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- PDB-7kr5: Cryo-EM structure of the CRAC channel Orai in an open conformatio... -

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Basic information

Entry
Database: PDB / ID: 7kr5
TitleCryo-EM structure of the CRAC channel Orai in an open conformation; H206A gain-of-function mutation in complex with an antibody
Components
  • 19B5 Fab heavy chain
  • 19B5 Fab light chain
  • Calcium release-activated calcium channel protein 1
KeywordsTRANSPORT PROTEIN/IMMUNE SYSTEM / ion channel / calcium / SOCE / CRAC / eukaryotic / open structure / MEMBRANE PROTEIN / amphiboles / cryo-EM / TRANSPORT PROTEIN-IMMUNE SYSTEM complex
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLong, S.B. / Hou, X. / Outhwaite, I.R.
Funding support United States, 2items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Calcium release-activated calcium channel protein 1
B: Calcium release-activated calcium channel protein 1
C: Calcium release-activated calcium channel protein 1
D: Calcium release-activated calcium channel protein 1
E: Calcium release-activated calcium channel protein 1
F: Calcium release-activated calcium channel protein 1
H: 19B5 Fab heavy chain
L: 19B5 Fab light chain
M: 19B5 Fab heavy chain
N: 19B5 Fab light chain
O: 19B5 Fab heavy chain
P: 19B5 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)302,42213
Polymers302,38212
Non-polymers401
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area23880 Å2
ΔGint-257 kcal/mol
Surface area60700 Å2

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Components

#1: Protein
Calcium release-activated calcium channel protein 1 / Protein orai


Mass: 24160.168 Da / Num. of mol.: 6 / Mutation: H206A, C224S, C283T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Orai, CRACM1, olf186-F, CG11430 / Production host: Komagataella pastoris (fungus) / References: UniProt: Q9U6B8
#2: Antibody 19B5 Fab heavy chain


Mass: 26886.477 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody 19B5 Fab light chain


Mass: 25587.295 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Orai channel, H206A mutant, in complex with three Fab fragmentsCOMPLEX#1-#30MULTIPLE SOURCES
2Orai channel, H206A mutantCOMPLEX#11RECOMBINANT
3Fab fragmentsCOMPLEX#2-#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Drosophila melanogaster (fruit fly)7227
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Komagataella pastoris (fungus)4922
23Mus musculus (house mouse)10090
Buffer solutionpH: 8.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 10 sec. / Electron dose: 76 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
4CTFFINDCTF correction
10cryoSPARC2initial Euler assignment
11cryoSPARC2final Euler assignment
13cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 236132
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85614 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410795
ELECTRON MICROSCOPYf_angle_d0.55914774
ELECTRON MICROSCOPYf_dihedral_angle_d15.7883465
ELECTRON MICROSCOPYf_chiral_restr0.0371798
ELECTRON MICROSCOPYf_plane_restr0.0041808

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