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- EMDB-45055: Pannexin 1 lacking C-terminal activating domain -

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Basic information

Entry
Database: EMDB / ID: EMD-45055
TitlePannexin 1 lacking C-terminal activating domain
Map data
Sample
  • Complex: frog pannexin 1
    • Protein or peptide: Pannexin
KeywordsATP release channel / nanodisc / large-pore / C-terminal activating domain / MEMBRANE PROTEIN
Function / homology
Function and homology information


positive regulation of interleukin-1 production / gap junction / monoatomic cation transport / channel activity / monoatomic ion transmembrane transport / endoplasmic reticulum membrane / plasma membrane
Similarity search - Function
Pannexin / Innexin / Innexin / Pannexin family profile.
Similarity search - Domain/homology
Biological speciesXenopus tropicalis (tropical clawed frog)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsEhrlich JJ / Kawate T
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM114379 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: The C-terminal activating domain promotes pannexin 1 channel opening.
Authors: Erik Henze / Jacqueline J Ehrlich / Janice L Robertson / Eric Gelsleichter / Toshimitsu Kawate /
Abstract: Pannexin 1 (Panx1) constitutes a large pore channel responsible for the release of adenosine triphosphate (ATP) from apoptotic cells. Strong evidence indicates that caspase-mediated cleavage of the C- ...Pannexin 1 (Panx1) constitutes a large pore channel responsible for the release of adenosine triphosphate (ATP) from apoptotic cells. Strong evidence indicates that caspase-mediated cleavage of the C-terminus promotes the opening of the Panx1 channel by unplugging the pore. However, this simple pore-plugging mechanism alone cannot account for the observation that a Panx1 construct ending before the caspase cleavage site remains closed. Here, we show that a helical region located immediately before the caspase cleavage site, referred to as the "C-terminal activating domain (CAD)", plays a pivotal role in facilitating Panx1 activation. Electrophysiology and mutagenesis studies uncovered that two conserved leucine residues within the CAD play a pivotal role. Cryoelectron microscopy (Cryo-EM) analysis of the construct ending before reaching the CAD demonstrated that the N terminus extends into an intracellular pocket. In contrast, the construct including the CAD revealed that this domain occupies the intracellular pocket, causing the N terminus to flip upward within the pore. Analysis of electrostatic free energy landscape in the closed conformation indicated that the intracellular side of the ion permeation pore may be occupied by anions like ATP, creating an electrostatic barrier for anions attempting to permeate the pore. When the N terminus flips up, it diminishes the positively charged surface, thereby reducing the drive to accumulate anions inside the pore. This dynamic change in the electrostatic landscape likely contributes to the selection of permeant ions. Collectively, these experiments put forth a mechanism in which C-terminal cleavage liberates the CAD, causing the repositioning of the N terminus to promote Panx1 channel opening.
History
DepositionMay 24, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45055.png

Downloads & links

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Map

FileDownload / File: emd_45055.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 208 pix.
= 222.56 Å		1.07 Å/pix.
x 208 pix.
= 222.56 Å		1.07 Å/pix.
x 208 pix.
= 222.56 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.019324228 - 0.051441755
Average (Standard dev.)0.000034869052 (±0.0023654767)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 222.56001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45055_msk_1.map
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Half map: #2

Fileemd_45055_half_map_1.map
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Half map: #1

Fileemd_45055_half_map_2.map
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Sample components

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Entire : frog pannexin 1

EntireName: frog pannexin 1
Components
  • Complex: frog pannexin 1
    • Protein or peptide: Pannexin

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Supramolecule #1: frog pannexin 1

SupramoleculeName: frog pannexin 1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Engineered frog pannexin 1 truncated after amino acid 357 in lipid nanodiscs
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)

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Macromolecule #1: Pannexin

MacromoleculeName: Pannexin / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Xenopus tropicalis (tropical clawed frog)
Molecular weightTheoretical: 39.757516 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: VFSDFLLKDP PESKYKGLRL ELAVDKLVSC IAVGLPLLLI SLAFAQEITL GSQISCFAPT SFSWRQAAYV DSFCWAAVQQ KHLSQSDSG NVPLWLHKFF PYILLLVAVL LYLPNLFWRF TAAPHLSSDL KFVMEELDKC YNRDIKDIKA ANNLNSSDKR D GLNSPVVS ...String:
VFSDFLLKDP PESKYKGLRL ELAVDKLVSC IAVGLPLLLI SLAFAQEITL GSQISCFAPT SFSWRQAAYV DSFCWAAVQQ KHLSQSDSG NVPLWLHKFF PYILLLVAVL LYLPNLFWRF TAAPHLSSDL KFVMEELDKC YNRDIKDIKA ANNLNSSDKR D GLNSPVVS ENLQQSLWEI PLSHYKYPIV EQYLKTKNNS YGLIIKYLIC RVVTLIIVFT ACIYLGYYIS LFSLTDEFTC NI RTGILRN DTALPPLVQC KLIAVGVFRL LSYINLIIYV LIMPFIIYAM LVPFRKTANV LKVYEVLPTF SVQQAPSKTY DDH SLFLLF LEENVSELKS YKFLKVLENI K

UniProtKB: Pannexin

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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

Concentration1.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride

Details: Sample was subject to size exclusion chromatography with HEPES/NaCl buffer.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 45 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288.15 K / Instrument: FEI VITROBOT MARK IV
Details: Grid was blotted for 4 seconds with a force of 7 before plunging into liquid ethane..
DetailsfrPanx1 in lipid nanodiscs containing POPC, POPG, POPE and cholesterol. Sample eluted in a single peak from size-exclusion and was pooled and concentrated.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8713 / Average exposure time: 3.81 sec. / Average electron dose: 50.0 e/Å2
Details: 50-frame movies were collected in counted super resolution mode.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 20.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6416000
Details: Particles picked with template picker in cryoSPARC and non-protein picks were purged using inspect particle picks
Startup modelType of model: INSILICO MODEL
In silico model: Used ab initio job in cryoSPARC to generate starting map
Details: Used 5 classes and imposed C7 symmetry
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Reconstruction generated in Relion using 3D classification and 3D autorefinement
Number images used: 25322
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 8
Details: Classification initially carried out with alignments, then when estimated accuracy angles was below 3 degrees, sorted particles without alignments
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6vd7


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9bz7:
Pannexin 1 lacking C-terminal activating domain

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