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- EMDB-27738: Negative stain EM map of the heterodimeric p110gamma-p84 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-27738
TitleNegative stain EM map of the heterodimeric p110gamma-p84 complex
Map dataNegative stain EM ab initio model of the heterodimeric p110gamma-p84 complex
Sample
  • Complex: Ternary complex of p110 gamma with p84
KeywordsPI3K / p110 / PIK3CG / phosphoinositide / PIK3R6 / p84/p87 / 3-kinase / PIP3 / IMMUNE SYSTEM / TRANSFERASE
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 17.1 Å
AuthorsBurke JE / Dalwadi U / Rathinaswamy MK / Yip CK / Nam SE
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)168998 Canada
CitationJournal: Cell Rep / Year: 2023
Title: Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.
Authors: Manoj K Rathinaswamy / Meredith L Jenkins / Benjamin R Duewell / Xuxiao Zhang / Noah J Harris / John T Evans / Jordan T B Stariha / Udit Dalwadi / Kaelin D Fleming / Harish Ranga-Prasad / ...Authors: Manoj K Rathinaswamy / Meredith L Jenkins / Benjamin R Duewell / Xuxiao Zhang / Noah J Harris / John T Evans / Jordan T B Stariha / Udit Dalwadi / Kaelin D Fleming / Harish Ranga-Prasad / Calvin K Yip / Roger L Williams / Scott D Hansen / John E Burke /
Abstract: Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled ...Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gβγ subunits, p110γ-p84 is weakly recruited to membranes by Gβγ subunits alone and requires recruitment by Ras to allow for Gβγ activation. We mapped two distinct Gβγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gβγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.
History
DepositionJul 28, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27738.png

Downloads & links

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Map

FileDownload / File: emd_27738.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM ab initio model of the heterodimeric p110gamma-p84 complex
Voxel sizeX=Y=Z: 4.67 Å
Density
Contour LevelBy AUTHOR: 4.42
Minimum - Maximum-0.11330669 - 8.215298000000001
Average (Standard dev.)0.11702216 (±0.6311536)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions727272
Spacing727272
CellA=B=C: 336.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27738_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_27738_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 : Ternary complex of p110 gamma with p84

EntireName: Ternary complex of p110 gamma with p84
Components
  • Complex: Ternary complex of p110 gamma with p84

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Supramolecule #1: Ternary complex of p110 gamma with p84

SupramoleculeName: Ternary complex of p110 gamma with p84 / type: complex / ID: 1 / Parent: 0 / Details: Both p110 and p84 subunits are from Homo sapiens
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 8.5
Component:
ConcentrationNameFormula
20.0 mMTris(hydroxymethyl)aminomethane-Hydrochloric acid
100.0 mMSodium ChlorideNaCl
50.0 mMAmmonium Sulfate(NH4)2SO4
0.5 mMTris(2-carboxyethyl)phosphine

Details: Freshly prepared gel filtration buffer, filtered through 0.22 um filter and degassed
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Negative stain EM samples prepares by adsorbing samples on grid for 15 second, followed by blotting of sample, 2 washes with water, 1 wash with stain, and a final 30 second soak in stain and blot.
GridModel: Homemade / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS
DetailsSpecimen was a 1:1 molar ratio of p110g to p84, purified to homogeneity by gel filtration.

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 50 / Average exposure time: 1.0 sec. / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 49000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

DetailsCTF estimation and manual particle picking were done using Relion 3.0.8. Manually picked particles were 2D classified into 4 classes and used as templates for autopicking. Autopicked particles were exported to cryoSPARC 2.14.2 and subjected to 2D classification. Particles which classified well were selected and used to generate an ab initio 3D reconstruction.
Particle selectionNumber selected: 20610
Details: Particles were picked using the cryoSPARC template picker.
Startup modelType of model: NONE / Details: Ab initio reconstruction
Final reconstructionResolution.type: BY AUTHOR / Resolution: 17.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14.3) / Number images used: 10344
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14.2) / Details: cryoSPARC SGD-based ab intio reconstruction
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0) / Details: cryoSPARC non-uniform refinement
Final 3D classificationNumber classes: 50 / Software - Name: cryoSPARC (ver. 2.14.2)
FSC plot (resolution estimation)

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