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- PDB-6r8f: Cryo-EM structure of the Human BRISC-SHMT2 complex -

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Entry
Database: PDB / ID: 6r8f
TitleCryo-EM structure of the Human BRISC-SHMT2 complex
Components
  • BRISC and BRCA1-A complex member 2,BRCC45 (BRE, BRISC and BRCA1-A complex member 2)
  • BRISC complex subunit Abraxas 2
  • Lys-63-specific deubiquitinase BRCC36
  • Serine hydroxymethyltransferase, mitochondrial
KeywordsSIGNALING PROTEIN / Complex / Deubiquitylation / Ubiquitin / Immune signalling / signaling protein
Function / homology
Function and homology information


G2/M DNA damage checkpoint / Metabolism of folate and pterines / Processing of DNA double-strand break ends / Nonhomologous End-Joining (NHEJ) / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Metalloprotease DUBs / nuclear ubiquitin ligase complex / ec:4.1.3.38: / 4-amino-4-deoxychorismate lyase activity / histone H2A K63-linked deubiquitination ...G2/M DNA damage checkpoint / Metabolism of folate and pterines / Processing of DNA double-strand break ends / Nonhomologous End-Joining (NHEJ) / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / Metalloprotease DUBs / nuclear ubiquitin ligase complex / ec:4.1.3.38: / 4-amino-4-deoxychorismate lyase activity / histone H2A K63-linked deubiquitination / regulation of mitochondrial translation / BRISC complex / peroxisome targeting sequence binding / L-allo-threonine aldolase activity / cellular response to tetrahydrofolate / serine binding / L-serine metabolic process / L-serine catabolic process / BRCA1-A complex / glycine metabolic process / regulation of oxidative phosphorylation / L-serine biosynthetic process / ec:2.1.2.1: / ec:3.4.19.-: / glycine hydroxymethyltransferase activity / glycine biosynthetic process from serine / Lys63-specific deubiquitinase activity / protein K63-linked deubiquitination / regulation of aerobic respiration / folic acid biosynthetic process / tetrahydrofolate metabolic process / response to type I interferon / tumor necrosis factor receptor binding / mitochondrial nucleoid / response to ionizing radiation / signal transduction involved in G2 DNA damage checkpoint / cobalt ion binding / tetrahydrofolate interconversion / folic acid metabolic process / amino acid binding / enzyme regulator activity / polyubiquitin modification-dependent protein binding / ubiquitin ligase complex / response to X-ray / thiol-dependent ubiquitin-specific protease activity / thiol-dependent ubiquitinyl hydrolase activity / positive regulation of DNA repair / one-carbon metabolic process / metallopeptidase activity / microtubule cytoskeleton / double-strand break repair / mitochondrial intermembrane space / spindle pole / protein tetramerization / chromatin organization / mitochondrial inner membrane / double-strand break repair via nonhomologous end joining / protein homotetramerization / pyridoxal phosphate binding / 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
Aminotransferase, class IV, conserved site / Aminodeoxychorismate lyase, class IV / Brcc36 isopeptidase / Aminotransferase-like, PLP-dependent enzymes / MPN domain / Serine hydroxymethyltransferase-like domain / BRCC36, C-terminal helical domain / Pyridoxal phosphate-dependent transferase / Pyridoxal phosphate-dependent transferase domain 1 / Pyridoxal phosphate-dependent transferase, major domain ...Aminotransferase, class IV, conserved site / Aminodeoxychorismate lyase, class IV / Brcc36 isopeptidase / Aminotransferase-like, PLP-dependent enzymes / MPN domain / Serine hydroxymethyltransferase-like domain / BRCC36, C-terminal helical domain / Pyridoxal phosphate-dependent transferase / Pyridoxal phosphate-dependent transferase domain 1 / Pyridoxal phosphate-dependent transferase, major domain / Serine hydroxymethyltransferase / Amino-transferase class IV / 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. / Aminotransferases class-IV signature. / MPN domain profile. / Serine hydroxymethyltransferase / JAB1/MPN/MOV34 metalloenzyme domain / Aminotransferase class IV / BRCA1-A complex subunit BRE / Serine hydroxymethyltransferase, pyridoxal phosphate binding site
Serine hydroxymethyltransferase, mitochondrial / Lys-63-specific deubiquitinase BRCC36 / 4-amino-4-deoxychorismate lyase / BRISC and BRCA1-A complex member 2
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWalden, M. / Hesketh, E. / Tian, L. / Ranson, N.A. / Greenberg, R.A. / Zeqiraj, E.
Funding supportUnited Kingdom , 1件
OrganizationGrant numberCountry
Wellcome Trust200523/Z/16/ZUnited Kingdom
CitationJournal: Nature / Year: 2019
Title: Metabolic control of BRISC-SHMT2 assembly regulates immune signalling.
Authors: Miriam Walden / Lei Tian / Rebecca L Ross / Upasana M Sykora / Dominic P Byrne / Emma L Hesketh / Safi K Masandi / Joel Cassel / Rachel George / James R Ault / Farid El Oualid / Krzysztof Pawłowski / Joseph M Salvino / Patrick A Eyers / Neil A Ranson / Francesco Del Galdo / Roger A Greenberg / Elton Zeqiraj /
Abstract: Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. ...Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 1, 2019 / Release: Jun 5, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 5, 2019Structure modelrepositoryInitial release
1.1Jun 12, 2019Structure modelData collection / Database referencescitation / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info_citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod

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Assembly

Deposited unit
A: Lys-63-specific deubiquitinase BRCC36
B: BRISC complex subunit Abraxas 2
K: Serine hydroxymethyltransferase, mitochondrial
C: Lys-63-specific deubiquitinase BRCC36
D: BRISC complex subunit Abraxas 2
L: Serine hydroxymethyltransferase, mitochondrial
E: BRISC and BRCA1-A complex member 2,BRCC45 (BRE, BRISC and BRCA1-A complex member 2)
G: BRISC and BRCA1-A complex member 2,BRCC45 (BRE, BRISC and BRCA1-A complex member 2)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)290,68810
Polymers290,5578
Non-polymers1312
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area34500 Å2
ΔGint-276 kcal/mol
Surface area94480 Å2
MethodPISA

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Components

#1: 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: 36119.918 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 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P46736, EC: 3.4.19.-
#2: Protein/peptide BRISC complex subunit Abraxas 2 / Abraxas brother protein 1 / Protein FAM175B


Mass: 31033.945 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ABRAXAS2, ABRO1, FAM175B, KIAA0157 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q15018
#3: Protein/peptide Serine hydroxymethyltransferase, mitochondrial / / SHMT / Glycine hydroxymethyltransferase / Serine methylase


Mass: 56097.902 Da / Num. of mol.: 2 / Mutation: A285T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SHMT2 / Production host: Escherichia coli (E. coli) / References: UniProt: P34897, EC: 2.1.2.1
#4: Protein/peptide BRISC and BRCA1-A complex member 2,BRCC45 (BRE, 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: 22026.717 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BABAM2, BRCC45, BRE, BABAM2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NXR7
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Zinc

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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-IDSourceDetails
1BRISC-SHMT21, 2, 3, 40MULTIPLE SOURCES
2MERIT40 (BRISC and BRCA1-A complex member 1)1, 2, 41RECOMBINANTMERIT40 also expressed along with other macromolecules, but no model is built into the low resolution density.
3Serine hydroxymethyltransferase, mitochondrial31RECOMBINANT
Molecular weightValue: 388.47 kDa/nm / Experimental value: YES
Source (natural)

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

IDEntity assembly-ID
22
33
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component

Buffer-ID: 1

IDConc.NameFormula
125 mMHEPES
2150 mMNaClSodium chloride
31 mMTCEP
SpecimenConc.: 0.051 mg/ml
Details: Specimen contained BRCC36, ABRAXAS2, BRCC45, MERIT40 and SHMT2 macromolecules
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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: 75000 X / Nominal defocus max: -1600 nm / Nominal defocus min: -3100 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 1.2 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 7494

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4RELIONCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 403499 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID3D fitting-ID
15CW31
26DK31

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