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- PDB-8skt: Structure of ternary complex of mouse cGAS with dsDNA and bound A... -

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Basic information

Entry
Database: PDB / ID: 8skt
TitleStructure of ternary complex of mouse cGAS with dsDNA and bound ATP with 5 mM Mn2+
Components
  • Cyclic GMP-AMP synthase
  • Palindromic DNA18
KeywordsTRANSFERASE/DNA / IMMUNE SYSTEM / TRANSFERASE-DNA complex
Function / homology
Function and homology information


2',3'-cyclic GMP-AMP synthase activity / cyclic GMP-AMP synthase / paracrine signaling / poly-ADP-D-ribose modification-dependent protein binding / regulation of type I interferon production / regulation of immunoglobulin production / cGAS/STING signaling pathway / regulation of T cell activation / negative regulation of cGAS/STING signaling pathway / negative regulation of DNA repair ...2',3'-cyclic GMP-AMP synthase activity / cyclic GMP-AMP synthase / paracrine signaling / poly-ADP-D-ribose modification-dependent protein binding / regulation of type I interferon production / regulation of immunoglobulin production / cGAS/STING signaling pathway / regulation of T cell activation / negative regulation of cGAS/STING signaling pathway / negative regulation of DNA repair / cGMP-mediated signaling / cellular response to exogenous dsRNA / positive regulation of type I interferon production / nucleosome binding / regulation of immune response / negative regulation of double-strand break repair via homologous recombination / positive regulation of defense response to virus by host / activation of innate immune response / phosphatidylinositol-4,5-bisphosphate binding / cAMP-mediated signaling / determination of adult lifespan / molecular condensate scaffold activity / positive regulation of cellular senescence / site of double-strand break / double-stranded DNA binding / defense response to virus / nuclear body / innate immune response / DNA repair / DNA damage response / chromatin binding / GTP binding / protein homodimerization activity / DNA binding / ATP binding / nucleus / metal ion binding / plasma membrane / cytosol
Similarity search - Function
Mab-21-like, nucleotidyltransferase domain / Mab-21-like / Mab-21-like, HhH/H2TH-like domain / Mab-21 protein HhH/H2TH-like domain / Mab-21 / Mab-21 protein nucleotidyltransferase domain
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / : / DNA / DNA (> 10) / Cyclic GMP-AMP synthase
Similarity search - Component
Biological speciesMus musculus (house mouse)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.69 Å
AuthorsWu, S. / Sohn, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nat Commun / Year: 2024
Title: The structural basis for 2'-5'/3'-5'-cGAMP synthesis by cGAS.
Authors: Wu, S. / Gabelli, S.B. / Sohn, J.
History
DepositionApr 20, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2May 29, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cyclic GMP-AMP synthase
C: Cyclic GMP-AMP synthase
E: Palindromic DNA18
F: Palindromic DNA18
I: Palindromic DNA18
J: Palindromic DNA18
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,70414
Polymers107,3396
Non-polymers1,3658
Water45025
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry, ATP.Mn binds to mcGAS catalytic domain and DNA complex nicely using ITC.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)78.026, 98.394, 142.589
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

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Protein / DNA chain , 2 types, 6 molecules ACEFIJ

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


Mass: 42640.254 Da / Num. of mol.: 2 / Fragment: catalytic domain (UNP residues 147-507)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Cgas, Mb21d1 / Plasmid: nHMT mCAT WT
Details (production host): His*6-MBP-Tev-AgeI-mcGAS CAT, Kanamycin resistance
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8C6L5, cyclic GMP-AMP synthase
#2: DNA chain
Palindromic DNA18


Mass: 5514.603 Da / Num. of mol.: 4 / Source method: obtained synthetically / Details: custom synthesized by IDT / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 33 molecules

#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 25 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.55 Å3/Da / Density % sol: 51.76 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 0.2 M ammonium acetate, 32% MPD, 0.1 M Bis-Tris, pH 6.5
PH range: 6.0-7.0

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: nitrogen gas stream / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-2 / Wavelength: 1.8914 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Mar 30, 2022
RadiationMonochromator: horizontal bounce Si(111) double crystal monochromator
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.8914 Å / Relative weight: 1
ReflectionResolution: 2.69→29.58 Å / Num. obs: 58811 / % possible obs: 99.6 % / Redundancy: 6.6 % / Biso Wilson estimate: 54.18 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.101 / Rpim(I) all: 0.042 / Rrim(I) all: 0.109 / Χ2: 1.04 / Net I/σ(I): 12.2
Reflection shellResolution: 2.69→2.82 Å / % possible obs: 97.6 % / Redundancy: 6.5 % / Rmerge(I) obs: 0.687 / Num. measured all: 25955 / Num. unique obs: 4001 / CC1/2: 0.824 / Rpim(I) all: 0.289 / Rrim(I) all: 0.747 / Χ2: 1.04 / Net I/σ(I) obs: 2.6

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Processing

Software
NameVersionClassification
XDS20210205data reduction
Aimless0.7.7data scaling
MOLREP11.7.03phasing
Coot0.9.6model building
PHENIX1.20.1_4487refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 4LEZ

4lez


Resolution: 2.69→29.58 Å / SU ML: 0.3271 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.094
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2467 2923 4.97 %
Rwork0.1987 55888 -
obs0.2012 58811 99.49 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 62.23 Å2
Refinement stepCycle: LAST / Resolution: 2.69→29.58 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5740 1464 68 25 7297
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00917566
X-RAY DIFFRACTIONf_angle_d1.072910488
X-RAY DIFFRACTIONf_chiral_restr0.05321141
X-RAY DIFFRACTIONf_plane_restr0.00991063
X-RAY DIFFRACTIONf_dihedral_angle_d24.65771454
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.69-2.730.36211150.30112480X-RAY DIFFRACTION91.96
2.73-2.780.32081400.28772686X-RAY DIFFRACTION100
2.78-2.830.42471660.31092625X-RAY DIFFRACTION99.75
2.83-2.880.36861460.28242720X-RAY DIFFRACTION99.9
2.88-2.940.35411400.2782603X-RAY DIFFRACTION99.85
2.94-30.32261450.26362712X-RAY DIFFRACTION99.9
3-3.070.33441280.25982681X-RAY DIFFRACTION99.93
3.07-3.150.32691340.24342672X-RAY DIFFRACTION99.93
3.15-3.240.30141400.25052685X-RAY DIFFRACTION100
3.24-3.330.25521300.22982663X-RAY DIFFRACTION99.93
3.33-3.440.25071560.20872635X-RAY DIFFRACTION99.96
3.44-3.560.23361120.19682709X-RAY DIFFRACTION99.96
3.56-3.70.27191380.20492709X-RAY DIFFRACTION99.89
3.7-3.870.26461170.19632687X-RAY DIFFRACTION99.96
3.87-4.080.26541350.17952671X-RAY DIFFRACTION99.96
4.08-4.330.22861350.17262687X-RAY DIFFRACTION100
4.33-4.660.21441710.15892625X-RAY DIFFRACTION99.96
4.66-5.130.20691570.16692652X-RAY DIFFRACTION99.96
5.13-5.870.20931470.1752658X-RAY DIFFRACTION99.96
5.87-7.370.26221280.19932685X-RAY DIFFRACTION99.65
7.38-29.580.14661430.13492643X-RAY DIFFRACTION98.86
Refinement TLS params.Method: refined / Origin x: -18.7762303418 Å / Origin y: -9.63462094297 Å / Origin z: 20.6768283631 Å
111213212223313233
T0.307805749312 Å2-0.00301994044027 Å2-0.00998875067604 Å2-0.363770834383 Å2-0.0227364294639 Å2--0.270959526269 Å2
L0.971876613224 °2-0.670265518376 °20.298426786493 °2-1.90568082254 °2-0.192281393226 °2--0.409532421607 °2
S-0.0771508424722 Å °-0.112080339614 Å °0.0720467994996 Å °0.157561752541 Å °0.0179002925563 Å °-0.0522959660914 Å °-0.0515188033345 Å °-0.0531450130847 Å °0.058986853704 Å °
Refinement TLS groupSelection details: all

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