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- PDB-6y5d: Structure of human cGAS (K394E) bound to the nucleosome -

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Basic information

Entry
Database: PDB / ID: 6y5d
TitleStructure of human cGAS (K394E) bound to the nucleosome
Components
  • (DNA (153-MER)) x 2
  • Cyclic GMP-AMP synthase
  • Histone H2A type 2-A
  • Histone H2B type 1-K
  • Histone H3.2
  • Histone H4
KeywordsIMMUNE SYSTEM / cGAS / STING / Nucleosome / Innate Immunity / cGMP-AMP
Function / homology
Function and homology information


water channel activity / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere ...water channel activity / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / Chromatin modifying enzymes / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / Deposition of new CENPA-containing nucleosomes at the centromere / nucleosomal DNA binding / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / Inhibition of DNA recombination at telomere / telomere organization / Meiotic synapsis / Interleukin-7 signaling / RNA Polymerase I Promoter Opening / Assembly of the ORC complex at the origin of replication / SUMOylation of chromatin organization proteins / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / DNA methylation / Condensation of Prophase Chromosomes / SIRT1 negatively regulates rRNA expression / Chromatin modifications during the maternal to zygotic transition (MZT) / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / HCMV Late Events / innate immune response in mucosa / PRC2 methylates histones and DNA / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / HDACs deacetylate histones / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / NoRC negatively regulates rRNA expression / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / G2/M DNA damage checkpoint / HDMs demethylate histones / B-WICH complex positively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / heterochromatin formation / PKMTs methylate histone lysines / Metalloprotease DUBs / Meiotic recombination / Pre-NOTCH Transcription and Translation / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / HCMV Early Events / Transcriptional regulation of granulopoiesis / structural constituent of chromatin / UCH proteinases / antimicrobial humoral immune response mediated by antimicrobial peptide / nucleosome / nucleosome assembly / E3 ubiquitin ligases ubiquitinate target proteins / antibacterial humoral response / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / chromatin organization / RUNX1 regulates transcription of genes involved in differentiation of HSCs / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones / Processing of DNA double-strand break ends / Senescence-Associated Secretory Phenotype (SASP) / Oxidative Stress Induced Senescence / defense response to Gram-negative bacterium / Estrogen-dependent gene expression / killing of cells of another organism / chromosome, telomeric region / Ub-specific processing proteases / defense response to Gram-positive bacterium / protein heterodimerization activity / Amyloid fiber formation / protein-containing complex / DNA binding / RNA binding / extracellular space / extracellular exosome / extracellular region / nucleoplasm / membrane / nucleus / plasma membrane / cytosol
Similarity search - Function
Mab-21 protein nucleotidyltransferase domain / Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Aquaporin-like / Histone H2B signature. / Histone H2B / Histone H2B ...Mab-21 protein nucleotidyltransferase domain / Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Aquaporin-like / Histone H2B signature. / Histone H2B / Histone H2B / Histone H2A conserved site / Histone H2A signature. / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / Histone H2A / Histone 2A / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 / Histone-fold
Similarity search - Domain/homology
PENTANEDIAL / DNA / DNA (> 10) / DNA (> 100) / Histone H2B type 1-K / Histone H4 / Histone H2A type 2-A / Histone H3.2 / Probable aquaporin AqpM
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsPathare, G.R. / Cavadini, S. / Kempf, G. / Thoma, N.H.
Funding support Switzerland, 4items
OrganizationGrant numberCountry
European Research Council (ERC)666068 Switzerland
European Research Council (ERC)724022 Switzerland
European Research Council (ERC)804933 Switzerland
Swiss National Science FoundationBSSGI0-155984 Switzerland
CitationJournal: Nature / Year: 2020
Title: Structural mechanism of cGAS inhibition by the nucleosome.
Authors: Ganesh R Pathare / Alexiane Decout / Selene Glück / Simone Cavadini / Kristina Makasheva / Ruud Hovius / Georg Kempf / Joscha Weiss / Zuzanna Kozicka / Baptiste Guey / Pauline Melenec / ...Authors: Ganesh R Pathare / Alexiane Decout / Selene Glück / Simone Cavadini / Kristina Makasheva / Ruud Hovius / Georg Kempf / Joscha Weiss / Zuzanna Kozicka / Baptiste Guey / Pauline Melenec / Beat Fierz / Nicolas H Thomä / Andrea Ablasser /
Abstract: The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer. Upon activation by double-stranded DNA, cytosolic cGAS ...The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer. Upon activation by double-stranded DNA, cytosolic cGAS produces 2'3' cGMP-AMP, which triggers the induction of inflammatory cytokines and type I interferons . cGAS is also present inside the cell nucleus, which is replete with genomic DNA, where chromatin has been implicated in restricting its enzymatic activity. However, the structural basis for inhibition of cGAS by chromatin remains unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also find cGAS engaged to a second nucleosome in trans. Mechanistically, binding of the nucleosome locks cGAS into a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash the activity of cGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction between cGAS and chromatin and details a mechanism that permits self-non-self discrimination of genomic DNA by cGAS.
History
DepositionFeb 25, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 23, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Histone H3.2
B: Histone H4
C: Histone H2A type 2-A
D: Histone H2B type 1-K
E: Histone H3.2
F: Histone H4
G: Histone H2A type 2-A
H: Histone H2B type 1-K
I: DNA (153-MER)
J: DNA (153-MER)
K: Cyclic GMP-AMP synthase
L: Cyclic GMP-AMP synthase
M: Histone H3.2
N: Histone H4
O: Histone H2A type 2-A
P: Histone H2B type 1-K
Q: Histone H3.2
R: Histone H4
S: Histone H2A type 2-A
T: Histone H2B type 1-K
U: DNA (153-MER)
V: DNA (153-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)478,34433
Polymers477,31222
Non-polymers1,03211
Water00
1


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

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Components

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Protein , 5 types, 18 molecules AEMQBFNRCGOSDHPTKL

#1: Protein
Histone H3.2 / Histone H3/m / Histone H3/o


Mass: 11456.345 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST2H3A, HIST2H3C, H3F2, H3FM, HIST2H3D / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q71DI3
#2: Protein
Histone H4


Mass: 11394.426 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, ...Gene: HIST1H4A, H4/A, H4FA, HIST1H4B, H4/I, H4FI, HIST1H4C, H4/G, H4FG, HIST1H4D, H4/B, H4FB, HIST1H4E, H4/J, H4FJ, HIST1H4F, H4/C, H4FC, HIST1H4H, H4/H, H4FH, HIST1H4I, H4/M, H4FM, HIST1H4J, H4/E, H4FE, HIST1H4K, H4/D, H4FD, HIST1H4L, H4/K, H4FK, HIST2H4A, H4/N, H4F2, H4FN, HIST2H4, HIST2H4B, H4/O, H4FO, HIST4H4
Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P62805
#3: Protein
Histone H2A type 2-A / Histone H2A.2 / Histone H2A/o


Mass: 14125.549 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST2H2AA3, H2AFO, HIST2H2AA, HIST2H2AA4 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6FI13
#4: Protein
Histone H2B type 1-K / H2B K / HIRA-interacting protein 1


Mass: 13921.213 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HIST1H2BK, H2BFT, HIRIP1 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: O60814
#7: Protein Cyclic GMP-AMP synthase / h-cGAS / 2'3'-cGAMP synthase / Mab-21 domain-containing protein 1


Mass: 42404.840 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CGAS, C6orf150, MB21D1 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: Q8N884, cyclic GMP-AMP synthase

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DNA chain , 2 types, 4 molecules IUJV

#5: DNA chain DNA (153-MER)


Mass: 46998.945 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA (153-MER)


Mass: 47457.234 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 11 molecules

#8: Chemical
ChemComp-PTD / PENTANEDIAL


Mass: 100.116 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C5H8O2
#9: Chemical ChemComp-ZN / ZINC ION


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

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Details

Has protein modificationY

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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
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of cGAS-NCP complexCOMPLEX#1-#70MULTIPLE SOURCES
2ProteinCOMPLEX#1-#2, #4, #71RECOMBINANT
3Histone H2A type 2-ACOMPLEX#31RECOMBINANT
4DNACOMPLEX#5-#61RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
24Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Escherichia coli (E. coli)562
34synthetic construct (others)32630
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Cs: 0.01 mm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13943 / Symmetry type: POINT

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