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- EMDB-0132: Cryo-EM structure of the BRISC complex bound to SHMT2 -

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Basic information

Entry
Database: EMDB / ID: EMD-0132
TitleCryo-EM structure of the BRISC complex bound to SHMT2
Map data
SampleBRISC complex bound to SHMT2 alpha
  • BRISC complex
  • SHMT2 alpha
  • BRISC complex subunit Abraxas 2
  • Lys-63-specific deubiquitinase BRCC36
  • (BRISC and BRCA1-A complex member ...) x 2
  • Serine hydroxymethyltransferase, mitochondrial
  • (ligand) x 2
Function / homology
Function and homology information


Metabolism of folate and pterines / Metalloprotease DUBs / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Nonhomologous End-Joining (NHEJ) / Processing of DNA double-strand break ends / G2/M DNA damage checkpoint / regulation of mitochondrial translation / histone H2A K63-linked deubiquitination / BRISC complex / peroxisome targeting sequence binding ...Metabolism of folate and pterines / Metalloprotease DUBs / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Nonhomologous End-Joining (NHEJ) / Processing of DNA double-strand break ends / G2/M DNA damage checkpoint / regulation of mitochondrial translation / histone H2A K63-linked deubiquitination / BRISC complex / peroxisome targeting sequence binding / nuclear ubiquitin ligase complex / L-allo-threonine aldolase activity / cellular response to tetrahydrofolate / attachment of spindle microtubules to kinetochore / serine binding / L-serine metabolic process / L-serine catabolic process / BRCA1-A complex / glycine metabolic process / regulation of oxidative phosphorylation / L-serine biosynthetic process / glycine hydroxymethyltransferase / Hydrolases, Acting on peptide bonds (peptidases), Omega peptidases / glycine hydroxymethyltransferase activity / glycine biosynthetic process from serine / Lys63-specific deubiquitinase activity / tetrahydrofolate metabolic process / regulation of aerobic respiration / protein K63-linked deubiquitination / response to type I interferon / folic acid metabolic process / tumor necrosis factor receptor binding / mitochondrial nucleoid / response to ionizing radiation / cobalt ion binding / signal transduction involved in G2 DNA damage checkpoint / tetrahydrofolate interconversion / amino acid binding / enzyme regulator activity / polyubiquitin modification-dependent protein binding / mitotic spindle assembly / ubiquitin ligase complex / response to X-ray / thiol-dependent ubiquitin-specific protease activity / thiol-dependent ubiquitinyl hydrolase activity / positive regulation of DNA repair / response to ischemia / one-carbon metabolic process / chromosome segregation / metallopeptidase activity / microtubule cytoskeleton / double-strand break repair / mitochondrial intermembrane space / spindle pole / protein tetramerization / chromatin organization / mitotic cell cycle / double-strand break repair via nonhomologous end joining / mitochondrial inner membrane / protein homotetramerization / pyridoxal phosphate binding / microtubule / microtubule binding / nuclear body / mitochondrial matrix / cell cycle / protein deubiquitination / cell division / DNA repair / apoptotic process / cellular response to DNA damage stimulus / chromatin binding / positive regulation of cell population proliferation / negative regulation of apoptotic process / signal transduction / mitochondrion / zinc ion binding / extracellular exosome / nucleoplasm / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
MPN domain / Pyridoxal phosphate-dependent transferase / FAM175 family / Pyridoxal phosphate-dependent transferase domain 1 / Pyridoxal phosphate-dependent transferase, major domain / BRCA1-A complex subunit BRE / FAM175 family, BRISC complex, Abro1 subunit / BRISC and BRCA1-A complex member 1 / Brcc36 isopeptidase / von Willebrand factor A-like domain superfamily ...MPN domain / Pyridoxal phosphate-dependent transferase / FAM175 family / Pyridoxal phosphate-dependent transferase domain 1 / Pyridoxal phosphate-dependent transferase, major domain / BRCA1-A complex subunit BRE / FAM175 family, BRISC complex, Abro1 subunit / BRISC and BRCA1-A complex member 1 / Brcc36 isopeptidase / von Willebrand factor A-like domain superfamily / Serine hydroxymethyltransferase / Serine hydroxymethyltransferase-like domain / BRCC36, C-terminal helical domain / Serine hydroxymethyltransferase / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Brain and reproductive organ-expressed protein (BRE) / BRCC36 C-terminal helical domain / Serine hydroxymethyltransferase pyridoxal-phosphate attachment site. / MPN domain profile. / JAB1/MPN/MOV34 metalloenzyme domain / Serine hydroxymethyltransferase, pyridoxal phosphate binding site
Serine hydroxymethyltransferase, mitochondrial / Lys-63-specific deubiquitinase BRCC36 / BRISC complex subunit Abraxas 2 / BRISC and BRCA1-A complex member 1 / BRISC and BRCA1-A complex member 2
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSchenk AD / Julius R / Cavadini S / Thoma NH
CitationJournal: Mol. Cell / Year: 2019
Title: Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.
Authors: Julius Rabl / Richard D Bunker / Andreas D Schenk / Simone Cavadini / Mark E Gill / Wassim Abdulrahman / Amparo Andrés-Pons / Martijn S Luijsterburg / Adel F M Ibrahim / Emma Branigan / Jacob D Aguirre / Aimee H Marceau / Claire Guérillon / Tewis Bouwmeester / Ulrich Hassiepen / Antoine H F M Peters / Martin Renatus / Laurent Gelman / Seth M Rubin / Niels Mailand / Haico van Attikum / Ronald T Hay / Nicolas H Thomä /
Abstract: In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. ...In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.
Validation ReportPDB-ID: 6h3c

SummaryFull reportAbout validation report
DateDeposition: Jul 18, 2018 / Header (metadata) release: Jul 3, 2019 / Map release: Jul 10, 2019 / Update: Jul 10, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00816
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.00816
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6h3c
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0132.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 350 pix.
= 301. Å
0.86 Å/pix.
x 350 pix.
= 301. Å
0.86 Å/pix.
x 350 pix.
= 301. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.00816 / Movie #1: 0.00816
Minimum - Maximum-0.06578454 - 0.1309982
Average (Standard dev.)0.0002689527 (±0.0028135402)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 301.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z301.000301.000301.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.0660.1310.000

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Supplemental data

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Mask #1

Fileemd_0132_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire BRISC complex bound to SHMT2 alpha

EntireName: BRISC complex bound to SHMT2 alpha / Number of components: 10

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Component #1: protein, BRISC complex bound to SHMT2 alpha

ProteinName: BRISC complex bound to SHMT2 alpha / Recombinant expression: No
MassExperimental: 438 kDa

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Component #2: protein, BRISC complex

ProteinName: BRISC complex / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper) / Vector: pFastBac / Strain: High Five

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Component #3: protein, SHMT2 alpha

ProteinName: SHMT2 alpha / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, BRISC complex subunit Abraxas 2

ProteinName: BRISC complex subunit Abraxas 2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 49.256246 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #5: protein, Lys-63-specific deubiquitinase BRCC36

ProteinName: Lys-63-specific deubiquitinase BRCC36 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 38.417402 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #6: protein, BRISC and BRCA1-A complex member 2

ProteinName: BRISC and BRCA1-A complex member 2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 45.890949 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #7: protein, BRISC and BRCA1-A complex member 1

ProteinName: BRISC and BRCA1-A complex member 1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 39.263824 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #8: protein, Serine hydroxymethyltransferase, mitochondrial

ProteinName: Serine hydroxymethyltransferase, mitochondrial / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 56.144711 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #9: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #10: ligand, water

LigandName: water / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 1.801505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.44 mg/mL / pH: 7.4
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 80 %
Details: A 4 ul sample was applied to the grid and a protocol consisting of 30 s pre-blot incubation, 2 s blotting and no post-blot incubation was utilized for vitrification..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal), 58140.0 X (calibrated) / Cs: 0 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1822 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 35595
Details: Drift correction was performed using Motioncor2. CTF was fitted using GCTF CryoFLARE was used for automation.
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION
CTF correction: CTF was determined using GCTF. CTF was corrected within RELION.
Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Correlation coefficient and / Refinement space: REAL
Details: Initial fitting with done using COOT, Rosetta was used for amino acid sequence threading and flexible fitting. COOT and ISOLDE were used for local rebuilding. Phenix was used for atom displacement parameter (ADP / B-factor) refinement.
Input PDB model: 6GVW, 5V7I
Chain ID: A

Overall bvalue: 117
Output model

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