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- EMDB-11006: Structure of human cGAS bound to the nucleosome -

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Basic information

Entry
Database: EMDB / ID: EMD-11006
TitleStructure of human cGAS bound to the nucleosome
Map data
Sample
  • Complex: Cryo EM structure of cGAS-NCP1 complex
    • Complex: Cryo EM structure of cGAS-NCP1 complex
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A type 2-C
      • Protein or peptide: Histone H2B type 1-K
      • RNA: Cyclic GMP-AMP synthase
    • Complex: DNA
      • RNA: DNA (153-MER)
      • RNA: DNA (153-MER)
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsPathare GR / Cavadini S / Kempf G / Thoma NH
Funding support Switzerland, 5 items
OrganizationGrant numberCountry
Swiss National Science FoundationBSSGI0-155984 Switzerland
European Research Council (ERC)666068 Switzerland
European Research Council (ERC)804933 Switzerland
Swiss National Science Foundation31003A_179541 Switzerland
European Research Council (ERC)724022 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
DepositionMay 6, 2020-
Header (metadata) releaseSep 23, 2020-
Map releaseSep 23, 2020-
UpdateDec 9, 2020-
Current statusDec 9, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11006.png

Downloads & links

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Map

FileDownload / File: emd_11006.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.68 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.1
Minimum - Maximum-0.10925462 - 0.7860728
Average (Standard dev.)0.006347958 (±0.040449716)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 272.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.680.680.68
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z272.000272.000272.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-31-35-52
NX/NY/NZ11798209
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.1090.7860.006

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Supplemental data

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Additional map: Local-resolution filtered and sharpened map.

Fileemd_11006_additional.map
AnnotationLocal-resolution filtered and sharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local-resolution filtered and sharpened map.

Fileemd_11006_additional_1.map
AnnotationLocal-resolution filtered and sharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo EM structure of cGAS-NCP1 complex

EntireName: Cryo EM structure of cGAS-NCP1 complex
Components
  • Complex: Cryo EM structure of cGAS-NCP1 complex
    • Complex: Cryo EM structure of cGAS-NCP1 complex
      • Protein or peptide: Histone H3.2
      • Protein or peptide: Histone H4
      • Protein or peptide: Histone H2A type 2-C
      • Protein or peptide: Histone H2B type 1-K
      • RNA: Cyclic GMP-AMP synthase
    • Complex: DNA
      • RNA: DNA (153-MER)
      • RNA: DNA (153-MER)

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Supramolecule #1: Cryo EM structure of cGAS-NCP1 complex

SupramoleculeName: Cryo EM structure of cGAS-NCP1 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 240 KDa

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Supramolecule #2: Cryo EM structure of cGAS-NCP1 complex

SupramoleculeName: Cryo EM structure of cGAS-NCP1 complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4, #7
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Histone H3.2

MacromoleculeName: Histone H3.2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
PHRYRPGTVA LREIRRYQKS TELLIRKLPF QRLVREIAQD FKTDLRFQSS AVMALQEASE AYLVGLFEDT NLAAIHAKRV TIMPKDIQL ARRIRGE

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Macromolecule #2: Histone H4

MacromoleculeName: Histone H4 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG

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Macromolecule #3: Histone H2A type 2-C

MacromoleculeName: Histone H2A type 2-C / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
RAKAKSRSSR AGLQFPVGRV HRLLRKGNYA ERVGAGAPVY MAAVLEYLTA EILELAGNAA RDNKKTRIIP RHLQLAIRND EELNKLLGK VTIAQGGVLP NIQAVLLP

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Macromolecule #4: Histone H2B type 1-K

MacromoleculeName: Histone H2B type 1-K / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
SRKESYSVYV YKVLKQVHPD TGISSKAMGI MNSFVNDIFE RIAGEASRLA HYNKRSTITS REIQTAVRLL LPGELAKHAV SEGTKAVTK YTSA

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Macromolecule #5: DNA (153-MER)

MacromoleculeName: DNA (153-MER) / type: rna / ID: 5
Source (natural)Organism: Homo sapiens (human)
SequenceString: (DA)(DT)(DC)(DC)(DT)(DG)(DG)(DA)(DG)(DA) (DA)(DT)(DC)(DC)(DC)(DG)(DG)(DT)(DG)(DC) (DC)(DG)(DA)(DG)(DG)(DC)(DC)(DG)(DC) (DT)(DC)(DA)(DA)(DT)(DT)(DG)(DG)(DT)(DC) (DG) (DT)(DA)(DG)(DA)(DC)(DA) ...String:
(DA)(DT)(DC)(DC)(DT)(DG)(DG)(DA)(DG)(DA) (DA)(DT)(DC)(DC)(DC)(DG)(DG)(DT)(DG)(DC) (DC)(DG)(DA)(DG)(DG)(DC)(DC)(DG)(DC) (DT)(DC)(DA)(DA)(DT)(DT)(DG)(DG)(DT)(DC) (DG) (DT)(DA)(DG)(DA)(DC)(DA)(DG)(DC) (DT)(DC)(DT)(DA)(DG)(DC)(DA)(DC)(DC)(DG) (DC)(DT) (DT)(DA)(DA)(DA)(DC)(DG)(DC) (DA)(DC)(DG)(DT)(DA)(DC)(DG)(DC)(DG)(DC) (DT)(DG)(DT) (DC)(DC)(DC)(DC)(DC)(DG) (DC)(DG)(DT)(DT)(DT)(DT)(DA)(DA)(DC)(DC) (DG)(DC)(DC)(DA) (DA)(DG)(DG)(DG)(DG) (DA)(DT)(DT)(DA)(DC)(DT)(DC)(DC)(DC)(DT) (DA)(DG)(DT)(DC)(DT) (DC)(DC)(DA)(DG) (DG)(DC)(DA)(DC)(DG)(DT)(DG)(DT)(DC)(DA) (DG)(DA)(DT)(DA)(DT)(DA) (DT)(DA)(DC) (DA)(DT)(DC)(DC)(DT)(DG)(DT)(DG)(DA)(DT)

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Macromolecule #6: DNA (153-MER)

MacromoleculeName: DNA (153-MER) / type: rna / ID: 6
Source (natural)Organism: Homo sapiens (human)
SequenceString: (DA)(DT)(DC)(DA)(DC)(DA)(DG)(DG)(DA)(DT) (DG)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT) (DG)(DA)(DC)(DA)(DC)(DG)(DT)(DG)(DC) (DC)(DT)(DG)(DG)(DA)(DG)(DA)(DC)(DT)(DA) (DG) (DG)(DG)(DA)(DG)(DT)(DA) ...String:
(DA)(DT)(DC)(DA)(DC)(DA)(DG)(DG)(DA)(DT) (DG)(DT)(DA)(DT)(DA)(DT)(DA)(DT)(DC)(DT) (DG)(DA)(DC)(DA)(DC)(DG)(DT)(DG)(DC) (DC)(DT)(DG)(DG)(DA)(DG)(DA)(DC)(DT)(DA) (DG) (DG)(DG)(DA)(DG)(DT)(DA)(DA)(DT) (DC)(DC)(DC)(DC)(DT)(DT)(DG)(DG)(DC)(DG) (DG)(DT) (DT)(DA)(DA)(DA)(DA)(DC)(DG) (DC)(DG)(DG)(DG)(DG)(DG)(DA)(DC)(DA)(DG) (DC)(DG)(DC) (DG)(DT)(DA)(DC)(DG)(DT) (DG)(DC)(DG)(DT)(DT)(DT)(DA)(DA)(DG)(DC) (DG)(DG)(DT)(DG) (DC)(DT)(DA)(DG)(DA) (DG)(DC)(DT)(DG)(DT)(DC)(DT)(DA)(DC)(DG) (DA)(DC)(DC)(DA)(DA) (DT)(DT)(DG)(DA) (DG)(DC)(DG)(DG)(DC)(DC)(DT)(DC)(DG)(DG) (DC)(DA)(DC)(DC)(DG)(DG) (DG)(DA)(DT) (DT)(DC)(DT)(DC)(DC)(DA)(DG)(DG)(DA)(DT)

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Macromolecule #7: Cyclic GMP-AMP synthase

MacromoleculeName: Cyclic GMP-AMP synthase / type: rna / ID: 7
Source (natural)Organism: Homo sapiens (human)
SequenceString: GASKLRAVLE KLKLSRDDIS TAAGMVKGVV DHLLLRLKCD SAFRGVGLLN TGSYYEHVKI SAPNEFDVMF KLEVPRIQLE EYSNTRAYY FVKFKRNPKE NPLSQFLEGE ILSASKMLSK FRKIIKEEIN DIKDTDVIMK RKRGGSPAVT LLISEKISVD I TLALESKS ...String:
GASKLRAVLE KLKLSRDDIS TAAGMVKGVV DHLLLRLKCD SAFRGVGLLN TGSYYEHVKI SAPNEFDVMF KLEVPRIQLE EYSNTRAYY FVKFKRNPKE NPLSQFLEGE ILSASKMLSK FRKIIKEEIN DIKDTDVIMK RKRGGSPAVT LLISEKISVD I TLALESKS SWPASTQEGL RIQNWLSAKV RKQLRLKPFY LVPKHAKEGN GFQEETWRLS FSHIEKEILN NHGKSKTCCE NK EEKCCRK DCLKLMKYLL EQLKERFKDK KHLDKFSSYH VKTAFFHVCT QNPQDSQWDR KDLGLCFDNC VTYFLQCLRT EKL ENYFIP EFNLFSSNLI DKRSKEFLTK QIEYERNNEF PVFDEF

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4 / Component - Concentration: 300.0 mM / Component - Formula: NaClSodium chloride / Component - Name: Sodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 1 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus min: 2.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus min: -0.0005 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 3 / Number real images: 5007 / Average electron dose: 35.0 e/Å2

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Image processing

Particle selectionNumber selected: 16000
CTF correctionSoftware - Name: cryoSPARC (ver. 2.14.2)
Startup modelType of model: OTHER / Details: Ab initio model
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2.14.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14) / Number images used: 142743

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