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- PDB-6x5a: The mouse cGAS catalytic domain binding to human nucleosome that ... -

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Basic information

Entry
Database: PDB / ID: 6x5a
TitleThe mouse cGAS catalytic domain binding to human nucleosome that purified from HEK293T cells
Components
  • (DNA (natural)) x 2
  • Cyclic GMP-AMP synthase
  • Histone H2A type 1
  • Histone H2B type 1-C/E/F/G/I
  • Histone H3.2
  • Histone H4
KeywordsDNA BINDING PROTEIN/DNA/TRANSFERASE / Immunity / DNA BINDING PROTEIN-DNA-TRANSFERASE complex
Function / homology
Function and homology information


cyclic GMP-AMP synthase / positive regulation of cGMP-mediated signaling / cyclic-GMP-AMP synthase activity / regulation of immunoglobulin production / paracrine signaling / regulation of type I interferon production / negative regulation of double-strand break repair via homologous recombination / regulation of T cell activation / positive regulation of cAMP-mediated signaling / cellular response to exogenous dsRNA ...cyclic GMP-AMP synthase / positive regulation of cGMP-mediated signaling / cyclic-GMP-AMP synthase activity / regulation of immunoglobulin production / paracrine signaling / regulation of type I interferon production / negative regulation of double-strand break repair via homologous recombination / regulation of T cell activation / positive regulation of cAMP-mediated signaling / cellular response to exogenous dsRNA / negative regulation of megakaryocyte differentiation / positive regulation of defense response to virus by host / CENP-A containing nucleosome assembly / activation of innate immune response / DNA replication-independent nucleosome assembly / telomere capping / interleukin-7-mediated signaling pathway / chromatin silencing / determination of adult lifespan / positive regulation of type I interferon production / telomere organization / DNA replication-dependent nucleosome assembly / innate immune response in mucosa / nuclear nucleosome / rDNA heterochromatin assembly / negative regulation of gene expression, epigenetic / regulation of gene silencing by miRNA / nuclear chromosome / DNA-templated transcription, initiation / regulation of megakaryocyte differentiation / phosphatidylinositol-4,5-bisphosphate binding / nucleosome assembly / nucleosome / site of double-strand break / positive regulation of cellular senescence / double-strand break repair via nonhomologous end joining / defense response to virus / double-stranded DNA binding / chromatin organization / regulation of immune response / nuclear chromosome, telomeric region / antibacterial humoral response / antimicrobial humoral immune response mediated by antimicrobial peptide / blood coagulation / protein ubiquitination / protein heterodimerization activity / defense response to Gram-positive bacterium / nuclear chromatin / DNA repair / protein domain specific binding / GTP binding / cellular response to DNA damage stimulus / chromatin binding / cellular protein metabolic process / innate immune response / host cell nucleus / enzyme binding / protein-containing complex / RNA binding / DNA binding / extracellular space / extracellular exosome / membrane / extracellular region / nucleoplasm / ATP binding / identical protein binding / plasma membrane / metal ion binding / nucleus / cytosol
Histone H2B / TATA box binding protein associated factor (TAF) / CENP-T/Histone H4, histone fold / Histone H2A conserved site / Histone H2A, C-terminal domain / Mab-21 domain / Histone H4, conserved site / Histone H3/CENP-A / Histone-fold / Histone H4 ...Histone H2B / TATA box binding protein associated factor (TAF) / CENP-T/Histone H4, histone fold / Histone H2A conserved site / Histone H2A, C-terminal domain / Mab-21 domain / Histone H4, conserved site / Histone H3/CENP-A / Histone-fold / Histone H4 / Histone H2A / Histone H2A/H2B/H3
Histone H2A type 1 / Histone H4 / Histone H2B type 1-C/E/F/G/I / Histone H3.2 / Cyclic GMP-AMP synthase
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.36 Å
AuthorsPengbiao, X. / Pingwei, L. / Baoyu, Z.
Funding support United States, 2items
OrganizationGrant numberCountry
Welch FoundationGrant A-1931-20170325 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)Grant R01 AI145287 United States
CitationJournal: Nature / Year: 2020
Title: The Molecular Basis of Tight Nuclear Tethering and Inactivation of cGAS.
Authors: Baoyu Zhao / Pengbiao Xu / Chesley M Rowlett / Tao Jing / Omkar Shinde / Yuanjiu Lei / A Phillip West / Wenshe Ray Liu / Pingwei Li /
Abstract: Pathogen-derived nucleic acids induce potent innate immune responses. Cyclic GMP-AMP synthase (cGAS) is a dsDNA sensor that catalyzes the synthesis of a cyclic dinucleotide cGAMP, which mediates the ...Pathogen-derived nucleic acids induce potent innate immune responses. Cyclic GMP-AMP synthase (cGAS) is a dsDNA sensor that catalyzes the synthesis of a cyclic dinucleotide cGAMP, which mediates the induction of type I interferons through the STING-TBK1-IRF3 signaling axis. It was widely accepted that cGAS is not reactive to self-DNA due to its cytosolic localization. However, recent studies revealed that cGAS is mostly localized in the nucleus and tight nuclear tethering keeps cGAS inactive. Here we show that cGAS binds to nucleosomes with nanomolar affinity and nucleosome binding potently inhibits the catalytic activity of cGAS. To elucidate the molecular basis of cGAS inactivation by nuclear tethering, we have determined the structure of mouse cGAS bound to human nucleosome by cryo-EM. The structure shows that cGAS binds to a negatively charged acidic patch formed by histone H2A and H2B via its second DNA binding site. High affinity nucleosome binding blocks dsDNA binding and keeps cGAS in an inactive conformation. Mutations of cGAS that disrupt nucleosome binding dramatically affect cGAS mediated signaling in cells.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 25, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 23, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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  • Deposited structure unit
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  • EMDB-22047
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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A type 1
D: Histone H2B type 1-C/E/F/G/I
E: Histone H3.2
F: Histone H4
G: Histone H2A type 1
H: Histone H2B type 1-C/E/F/G/I
I: DNA (natural)
J: DNA (natural)
K: Cyclic GMP-AMP synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)242,90312
Polymers242,83811
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, surface plasmon resonance
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TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 9 molecules AEBFCGDHK

#1: Protein Histone H3.2 / H3-clustered histone 13 / H3-clustered histone 14 / H3-clustered histone 15 / Histone H3/m / Histone H3/o


Mass: 15257.838 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293T / References: UniProt: Q71DI3
#2: Protein Histone H4 /


Mass: 11263.231 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293T / References: UniProt: P62805
#3: Protein Histone H2A type 1 / H2A.1 / Histone H2A/ptl


Mass: 13990.342 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293T / References: UniProt: P0C0S8
#4: Protein Histone H2B type 1-C/E/F/G/I / Histone H2B.1 A / Histone H2B.a / H2B/a / Histone H2B.g / H2B/g / Histone H2B.h / H2B/h / Histone ...Histone H2B.1 A / Histone H2B.a / H2B/a / Histone H2B.g / H2B/g / Histone H2B.h / H2B/h / Histone H2B.k / H2B/k / Histone H2B.l / H2B/l


Mass: 13708.903 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293T / References: UniProt: P62807
#7: Protein Cyclic GMP-AMP synthase / / m-cGAS / 2'3'-cGAMP synthase / Mab-21 domain-containing protein 1


Mass: 43647.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Cgas, Mb21d1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8C6L5, cyclic GMP-AMP synthase

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain DNA (natural)


Mass: 45145.754 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293T
#6: DNA chain DNA (natural)


Mass: 45604.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293T

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Non-polymers , 1 types, 1 molecules

#8: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

ComponentName: cGAS-nucleosome complex / Type: COMPLEX
Details: The mouse cGAS catalytic domain binding to human nucleosome that purified from HEK293T cells
Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 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 defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
4GctfCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23463 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00715829
ELECTRON MICROSCOPYf_angle_d0.88722591
ELECTRON MICROSCOPYf_dihedral_angle_d32.3444153
ELECTRON MICROSCOPYf_chiral_restr0.0482540
ELECTRON MICROSCOPYf_plane_restr0.0071858

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