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- EMDB-14999: ARISC-RAP80 complex, Map 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-14999
TitleARISC-RAP80 complex, Map 1
Map data
Sample
  • Complex: Complex containing the four-subunit ARISC together with FL RAP80
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsFoglizzo M / Zeqiraj E
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/T029471/1 United Kingdom
Wellcome Trust200523/Z/16/Z United Kingdom
Wellcome Trust222531/Z/21/Z United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
CitationJournal: J Cell Biol / Year: 2022
Title: Autologous K63 deubiquitylation within the BRCA1-A complex licenses DNA damage recognition.
Authors: Qinqin Jiang / Martina Foglizzo / Yaroslav I Morozov / Xuejiao Yang / Arindam Datta / Lei Tian / Vaughn Thada / Weihua Li / Elton Zeqiraj / Roger A Greenberg /
Abstract: The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains ...The BRCA1-A complex contains matching lysine-63 ubiquitin (K63-Ub) binding and deubiquitylating activities. How these functionalities are coordinated to effectively respond to DNA damage remains unknown. We generated Brcc36 deubiquitylating enzyme (DUB) inactive mice to address this gap in knowledge in a physiologic system. DUB inactivation impaired BRCA1-A complex damage localization and repair activities while causing early lethality when combined with Brca2 mutation. Damage response dysfunction in DUB-inactive cells corresponded to increased K63-Ub on RAP80 and BRCC36. Chemical cross-linking coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and cryogenic-electron microscopy (cryo-EM) analyses of isolated BRCA1-A complexes demonstrated the RAP80 ubiquitin interaction motifs are occupied by ubiquitin exclusively in the DUB-inactive complex, linking auto-inhibition by internal K63-Ub chains to loss of damage site ubiquitin recognition. These findings identify RAP80 and BRCC36 as autologous DUB substrates in the BRCA1-A complex, thus explaining the evolution of matching ubiquitin-binding and hydrolysis activities within a single macromolecular assembly.
History
DepositionMay 19, 2022-
Header (metadata) releaseJul 27, 2022-
Map releaseJul 27, 2022-
UpdateAug 17, 2022-
Current statusAug 17, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14999.png

Downloads & links

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Map

FileDownload / File: emd_14999.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 320 pix.
= 340.8 Å		1.07 Å/pix.
x 320 pix.
= 340.8 Å		1.07 Å/pix.
x 320 pix.
= 340.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.065 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0115
Minimum - Maximum-0.037743453 - 0.07846526
Average (Standard dev.)9.522706e-05 (±0.003334287)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 340.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14999_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14999_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14999_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex containing the four-subunit ARISC together with FL RAP80

EntireName: Complex containing the four-subunit ARISC together with FL RAP80
Components
  • Complex: Complex containing the four-subunit ARISC together with FL RAP80

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Supramolecule #1: Complex containing the four-subunit ARISC together with FL RAP80

SupramoleculeName: Complex containing the four-subunit ARISC together with FL RAP80
type: complex / Chimera: Yes / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightExperimental: 235 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

Concentration0.16 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
0.025 MHEPES2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
0.15 MNaClSodium Chloride
0.001 MTCEPTris(2-carboxyethyl)phosphine

Details: The sample was supplemented with 2 mM EDTA/EGTA mixture immediately before grids preparation
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: The sample was blotted for 6 seconds and with a blot force of 6 before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4392 / Average exposure time: 70.0 sec. / Average electron dose: 1.46 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 904273
CTF correctionSoftware - Name: Gctf
Startup modelType of model: INSILICO MODEL
In silico model: Models for the five subunits constituting the complex (BRCC36, Abraxas1, BRCC45, MERIT40 and RAP80) were generated using SWISS-MODEL (Bertoni et al., 2018)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 92035
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 6 / Software - Name: RELION

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