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- EMDB-32768: Cryo-EM structure of the N-terminal deletion mutant of human pann... -

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Basic information

Entry
Database: EMDB / ID: EMD-32768
TitleCryo-EM structure of the N-terminal deletion mutant of human pannexin-1 in a nanodisc
Map data3D map of the N-terminal deletion mutant of human pannexin-1
Sample
  • Complex: Heptamar of N-terminal deleted human pannexin-1 in a nanodisc
    • Protein or peptide: Pannexin-1
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)
Methodsingle 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, 3 items
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, 2022-
SupersessionFeb 16, 2022ID: EMD-31492
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateFeb 23, 2022-
Current statusFeb 23, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7wsv
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_32768.png

Downloads & links

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Map

FileDownload / File: emd_32768.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of the N-terminal deletion mutant of human pannexin-1
Voxel sizeX=Y=Z: 1.232 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.10664548 - 0.14792345
Average (Standard dev.)2.9632942e-05 (±0.008361217)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 197.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2321.2321.232
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z197.120197.120197.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1070.1480.000

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Supplemental data

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Mask #1

Fileemd_32768_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_32768_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32768_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Heptamar of N-terminal deleted human pannexin-1 in a nanodisc

EntireName: Heptamar of N-terminal deleted human pannexin-1 in a nanodisc
Components
  • Complex: Heptamar of N-terminal deleted human pannexin-1 in a nanodisc
    • Protein or peptide: Pannexin-1

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Supramolecule #1: Heptamar of N-terminal deleted human pannexin-1 in a nanodisc

SupramoleculeName: Heptamar of N-terminal deleted human pannexin-1 in a nanodisc
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Pannexin-1

MacromoleculeName: Pannexin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 45.955438 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTEPKFKGLR LELAVDKMVT CIAVGLPLLL ISLAFAQEIS IGTQISCFSP SSFSWRQAAF VDSYCWAAVQ QKNSLQSESG NLPLWLHKF FPYILLLFAI LLYLPPLFWR FAAAPHICSD LKFIMEELDK VYNRAIKAAK SARDLDMRDG ACSVPGVTEN L GQSLWEVS ...String:
MTEPKFKGLR LELAVDKMVT CIAVGLPLLL ISLAFAQEIS IGTQISCFSP SSFSWRQAAF VDSYCWAAVQ QKNSLQSESG NLPLWLHKF FPYILLLFAI LLYLPPLFWR FAAAPHICSD LKFIMEELDK VYNRAIKAAK SARDLDMRDG ACSVPGVTEN L GQSLWEVS ESHFKYPIVE QYLKTKKNSN NLIIKYISCR LLTLIIILLA CIYLGYYFSL SSLSDEFVCS IKSGILRNDS TV PDQFQCK LIAVGIFQLL SVINLVVYVL LAPVVVYTLF VPFRQKTDVL KVYEILPTFD VLHFKSEGYN DLSLYNLFLE ENI SEVKSY KCLKVLENIK SSGQGIDPML LLTNLGMIKM DVVDGKTPMS AEMREEQGNQ TAELQGMNID SETKANNGEK NARQ RLLDS SC

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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

Concentration3.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMHEPES2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid
300.0 mMNaClSodium chloridesodium chloride

Details: pH 7.5 was used.
GridModel: Quantifoil R2/2 / Material: MOLYBDENUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: LEICA KF80
Details: Blot for 10 seconds at room temperature followed by plunge freezing. Humidity and temperature are not controlled..

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Electron microscopy

MicroscopeJEOL 3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.5 µm / Calibrated defocus min: 1.4000000000000001 µm / Calibrated magnification: 40600 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: JEOL / Cooling holder cryogen: HELIUM
TemperatureMin: 80.0 K / Max: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Number grids imaged: 1 / Number real images: 3587 / Average exposure time: 8.0 sec. / Average electron dose: 56.0 e/Å2

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Image processing

Particle selectionNumber selected: 3005895
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 98796
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 344.6
Output model

PDB-7wsv:
Cryo-EM structure of the N-terminal deletion mutant of human pannexin-1 in a nanodisc

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