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- EMDB-4727: Cryo-EM Structure of the PI3-Kinase SH3 Domain Amyloid Fibril -

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Basic information

Entry
Database: EMDB / ID: EMD-4727
TitleCryo-EM Structure of the PI3-Kinase SH3 Domain Amyloid Fibril
Map dataEM reconstruction of sh3 fibril, map sharpened with a B-factor of -150 A2 and low-pass filtered to 3.0 A.
Sample
  • Complex: Fibril of PI3K-SH3 domains
    • Protein or peptide: Phosphatidylinositol 3-kinase regulatory subunit alpha
Keywordsamyloid fibril / sh3 domain / PROTEIN FIBRIL
Function / homology
Function and homology information


Signaling by ALK / MET activates PI3K/AKT signaling / RHOC GTPase cycle / CDC42 GTPase cycle / RAC1 GTPase cycle / RAC2 GTPase cycle / RHOD GTPase cycle / RHOJ GTPase cycle / RAC3 GTPase cycle / RHOF GTPase cycle ...Signaling by ALK / MET activates PI3K/AKT signaling / RHOC GTPase cycle / CDC42 GTPase cycle / RAC1 GTPase cycle / RAC2 GTPase cycle / RHOD GTPase cycle / RHOJ GTPase cycle / RAC3 GTPase cycle / RHOF GTPase cycle / FLT3 Signaling / RND3 GTPase cycle / RND2 GTPase cycle / RND1 GTPase cycle / PI3K events in ERBB4 signaling / Interleukin-7 signaling / GAB1 signalosome / PI3K events in ERBB2 signaling / Erythropoietin activates Phosphoinositide-3-kinase (PI3K) / IRS-mediated signalling / GPVI-mediated activation cascade / Signaling by SCF-KIT / Downstream signal transduction / PI3K/AKT activation / Role of phospholipids in phagocytosis / Tie2 Signaling / Role of LAT2/NTAL/LAB on calcium mobilization / Costimulation by the CD28 family / CD28 dependent PI3K/Akt signaling / RAF/MAP kinase cascade / Interleukin receptor SHC signaling / PI-3K cascade:FGFR1 / PI-3K cascade:FGFR2 / PI-3K cascade:FGFR3 / PI-3K cascade:FGFR4 / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / PI3K Cascade / PIP3 activates AKT signaling / GP1b-IX-V activation signalling / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / Synthesis of PIPs at the plasma membrane / RHOA GTPase cycle / DAP12 signaling / RHOU GTPase cycle / RHOV GTPase cycle / Regulation of signaling by CBL / Downstream TCR signaling / RHOG GTPase cycle / RET signaling / Interleukin-3, Interleukin-5 and GM-CSF signaling / VEGFA-VEGFR2 Pathway / phosphatidylinositol 3-kinase regulator activity / phosphatidylinositol 3-kinase activator activity / 1-phosphatidylinositol-3-kinase regulator activity / ErbB-3 class receptor binding / transmembrane receptor protein tyrosine kinase adaptor activity / positive regulation of endoplasmic reticulum unfolded protein response / enzyme-substrate adaptor activity / phosphatidylinositol 3-kinase complex / Extra-nuclear estrogen signaling / phosphatidylinositol 3-kinase complex, class IA / G alpha (q) signalling events / intracellular glucose homeostasis / phosphatidylinositol phosphate biosynthetic process / insulin receptor substrate binding / phosphatidylinositol 3-kinase binding / insulin-like growth factor receptor binding / response to endoplasmic reticulum stress / substrate adhesion-dependent cell spreading / positive regulation of RNA splicing / insulin-like growth factor receptor signaling pathway / phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of protein localization to plasma membrane / positive regulation of glucose import / insulin receptor binding / positive regulation of protein import into nucleus / cellular response to insulin stimulus / protein transport / insulin receptor signaling pathway / protein stabilization / negative regulation of apoptotic process / positive regulation of transcription by RNA polymerase II / identical protein binding / nucleus
Similarity search - Function
Phosphatidylinositol 3-kinase regulatory subunit alpha, SH3 domain / PIK3R1, inter-SH2 domain / PI3K p85 subunit, C-terminal SH2 domain / PI3K regulatory subunit p85-related , inter-SH2 domain / PI3K p85 subunit, N-terminal SH2 domain / Phosphatidylinositol 3-kinase regulatory subunit P85 inter-SH2 domain / Rho GTPase-activating protein domain / RhoGAP domain / Rho GTPase-activating proteins domain profile. / GTPase-activator protein for Rho-like GTPases ...Phosphatidylinositol 3-kinase regulatory subunit alpha, SH3 domain / PIK3R1, inter-SH2 domain / PI3K p85 subunit, C-terminal SH2 domain / PI3K regulatory subunit p85-related , inter-SH2 domain / PI3K p85 subunit, N-terminal SH2 domain / Phosphatidylinositol 3-kinase regulatory subunit P85 inter-SH2 domain / Rho GTPase-activating protein domain / RhoGAP domain / Rho GTPase-activating proteins domain profile. / GTPase-activator protein for Rho-like GTPases / Rho GTPase activation protein / SH2 domain / Src homology 2 (SH2) domain profile. / Src homology 2 domains / SH2 domain / Src homology 3 domains / SH2 domain superfamily / SH3-like domain superfamily / Src homology 3 (SH3) domain profile. / SH3 domain
Similarity search - Domain/homology
Phosphatidylinositol 3-kinase regulatory subunit alpha
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodhelical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRoeder C / Vettore N
Funding support Germany, 4 items
OrganizationGrant numberCountry
European Commission726368 Germany
German Research FoundationSFB 974 Germany
German Research FoundationSFB 1208 Germany
German Research Foundation Germany
CitationJournal: Nat Commun / Year: 2019
Title: Atomic structure of PI3-kinase SH3 amyloid fibrils by cryo-electron microscopy.
Authors: Christine Röder / Nicola Vettore / Lena N Mangels / Lothar Gremer / Raimond B G Ravelli / Dieter Willbold / Wolfgang Hoyer / Alexander K Buell / Gunnar F Schröder /
Abstract: High resolution structural information on amyloid fibrils is crucial for the understanding of their formation mechanisms and for the rational design of amyloid inhibitors in the context of protein ...High resolution structural information on amyloid fibrils is crucial for the understanding of their formation mechanisms and for the rational design of amyloid inhibitors in the context of protein misfolding diseases. The Src-homology 3 domain of phosphatidyl-inositol-3-kinase (PI3K-SH3) is a model amyloid system that plays a pivotal role in our basic understanding of protein misfolding and aggregation. Here, we present the atomic model of the PI3K-SH3 amyloid fibril with a resolution determined to 3.4 Å by cryo-electron microscopy (cryo-EM). The fibril is composed of two intertwined protofilaments that create an interface spanning 13 residues from each monomer. The model comprises residues 1-77 out of 86 amino acids in total, with the missing residues located in the highly flexible C-terminus. The fibril structure allows us to rationalise the effects of chemically conservative point mutations as well as of the previously reported sequence perturbations on PI3K-SH3 fibril formation and growth.
History
DepositionMar 23, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseAug 28, 2019-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6r4r
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6r4r
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4727.png

Downloads & links

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Map

FileDownload / File: emd_4727.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM reconstruction of sh3 fibril, map sharpened with a B-factor of -150 A2 and low-pass filtered to 3.0 A.
Voxel sizeX=Y=Z: 0.935 Å
Density
Contour LevelBy AUTHOR: 1.1 / Movie #1: 1.1
Minimum - Maximum-3.4095263 - 3.3725786
Average (Standard dev.)0.010784938 (±0.46720803)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 205.7 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9350.9350.935
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z205.700205.700205.700
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ132132232
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-3.4103.3730.011

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Supplemental data

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Half map: #1

Fileemd_4727_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_4727_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 : Fibril of PI3K-SH3 domains

EntireName: Fibril of PI3K-SH3 domains
Components
  • Complex: Fibril of PI3K-SH3 domains
    • Protein or peptide: Phosphatidylinositol 3-kinase regulatory subunit alpha

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Supramolecule #1: Fibril of PI3K-SH3 domains

SupramoleculeName: Fibril of PI3K-SH3 domains / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 34.55 kDa/nm

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Macromolecule #1: Phosphatidylinositol 3-kinase regulatory subunit alpha

MacromoleculeName: Phosphatidylinositol 3-kinase regulatory subunit alpha
type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 9.649585 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
GSMSAEGYQY RALYDYKKER EEDIDLHLGD ILTVNKGSLV ALGFSDGQEA KPEEIGWLNG YNETTGERGD FPGTYVEYIG RKKISP

UniProtKB: Phosphatidylinositol 3-kinase regulatory subunit alpha

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.62 mg/mL
BufferpH: 2.5 / Component - Concentration: 0.11 mg/mL / Component - Formula: C2H6ClNO2 / Component - Name: glycin-hydrochloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 622 / Average exposure time: 65.0 sec. / Average electron dose: 26.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 103733 / Details: Filaments were picked manually in Relion2
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2)
Final reconstructionApplied symmetry - Helical parameters - Δz: 2.3548 Å
Applied symmetry - Helical parameters - Δ&Phi: 179.436 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2)
Details: For the even/odd test, the segment images were split by entire fibrils and refined separately for 25 iterations using the same reference density (low-passed filtered to 20 A).
Number images used: 27681
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6r4r:
Cryo-EM Structure of the PI3-Kinase SH3 Domain Amyloid Fibril

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