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- PDB-7wsv: Cryo-EM structure of the N-terminal deletion mutant of human pann... -

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Basic information

Entry
Database: PDB / ID: 7wsv
TitleCryo-EM structure of the N-terminal deletion mutant of human pannexin-1 in a nanodisc
ComponentsPannexin-1
KeywordsMEMBRANE PROTEIN / ATP release channel / vertebrate innexin homolog
Function / homology
Function and homology information


Electric Transmission Across Gap Junctions / leak channel activity / positive regulation of interleukin-1 alpha production / bleb / wide pore channel activity / gap junction channel activity / gap junction / positive regulation of macrophage cytokine production / oogenesis / response to ATP ...Electric Transmission Across Gap Junctions / leak channel activity / positive regulation of interleukin-1 alpha production / bleb / wide pore channel activity / gap junction channel activity / gap junction / positive regulation of macrophage cytokine production / oogenesis / response to ATP / monoatomic cation transport / The NLRP3 inflammasome / positive regulation of interleukin-1 beta production / response to ischemia / calcium channel activity / calcium ion transport / actin filament binding / cell-cell signaling / scaffold protein binding / protease binding / transmembrane transporter binding / signaling receptor binding / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / protein-containing complex / membrane / identical protein binding / plasma membrane
Similarity search - Function
Pannexin / Innexin / Innexin / Pannexin family profile.
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsKuzuya, M. / Hirano, H. / Hayashida, K. / Watanabe, M. / Kobayashi, K. / Tani, K. / Fujiyoshi, Y. / Oshima, A.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP20ae0101051 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)JP20H03165 Japan
New Energy and Industrial Technology Development Organization (NEDO)JP20am0101118 Japan
CitationJournal: Sci Signal / Year: 2022
Title: Structures of human pannexin-1 in nanodiscs reveal gating mediated by dynamic movement of the N terminus and phospholipids.
Authors: Maki Kuzuya / Hidemi Hirano / Kenichi Hayashida / Masakatsu Watanabe / Kazumi Kobayashi / Tohru Terada / Md Iqbal Mahmood / Florence Tama / Kazutoshi Tani / Yoshinori Fujiyoshi / Atsunori Oshima /
Abstract: Pannexin (PANX) family proteins form large-pore channels that mediate purinergic signaling. We analyzed the cryo-EM structures of human PANX1 in lipid nanodiscs to elucidate the gating mechanism and ...Pannexin (PANX) family proteins form large-pore channels that mediate purinergic signaling. We analyzed the cryo-EM structures of human PANX1 in lipid nanodiscs to elucidate the gating mechanism and its regulation by the amino terminus in phospholipids. The wild-type channel has an amino-terminal funnel in the pore, but in the presence of the inhibitor probenecid, a cytoplasmically oriented amino terminus and phospholipids obstruct the pore. Functional analysis using whole-cell patch-clamp and oocyte voltage clamp showed that PANX1 lacking the amino terminus did not open and had a dominant negative effect on channel activity, thus confirming that the amino-terminal domain played an essential role in channel opening. These observations suggest that dynamic conformational changes in the amino terminus of human PANX1 are associated with lipid movement in and out of the pore. Moreover, the data provide insight into the gating mechanism of PANX1 and, more broadly, other large-pore channels.
History
DepositionFeb 1, 2022Deposition site: PDBJ / Processing site: PDBJ
SupersessionFeb 16, 2022ID: 7F8Q
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Pannexin-1
B: Pannexin-1
C: Pannexin-1
D: Pannexin-1
E: Pannexin-1
F: Pannexin-1
G: Pannexin-1


Theoretical massNumber of molelcules
Total (without water)321,6887
Polymers321,6887
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1PHESERA1 - 293
d_21ens_1PHESERB1 - 293
d_31ens_1PHESERC1 - 293
d_41ens_1PHESERD1 - 293
d_51ens_1PHESERE1 - 293
d_61ens_1PHESERF1 - 293
d_71ens_1PHESERG1 - 293

NCS oper:
IDCodeMatrixVector
1given(0.613629934666, 0.789591833343, 0.00174355977602), (-0.789593600655, 0.613626920307, 0.00198707783369), (0.00049908521397, -0.00259603408276, 0.999996505754)-39.8047144705, 116.157760875, 0.260206785406
2given(-0.229200303237, 0.973320428814, -0.0107034503074), (-0.973378490432, -0.229200258103, 0.00124741743578), (-0.00123909669955, 0.0107044167572, 0.999941938365)25.7963728035, 217.170796871, -1.05614944145
3given(-0.900777886931, 0.434277553425, -0.00148492693536), (-0.434276511536, -0.900779079287, -0.00098073788561), (-0.00176350356716, -0.000238558110858, 0.999998416571)144.581604483, 230.099553543, 0.0372880652177
4given(-0.90715832828, -0.420779400902, -0.00290915939509), (0.420785177567, -0.907158603104, -0.00176157732477), (-0.00189783352169, -0.00282216069371, 0.999994216802)229.173607694, 146.780154921, 0.549443069318
5given(-0.246244026463, -0.969192986796, 0.00536970910728), (0.969199894614, -0.246215758733, 0.00541889584657), (-0.00392984884864, 0.00653869223313, 0.999970900473)218.224593051, 27.4460860183, -0.292758491147
6given(0.612714368642, -0.79030289823, -0.00155932968714), (0.790300666127, 0.612704158045, 0.00429788711385), (-0.00244122485927, -0.00386571647991, 0.999989548274)116.270376641, -39.6699957499, 0.761742176078

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Components

#1: Protein
Pannexin-1 /


Mass: 45955.438 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PANX1, MRS1, UNQ2529/PRO6028 / Production host: Homo sapiens (human) / References: UniProt: Q96RD7

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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: Heptamar of N-terminal deleted human pannexin-1 in a nanodisc
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5 / Details: pH 7.5 was used.
Buffer component
IDConc.NameFormulaBuffer-ID
110 mM2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acidHEPES1
2300 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 3.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: MOLYBDENUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA KF80 / Cryogen name: ETHANE
Details: Blot for 10 seconds at room temperature followed by plunge freezing. Humidity and temperature are not controlled.

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Electron microscopy imaging

MicroscopyModel: JEOL 3000SFF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 30000 X / Calibrated magnification: 40600 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1400 nm / Calibrated defocus min: 1400 nm / Calibrated defocus max: 3500 nm / Cs: 1.6 mm
Specimen holderCryogen: HELIUM / Specimen holder model: JEOL / Temperature (max): 100 K / Temperature (min): 80 K
Image recordingAverage exposure time: 8 sec. / Electron dose: 56 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3587
Image scansWidth: 3710 / Height: 3838

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
9RELION3initial Euler assignment
12RELION33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 3005895
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98796 / Symmetry type: POINT
Atomic model buildingB value: 344.6 / Protocol: RIGID BODY FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 94.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316807
ELECTRON MICROSCOPYf_angle_d0.717222799
ELECTRON MICROSCOPYf_chiral_restr0.04212716
ELECTRON MICROSCOPYf_plane_restr0.00382751
ELECTRON MICROSCOPYf_dihedral_angle_d3.83232184
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000708508705703
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.0998668770296
ens_1d_4AELECTRON MICROSCOPYNCS constraints0.000710370377874
ens_1d_5AELECTRON MICROSCOPYNCS constraints0.0655880660261
ens_1d_6AELECTRON MICROSCOPYNCS constraints0.000709184332723
ens_1d_7AELECTRON MICROSCOPYNCS constraints0.000710600012715

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