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

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Basic information

Entry
Database: PDB / ID: 6r4r
TitleCryo-EM Structure of the PI3-Kinase SH3 Domain Amyloid Fibril
ComponentsPhosphatidylinositol 3-kinase regulatory subunit alpha
KeywordsPROTEIN FIBRIL / amyloid fibril / sh3 domain
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 / FLT3 Signaling ...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 / 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 / RHOF 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, class IA / phosphatidylinositol 3-kinase complex / Extra-nuclear estrogen signaling / G alpha (q) signalling events / intracellular glucose homeostasis / phosphatidylinositol phosphate biosynthetic process / insulin receptor substrate binding / phosphatidylinositol 3-kinase binding / response to endoplasmic reticulum stress / phosphatidylinositol 3-kinase/protein kinase B signal transduction / substrate adhesion-dependent cell spreading / positive regulation of RNA splicing / insulin-like growth factor receptor signaling pathway / positive regulation of glucose import / positive regulation of protein localization to plasma membrane / insulin-like growth factor receptor binding / 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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRoeder, C. / Vettore, N. / Mangels, L.N. / Gremer, L. / Ravelli, R.B.G. / Willbold, D. / Hoyer, W. / Buell, A.K. / Schroder, G.F.
Funding support Germany, 4items
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, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 28, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.3May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Phosphatidylinositol 3-kinase regulatory subunit alpha
B: Phosphatidylinositol 3-kinase regulatory subunit alpha
C: Phosphatidylinositol 3-kinase regulatory subunit alpha
D: Phosphatidylinositol 3-kinase regulatory subunit alpha
E: Phosphatidylinositol 3-kinase regulatory subunit alpha
F: Phosphatidylinositol 3-kinase regulatory subunit alpha
G: Phosphatidylinositol 3-kinase regulatory subunit alpha


Theoretical massNumber of molelcules
Total (without water)67,5477
Polymers67,5477
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: scanning transmission electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area41270 Å2
ΔGint-174 kcal/mol
Surface area23660 Å2
MethodPISA

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Components

#1: Protein
Phosphatidylinositol 3-kinase regulatory subunit alpha / PtdIns-3-kinase regulatory subunit alpha / Phosphatidylinositol 3-kinase 85 kDa regulatory subunit ...PtdIns-3-kinase regulatory subunit alpha / Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha / PtdIns-3-kinase regulatory subunit p85-alpha


Mass: 9649.585 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: PIK3R1 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P23727

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Fibril of PI3K-SH3 domains / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 34.55 kDa/nm / Experimental value: NO
Source (natural)Organism: Bos taurus (cattle)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 2.5
Buffer componentConc.: 0.11 mg/mL / Name: glycin-hydrochloride / Formula: C2H6ClNO2
SpecimenConc.: 1.62 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm
Image recordingAverage exposure time: 65 sec. / Electron dose: 26 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 622
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
2EPU1.2.0.16image acquisition
4CTFFIND4.1.10CTF correction
7Cootmodel fitting
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13PHENIX1.13_2998model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 179.436 ° / Axial rise/subunit: 2.3548 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 103733 / Details: Filaments were picked manually in Relion2
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27681
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).
Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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