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- PDB-6uiw: Cryo-EM structure of human CALHM2 in a ruthenium red-bound inhibi... -

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Basic information

Entry
Database: PDB / ID: 6uiw
TitleCryo-EM structure of human CALHM2 in a ruthenium red-bound inhibited state
ComponentsCalcium homeostasis modulator protein 2
KeywordsTRANSPORT PROTEIN/INHIBITOR / calcium homeostasis modulator / CALHM2 / ruthenium red / TRANSPORT PROTEIN-INHIBITOR complex
Function / homologyCalcium homeostasis modulator family / Calcium homeostasis modulator protein 2 / Calcium homeostasis modulator / cation channel activity / positive regulation of apoptotic process / integral component of plasma membrane / Calcium homeostasis modulator protein 2
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsLu, W. / Du, J. / Choi, W.
CitationJournal: Nature / Year: 2019
Title: The structures and gating mechanism of human calcium homeostasis modulator 2.
Authors: Wooyoung Choi / Nicolina Clemente / Weinan Sun / Juan Du / Wei Lü /
Abstract: Calcium homeostasis modulators (CALHMs) are voltage-gated, Ca-inhibited nonselective ion channels that act as major ATP release channels, and have important roles in gustatory signalling and neuronal ...Calcium homeostasis modulators (CALHMs) are voltage-gated, Ca-inhibited nonselective ion channels that act as major ATP release channels, and have important roles in gustatory signalling and neuronal toxicity. Dysfunction of CALHMs has previously been linked to neurological disorders. Here we present cryo-electron microscopy structures of the human CALHM2 channel in the Ca-free active or open state and in the ruthenium red (RUR)-bound inhibited state, at resolutions up to 2.7 Å. Our work shows that purified CALHM2 channels form both gap junctions and undecameric hemichannels. The protomer shows a mirrored arrangement of the transmembrane domains (helices S1-S4) relative to other channels with a similar topology, such as connexins, innexins and volume-regulated anion channels. Upon binding to RUR, we observed a contracted pore with notable conformational changes of the pore-lining helix S1, which swings nearly 60° towards the pore axis from a vertical to a lifted position. We propose a two-section gating mechanism in which the S1 helix coarsely adjusts, and the N-terminal helix fine-tunes, the pore size. We identified a RUR-binding site near helix S1 that may stabilize this helix in the lifted conformation, giving rise to channel inhibition. Our work elaborates on the principles of CALHM2 channel architecture and symmetry, and the mechanism that underlies channel inhibition.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 1, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 27, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Dec 11, 2019Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Dec 18, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Calcium homeostasis modulator protein 2
B: Calcium homeostasis modulator protein 2
C: Calcium homeostasis modulator protein 2
D: Calcium homeostasis modulator protein 2
E: Calcium homeostasis modulator protein 2
F: Calcium homeostasis modulator protein 2
G: Calcium homeostasis modulator protein 2
H: Calcium homeostasis modulator protein 2
I: Calcium homeostasis modulator protein 2
J: Calcium homeostasis modulator protein 2
K: Calcium homeostasis modulator protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)415,34022
Polymers409,18511
Non-polymers6,15511
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Calcium homeostasis modulator protein 2 / Protein FAM26B


Mass: 37198.676 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CALHM2, FAM26B / Production host: Homo sapiens (human) / References: UniProt: Q9HA72
#2: Chemical
ChemComp-R2R / ruthenium(6+) azanide pentaamino(oxido)ruthenium (1/4/2) / Ruthenium red


Mass: 559.525 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: H28N14O2Ru3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

ComponentName: human CALHM2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 54.4 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEM3.7image acquisition
4GctfCTF correction
9RELION3initial Euler assignment
10RELION300final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.16model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 716120
SymmetryPoint symmetry: C11 (11 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83728 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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