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Yorodumi- EMDB-23002: Cryo-EM structure of the CRAC channel Orai in an open conformatio... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23002 | |||||||||
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Title | Cryo-EM structure of the CRAC channel Orai in an open conformation; H206A gain-of-function mutation in complex with an antibody | |||||||||
Map data | cryo-EM map | |||||||||
Sample |
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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 | |||||||||
Biological species | Drosophila melanogaster (fruit fly) / Mus musculus (house mouse) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
Authors | Long SB / Hou X / Outhwaite IR | |||||||||
Funding support | United States, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_23002.map.gz | 67.3 MB | EMDB map data format | |
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Header (meta data) | emd-23002-v30.xml emd-23002.xml | 15.3 KB 15.3 KB | Display Display | EMDB header |
Images | emd_23002.png | 199.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23002 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23002 | HTTPS FTP |
-Related structure data
Related structure data | 7kr5MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_23002.map.gz / Format: CCP4 / Size: 72.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | cryo-EM map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.87078 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Orai channel, H206A mutant, in complex with three Fab fragments
Entire | Name: Orai channel, H206A mutant, in complex with three Fab fragments |
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Components |
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-Supramolecule #1: Orai channel, H206A mutant, in complex with three Fab fragments
Supramolecule | Name: 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
Supramolecule | Name: Orai channel, H206A mutant / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Drosophila melanogaster (fruit fly) |
Recombinant expression | Organism: Komagataella pastoris (fungus) |
-Supramolecule #3: Fab fragments
Supramolecule | Name: Fab fragments / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Mus musculus (house mouse) |
-Macromolecule #1: Calcium release-activated calcium channel protein 1
Macromolecule | Name: Calcium release-activated calcium channel protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Drosophila melanogaster (fruit fly) |
Molecular weight | Theoretical: 24.160168 KDa |
Recombinant expression | Organism: Komagataella pastoris (fungus) |
Sequence | String: 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 |
-Macromolecule #2: 19B5 Fab heavy chain
Macromolecule | Name: 19B5 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 26.886477 KDa |
Recombinant expression | Organism: Mus musculus (house mouse) |
Sequence | String: 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 |
-Macromolecule #3: 19B5 Fab light chain
Macromolecule | Name: 19B5 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 25.587295 KDa |
Recombinant expression | Organism: Mus musculus (house mouse) |
Sequence | String: 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 |
-Macromolecule #4: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8.5 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy |
Image recording | Film 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 |
-Image processing
Particle selection | Number selected: 236132 |
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CTF correction | Software - Name: CTFFIND |
Startup model | Type of model: INSILICO MODEL / In silico model: abinitio |
Initial angle assignment | Type: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2) |
Final 3D classification | Number classes: 1 |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2) |
Final reconstruction | Number 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 |