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- PDB-6h3c: Cryo-EM structure of the BRISC complex bound to SHMT2 -

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Basic information

Entry
Database: PDB / ID: 6h3c
TitleCryo-EM structure of the BRISC complex bound to SHMT2
Components
  • (BRISC and BRCA1-A complex member ...) x 2
  • BRISC complex subunit Abraxas 2
  • Lys-63-specific deubiquitinase BRCC36
  • Serine hydroxymethyltransferase, mitochondrial
KeywordsSIGNALING PROTEIN / Deubiquitinase complex / DUB / Lysine-63 linkage specific / BRCC36-containing / BRCA1A binding
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBunker, R.D. / Rabl, J. / Thoma, N.H.
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 Report
SummaryFull reportAbout validation report
DateDeposition: Jul 18, 2018 / Release: Jul 10, 2019
RevisionDateData content typeProviderType
1.0Jul 10, 2019Structure modelrepositoryInitial release

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Structure visualization

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Assembly

Deposited unit
A: BRISC complex subunit Abraxas 2
B: Lys-63-specific deubiquitinase BRCC36
C: BRISC and BRCA1-A complex member 2
D: BRISC and BRCA1-A complex member 1
F: BRISC complex subunit Abraxas 2
G: Lys-63-specific deubiquitinase BRCC36
H: BRISC and BRCA1-A complex member 2
I: BRISC and BRCA1-A complex member 1
E: Serine hydroxymethyltransferase, mitochondrial
J: Serine hydroxymethyltransferase, mitochondrial
hetero molecules


Theoretical massNumber of molelcules
Total (without water)458,07712
Polymers457,94610
Non-polymers1312
Water362
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, Assembly can be purified by glycerol gradient centrifugation or size exclusion chromatography. Affinity between BRISC and SHMT2 was determined by biolayer interferometry (Kd ~46 nM).
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area43420 Å2
ΔGint-322 kcal/mol
Surface area129910 Å2
MethodPISA

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Components

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Protein/peptide , 3 types, 6 molecules AFBGEJ

#1: Protein/peptide BRISC complex subunit Abraxas 2 / Abraxas brother protein 1 / Protein FAM175B


Mass: 49256.246 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABRAXAS2, ABRO1, FAM175B, KIAA0157 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15018
#2: Protein/peptide Lys-63-specific deubiquitinase BRCC36 / BRCA1-A complex subunit BRCC36 / BRCA1/BRCA2-containing complex subunit 3 / BRCA1/BRCA2-containing complex subunit 36 / BRISC complex subunit BRCC36


Mass: 38417.402 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRCC3, BRCC36, C6.1A, CXorf53 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: P46736, Hydrolases, Acting on peptide bonds (peptidases), Omega peptidases
#5: Protein/peptide Serine hydroxymethyltransferase, mitochondrial / / SHMT / Glycine hydroxymethyltransferase / Serine methylase


Mass: 56144.711 Da / Num. of mol.: 2 / Mutation: A264T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SHMT2 / Plasmid: p28a-LIC
Details (production host): SGC Clone Accession: SHMT2:APC005_8-E02:C16046 (undocumented point mutation A285T (A264T for isoform 3))
Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P34897, glycine hydroxymethyltransferase

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BRISC and BRCA1-A complex member ... , 2 types, 4 molecules CHDI

#3: Protein/peptide BRISC and BRCA1-A complex member 2 / BRCA1-A complex subunit BRE / BRCA1/BRCA2-containing complex subunit 45 / Brain and reproductive organ-expressed protein


Mass: 45890.949 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BABAM2, BRCC45, BRE / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NXR7
#4: Protein/peptide BRISC and BRCA1-A complex member 1 / Mediator of RAP80 interactions and targeting subunit of 40 kDa / New component of the BRCA1-A complex


Mass: 39263.824 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BABAM1, C19orf62, MERIT40, NBA1, HSPC142 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NWV8

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Non-polymers , 2 types, 4 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Zinc
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O / Water

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component

Type: COMPLEX

IDNameEntity IDParent-IDSource
1BRISC complex bound to SHMT2 alpha1,2,3,4,50MULTIPLE SOURCES
2BRISC complex1,2,3,41RECOMBINANT
3SHMT2 alpha51RECOMBINANT
Molecular weightValue: 0.438 MDa / Experimental value: YES
Source (natural)

Ncbi tax-ID: 9606 / Organism: Homo sapiens (human)

IDEntity assembly-ID
22
33
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
22Trichoplusia ni (cabbage looper)7111High FivepFastBac
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.4
Buffer component

Buffer-ID: 1

IDConc.NameFormula
150 mMHEPES
2150 mMsodium chlorideNaClSodium chloride
30.2 mMTCEP
SpecimenConc.: 0.44 mg/ml
Details: Sample was purified by gel filtration over a Superose 6 column.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K
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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 58140 X / Calibrated defocus min: 500 nm / Calibrated defocus max: 5000 nm / Cs: 0 mm / C2 aperture diameter: 50 µns / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1822
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV / Phase plate: not used
Spherical aberration corrector: Microscope was equipped with a CEOS Cs-corrector
Image scansSampling size: 5 µns / Width: 3710 / Height: 3838 / Movie frames/image: 40 / Used frames/image: 4-20

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatch0.53particle selection
2EPU1.1.0.30image acquisitionCryoFLARE was used for automation of the drift correction, particle picking and CTF determination during data aquisition
4RELION2.1CTF correction
7Coot0.8.9model fitting
8Rosetta2018.09.60072model fitting
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
20Rosetta2018.09.60072model refinement
21PHENIX1.15-3459model refinement
Image processingDetails: Drift correction was performed using Motioncor2. CTF was fitted using GCTF CryoFLARE was used for automation.
CTF correctionDetails: CTF was determined using GCTF. CTF was corrected within RELION.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 332598 / Details: Particles were auto-picked.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35595 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 117 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient and
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.
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-ID
16GVW
25V7IA

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