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- EMDB-20390: Activated Class III PI-3 Kinase by NRBF2 -

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Basic information

Entry
Database: EMDB / ID: EMD-20390
TitleActivated Class III PI-3 Kinase by NRBF2
Map dataFull length NRBF2 pushes PI3KC3-C1 to an activate conformation in which peripheral membrane binding protein VPS34 is posed to phosphorylate substrate phosphatidylinositol.
Sample
  • Complex: Class III PI 3-Kinase Complex 1 containing NRBF2-MIT-linker-BECN1, ATG14, VPS34, VPS15
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsYoung LN / Goerdeler F / Hurley JH
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer InstituteF99 CA2230329 United States
National Institutes of Health/National Institute of General Medical Sciences051487 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Structural pathway for allosteric activation of the autophagic PI 3-kinase complex I.
Authors: Lindsey N Young / Felix Goerdeler / James H Hurley /
Abstract: Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is ...Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol (PI) 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the proautophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryoelectron microscopy was used to visualize a 2-step PI3KC3-C1 activation pathway driven by NRFB2 MIT domain binding. Binding of a single NRBF2 MIT domain bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.
History
DepositionJul 1, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseOct 2, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0127
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0127
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20390.png

Downloads & links

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Map

FileDownload / File: emd_20390.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull length NRBF2 pushes PI3KC3-C1 to an activate conformation in which peripheral membrane binding protein VPS34 is posed to phosphorylate substrate phosphatidylinositol.
Voxel sizeX=Y=Z: 1.137 Å
Density
Contour LevelBy AUTHOR: 0.0127 / Movie #1: 0.0127
Minimum - Maximum-0.022104606 - 0.05694269
Average (Standard dev.)0.00011574636 (±0.0014363729)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 400.224 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1371.1371.137
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z400.224400.224400.224
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.0220.0570.000

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Supplemental data

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Mask #1

Fileemd_20390_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map.

Fileemd_20390_additional.map
AnnotationUnsharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map.

Fileemd_20390_additional_1.map
AnnotationUnsharpened 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 : Class III PI 3-Kinase Complex 1 containing NRBF2-MIT-linker-BECN1...

EntireName: Class III PI 3-Kinase Complex 1 containing NRBF2-MIT-linker-BECN1, ATG14, VPS34, VPS15
Components
  • Complex: Class III PI 3-Kinase Complex 1 containing NRBF2-MIT-linker-BECN1, ATG14, VPS34, VPS15

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Supramolecule #1: Class III PI 3-Kinase Complex 1 containing NRBF2-MIT-linker-BECN1...

SupramoleculeName: Class III PI 3-Kinase Complex 1 containing NRBF2-MIT-linker-BECN1, ATG14, VPS34, VPS15
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK 293 GNTI / Recombinant cell: Kidney cells / Recombinant plasmid: pCAG vectors
Molecular weightTheoretical: 400 KDa

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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

Concentration0.2 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 88 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 6.4 sec. / Average electron dose: 0.98 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER / Details: Ab initio from cryosparc
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.4) / Details: Local resolution: 5.4-18 angstroms. / Number images used: 86218
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.4) / Details: from Relion
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

5dfz

,

4ph4

,

4zey

,

4pph

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 97

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