptide: Calcium release-activated calcium channel protein 1
    • Complex: Fab fragments
      • Protein or peptide: 19B5 Fab heavy chain
      • Protein or peptide: 19B5 Fab light chain
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Orai channel, H206A mutant, in complex with three Fab fragments

SupramoleculeName: Orai channel, H206A mutant, in complex with three Fab fragments
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: Orai channel, H206A mutant

SupramoleculeName: Orai channel, H206A mutant / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Recombinant expressionOrganism: Komagataella pastoris (fungus)

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Supramolecule #3: Fab fragments

SupramoleculeName: Fab fragments / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Mus musculus (house mouse)

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Macromolecule #1: Calcium release-activated calcium channel protein 1

MacromoleculeName: Calcium release-activated calcium channel protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 24.160168 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MSQSGEDLHS PTYLSWRKLQ LSRAKLKASS KTSALLSGFA MVAMVEVQLD HDTNVPPGML IAFAICTTLL VAVAMLALMI STCILPNIE TVSNLHSISL VHESPHERLH WYIETAWAFS TLLGLILFLL EIAILCWVKF YDLSPPAAWS ATVVLIPVMI I FMAFAIHF ...String:
MSQSGEDLHS PTYLSWRKLQ LSRAKLKASS KTSALLSGFA MVAMVEVQLD HDTNVPPGML IAFAICTTLL VAVAMLALMI STCILPNIE TVSNLHSISL VHESPHERLH WYIETAWAFS TLLGLILFLL EIAILCWVKF YDLSPPAAWS ATVVLIPVMI I FMAFAIHF YRSLVSHKYE VTVSGIRELE MLKEQMEQDH LEHHNNIRNN GEGEEF

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Macromolecule #2: 19B5 Fab heavy chain

MacromoleculeName: 19B5 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.886477 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MTLNMLLGLK WVFFVVFYQG VHCEVQLVES GGGLVQPKGS LKLSCAASGF TFNTYAMHWV RQAPGKGLEW VARIRTKSNN YATYYADSV KDRFTISRDD SQSMLYLQMN NLKTEDTAMY YCVRQKYGNY FDYWGQGTTL TVSSAKTTPP SVYPLAPGSA A QTNSMVTL ...String:
MTLNMLLGLK WVFFVVFYQG VHCEVQLVES GGGLVQPKGS LKLSCAASGF TFNTYAMHWV RQAPGKGLEW VARIRTKSNN YATYYADSV KDRFTISRDD SQSMLYLQMN NLKTEDTAMY YCVRQKYGNY FDYWGQGTTL TVSSAKTTPP SVYPLAPGSA A QTNSMVTL GCLVKGYFPE PVTVTWNSGS LSSGVHTFPA VLQSDLYTLS SSVTVPSSSW PSETVTCNVA HPASSTKVDK KI VPRD

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Macromolecule #3: 19B5 Fab light chain

MacromoleculeName: 19B5 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.587295 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MESQTQVFVY MLLWLSGVDG DVVMTQSQKF MSTSVGDRVS VTCKASQNVG INVAWYQQKP GQSPKALINS ASYRNSGVPD RFTGGGSGT DFTLTINNVQ SEDLAEYFCQ QCNSYPLTFG AGTKLELRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF Y PKDINVKW ...String:
MESQTQVFVY MLLWLSGVDG DVVMTQSQKF MSTSVGDRVS VTCKASQNVG INVAWYQQKP GQSPKALINS ASYRNSGVPD RFTGGGSGT DFTLTINNVQ SEDLAEYFCQ QCNSYPLTFG AGTKLELRRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF Y PKDINVKW KIDGSERQNG VLNSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRN

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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: 8.5
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Average exposure time: 10.0 sec. / Average electron dose: 76.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 236132
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: INSILICO MODEL / In silico model: abinitio
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2)
Final 3D classificationNumber classes: 1
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 85614

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