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- PDB-7a08: CryoEM Structure of cGAS Nucleosome complex -

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Basic information

Entry
Database: PDB / ID: 7a08
TitleCryoEM Structure of cGAS Nucleosome complex
Components
  • (Nucleosomal DNA strand ...) x 2
  • Cyclic GMP-AMP synthase
  • Histone H2A type 1-C
  • Histone H2B type 1-C/E/F/G/I
  • Histone H3.3
  • Histone H4
KeywordsTRANSFERASE / complex / immune protein / nuclear protein
Function / homology
Function and homology information


negative regulation of chromosome condensation / Barr body / : / 2',3'-cyclic GMP-AMP synthase activity / cyclic GMP-AMP synthase / regulation of type I interferon production / pericentric heterochromatin formation / inner kinetochore / paracrine signaling / poly-ADP-D-ribose modification-dependent protein binding ...negative regulation of chromosome condensation / Barr body / : / 2',3'-cyclic GMP-AMP synthase activity / cyclic GMP-AMP synthase / regulation of type I interferon production / pericentric heterochromatin formation / inner kinetochore / paracrine signaling / poly-ADP-D-ribose modification-dependent protein binding / muscle cell differentiation / regulation of immunoglobulin production / negative regulation of DNA repair / cGAS/STING signaling pathway / oocyte maturation / regulation of T cell activation / : / nucleosomal DNA binding / negative regulation of cGAS/STING signaling pathway / cellular response to exogenous dsRNA / nucleus organization / spermatid development / positive regulation of type I interferon production / regulation of immune response / single fertilization / negative regulation of double-strand break repair via homologous recombination / subtelomeric heterochromatin formation / : / RNA polymerase II core promoter sequence-specific DNA binding / negative regulation of megakaryocyte differentiation / protein localization to CENP-A containing chromatin / nucleosome binding / Replacement of protamines by nucleosomes in the male pronucleus / CENP-A containing nucleosome / Packaging Of Telomere Ends / positive regulation of defense response to virus by host / phosphatidylinositol-4,5-bisphosphate binding / Recognition and association of DNA glycosylase with site containing an affected purine / Cleavage of the damaged purine / embryo implantation / Deposition of new CENPA-containing nucleosomes at the centromere / Recognition and association of DNA glycosylase with site containing an affected pyrimidine / Cleavage of the damaged pyrimidine / activation of innate immune response / telomere organization / Inhibition of DNA recombination at telomere / RNA Polymerase I Promoter Opening / Meiotic synapsis / 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 / Chromatin modifications during the maternal to zygotic transition (MZT) / SIRT1 negatively regulates rRNA expression / HCMV Late Events / innate immune response in mucosa / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / determination of adult lifespan / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / HDACs deacetylate histones / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / Nonhomologous End-Joining (NHEJ) / RNA Polymerase I Promoter Escape / molecular condensate scaffold activity / Transcriptional regulation by small RNAs / Formation of the beta-catenin:TCF transactivating complex / Activated PKN1 stimulates transcription of AR (androgen receptor) regulated genes KLK2 and KLK3 / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / HDMs demethylate histones / G2/M DNA damage checkpoint / NoRC negatively regulates rRNA expression / DNA Damage/Telomere Stress Induced Senescence / B-WICH complex positively regulates rRNA expression / PKMTs methylate histone lysines / Meiotic recombination / Pre-NOTCH Transcription and Translation / Metalloprotease DUBs / multicellular organism growth / male gonad development / RMTs methylate histone arginines / Activation of anterior HOX genes in hindbrain development during early embryogenesis / Transcriptional regulation of granulopoiesis / HCMV Early Events / antimicrobial humoral immune response mediated by antimicrobial peptide / osteoblast differentiation / structural constituent of chromatin / UCH proteinases / positive regulation of cellular senescence / antibacterial humoral response / nucleosome / heterochromatin formation / nucleosome assembly / E3 ubiquitin ligases ubiquitinate target proteins / Recruitment and ATM-mediated phosphorylation of repair and signaling proteins at DNA double strand breaks / site of double-strand break / HATs acetylate histones / RUNX1 regulates transcription of genes involved in differentiation of HSCs
Similarity search - Function
Mab-21-like, nucleotidyltransferase domain / Mab-21 protein nucleotidyltransferase domain / Mab-21-like / Mab-21-like, HhH/H2TH-like domain / Mab-21 protein HhH/H2TH-like domain / Mab-21 / : / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. ...Mab-21-like, nucleotidyltransferase domain / Mab-21 protein nucleotidyltransferase domain / Mab-21-like / Mab-21-like, HhH/H2TH-like domain / Mab-21 protein HhH/H2TH-like domain / Mab-21 / : / Histone H2B signature. / Histone H2A conserved site / Histone H2A signature. / Histone H2B / Histone H2B / Histone H2A, C-terminal domain / C-terminus of histone H2A / Histone 2A / Histone H2A / TATA box binding protein associated factor / TATA box binding protein associated factor (TAF), histone-like fold domain / Histone H4, conserved site / Histone H4 signature. / Histone H4 / Histone H4 / CENP-T/Histone H4, histone fold / Centromere kinetochore component CENP-T histone fold / Histone H3 signature 1. / Histone H3 signature 2. / Histone H3 / Histone H3/CENP-A / Histone H2A/H2B/H3 / Core histone H2A/H2B/H3/H4 domain / Histone-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Histone H4 / Histone H2B type 1-C/E/F/G/I / Histone H3.3 / Cyclic GMP-AMP synthase / Histone H2A type 1-C
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsMichalski, S. / de Oliveira Mann, C.C. / Witte, G. / Bartho, J. / Lammens, K. / Hopfner, K.P.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)CRC/TRR237 Germany
CitationJournal: Nature / Year: 2020
Title: Structural basis for sequestration and autoinhibition of cGAS by chromatin.
Authors: Sebastian Michalski / Carina C de Oliveira Mann / Che A Stafford / Gregor Witte / Joseph Bartho / Katja Lammens / Veit Hornung / Karl-Peter Hopfner /
Abstract: Cyclic GMP-AMP synthase (cGAS) is an innate immune sensor for cytosolic microbial DNA. After binding DNA, cGAS synthesizes the messenger 2'3'-cyclic GMP-AMP (cGAMP), which triggers cell-autonomous ...Cyclic GMP-AMP synthase (cGAS) is an innate immune sensor for cytosolic microbial DNA. After binding DNA, cGAS synthesizes the messenger 2'3'-cyclic GMP-AMP (cGAMP), which triggers cell-autonomous defence and the production of type I interferons and pro-inflammatory cytokines via the activation of STING. In addition to responding to cytosolic microbial DNA, cGAS also recognizes mislocalized cytosolic self-DNA and has been implicated in autoimmunity and sterile inflammation. Specificity towards pathogen- or damage-associated DNA was thought to be caused by cytosolic confinement. However, recent findings place cGAS robustly in the nucleus, where tight tethering of chromatin is important to prevent autoreactivity to self-DNA. Here we show how cGAS is sequestered and inhibited by chromatin. We provide a cryo-electron microscopy structure of the cGAS catalytic domain bound to a nucleosome, which shows that cGAS does not interact with the nucleosomal DNA, but instead interacts with histone 2A-histone 2B, and is tightly anchored to the 'acidic patch'. The interaction buries the cGAS DNA-binding site B, and blocks the formation of active cGAS dimers. The acidic patch robustly outcompetes agonistic DNA for binding to cGAS, which suggests that nucleosome sequestration can efficiently inhibit cGAS, even when accessible DNA is nearby, such as in actively transcribed genomic regions. Our results show how nuclear cGAS is sequestered by chromatin and provides a mechanism for preventing autoreactivity to nuclear self-DNA.
History
DepositionAug 7, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 23, 2020Provider: repository / Type: Initial release
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Revision 1.2Feb 10, 2021Group: Source and taxonomy / Category: em_entity_assembly_naturalsource / Item: _em_entity_assembly_naturalsource.organism
Revision 1.3Oct 9, 2024Group: Data collection / Database references / Structure summary
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Structure visualization

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Assembly

Deposited unit
a: Cyclic GMP-AMP synthase
I: Nucleosomal DNA strand 1
J: Nucleosomal DNA strand 2
b: Histone H2A type 1-C
c: Histone H2B type 1-C/E/F/G/I
d: Histone H3.3
e: Histone H4
f: Histone H2A type 1-C
g: Histone H2B type 1-C/E/F/G/I
h: Histone H3.3
i: Histone H4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)242,82312
Polymers242,75811
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: surface plasmon resonance
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area51640 Å2
ΔGint-360 kcal/mol
Surface area85130 Å2

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Components

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Protein , 5 types, 9 molecules abfcgdhei

#1: Protein Cyclic GMP-AMP synthase / m-cGAS / 2'3'-cGAMP synthase / Mab-21 domain-containing protein 1


Mass: 43401.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Mb21d1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8C6L5, cyclic GMP-AMP synthase
#4: Protein Histone H2A type 1-C / H2A-clustered histone 6 / Histone H2A/l


Mass: 14004.329 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H2AC6, H2AFL, HIST1H2AC / Production host: Escherichia coli (E. coli) / References: UniProt: Q93077
#5: 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: 13806.018 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: HIST1H2BC, H2BFL, HIST1H2BE, H2BFH, HIST1H2BF, H2BFG, HIST1H2BG, H2BFA, HIST1H2BI, H2BFK
Production host: Escherichia coli (E. coli) / References: UniProt: P62807
#6: Protein Histone H3.3


Mass: 15229.787 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: H3-3A, H3.3A, H3F3, H3F3A, PP781, H3-3B, H3.3B, H3F3B / Production host: Escherichia coli (E. coli) / References: UniProt: P84243
#7: Protein Histone H4


Mass: 11263.231 Da / Num. of mol.: 2
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 (E. coli) / References: UniProt: P62805

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Nucleosomal DNA strand ... , 2 types, 2 molecules IJ

#2: DNA chain Nucleosomal DNA strand 1


Mass: 45145.754 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain Nucleosomal DNA strand 2


Mass: 45604.047 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 1 types, 1 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
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
1cGAS, Nucleosome and DNACOMPLEX#1-#70MULTIPLE SOURCES
2NucleosomeCOMPLEX#4-#71RECOMBINANT
3Nucleosomal DNA strands 1 and 2COMPLEX#2-#31RECOMBINANT
4cGASCOMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDUnitsExperimental value
11KILODALTONS/NANOMETERNO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic construct (others)32630
34Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 44.8 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172977 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00614749
ELECTRON MICROSCOPYf_angle_d0.6421019
ELECTRON MICROSCOPYf_dihedral_angle_d31.1583820
ELECTRON MICROSCOPYf_chiral_restr0.0432366
ELECTRON MICROSCOPYf_plane_restr0.0031753

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