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- EMDB-8993: Class III PI3K Complex 2 with inhibitor Rubicon -

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Basic information

Entry
Database: EMDB / ID: 8993
TitleClass III PI3K Complex 2 with inhibitor Rubicon
Map dataCryoEM map between 6-8 angstroms of the Class III PI3K Complex 2, bound to an inhibitor from Rubicon
SampleCore scaffold of PI3K Complex 2 bound to inhibitor Rubicon:
SourceHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / 6.8 Å resolution
AuthorsYoung LN / Morris KL / Hurley JH
CitationJournal: Mol. Cell / Year: 2019
Title: Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.
Authors: Chunmei Chang / Lindsey N Young / Kyle L Morris / Sören von Bülow / Johannes Schöneberg / Hitomi Yamamoto-Imoto / Yukako Oe / Kentaro Yamamoto / Shuhei Nakamura / Goran Stjepanovic / Gerhard Hummer / Tamotsu Yoshimori / James H Hurley
Abstract: Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed ...Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first β sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA β sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 β sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.
DateDeposition: Jul 25, 2018 / Header (metadata) release: Aug 29, 2018 / Map release: Jan 23, 2019 / Last update: Feb 6, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8993.map.gz (map file in CCP4 format, 174457 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
352 pix
1.15 Å/pix.
= 404.8 Å
352 pix
1.15 Å/pix.
= 404.8 Å
352 pix
1.15 Å/pix.
= 404.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density
Contour Level:0.03 (by author), 0.03 (movie #1):
Minimum - Maximum-0.034428053 - 0.09238863
Average (Standard dev.)0.00019581411 (0.002899992)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions352352352
Origin0.00.00.0
Limit351.0351.0351.0
Spacing352352352
CellA=B=C: 404.8 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z404.800404.800404.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-16-63-38
NX/NY/NZ727975
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.0340.0920.000

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Supplemental data

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Mask #1

Fileemd_8993_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire Core scaffold of PI3K Complex 2 bound to inhibitor Rubicon

EntireName: Core scaffold of PI3K Complex 2 bound to inhibitor Rubicon
Number of components: 1

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Component #1: protein, Core scaffold of PI3K Complex 2 bound to inhibitor Rubicon

ProteinName: Core scaffold of PI3K Complex 2 bound to inhibitor Rubicon
Recombinant expression: No
MassTheoretical: 360 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Strain: HEK 293 GNTI

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.2 mg/ml / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: OTHER / Temperature: 288 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 59.7 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 42708
3D reconstructionSoftware: RELION / CTF correction: GCTF / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible
Input PDB model: 5DFZ, 4DDP

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