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- PDB-8eae: Structure of Ternary Complex of cGAS with dsDNA and Bound 5-pppG(... -

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Basic information

Entry
Database: PDB / ID: 8eae
TitleStructure of Ternary Complex of cGAS with dsDNA and Bound 5-pppG(2,5)pI
Components
  • Cyclic GMP-AMP synthase
  • Palindromic DNA18
KeywordsTRANSFERASE/DNA / Transferase-DNA complex / cGAS / pppG(2 / 5)pI / IMMUNE SYSTEM
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
Chem-VLO / DNA / DNA (> 10) / Cyclic GMP-AMP synthase
Similarity search - Component
Biological speciesMus musculus (house mouse)
DNA molecule (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.56 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
DepositionAug 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 25, 2023Provider: 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)109,22512
Polymers107,3396
Non-polymers1,8866
Water1,06359
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: HPLC shows that cGAS reacts with GTP and ITP to produce a single dinucleotide but without cyclized product.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9570 Å2
ΔGint-52 kcal/mol
Surface area41760 Å2
MethodPISA
Unit cell
Length a, b, c (Å)77.428, 99.322, 141.723
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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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
Source method: isolated from a genetically manipulated source
Details: Mouse Cyclic GMP-AMP synthase catalytic domain 147-507; three extra residues at the N terminus (G from TEV site, TG from AgeI site).
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: isolated from a natural source / Source: (natural) DNA molecule (others) / Plasmid details: IDT

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Non-polymers , 4 types, 65 molecules

#3: Chemical ChemComp-VLO / [[(2~{R},3~{R},4~{R},5~{R})-5-(2-azanyl-6-oxidanylidene-1~{H}-purin-9-yl)-4-[[(2~{R},3~{S},4~{R},5~{R})-3,4-bis(oxidanyl)-5-(6-oxidanylidene-1~{H}-purin-9-yl)oxolan-2-yl]methoxy-oxidanyl-phosphoryl]oxy-3-oxidanyl-oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate


Mass: 853.371 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H27N9O21P4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 59 / 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: 2.54 Å3/Da / Density % sol: 51.55 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 0.2 M ammonium acetate, 32% MPD, with 0.1 M Bis-Tris pH 6.5
PH range: 6.0-7.0 / Temp details: 4-degree Celsius in cold room

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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-1 / Wavelength: 0.920119 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jul 25, 2022
RadiationMonochromator: double crystal Si(111) monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.920119 Å / Relative weight: 1
ReflectionResolution: 2.56→29.76 Å / Num. obs: 35867 / % possible obs: 99.2 % / Redundancy: 5.6 % / CC1/2: 0.998 / Rmerge(I) obs: 0.089 / Rpim(I) all: 0.041 / Rrim(I) all: 0.099 / Net I/σ(I): 11.5 / Num. measured all: 201302 / Scaling rejects: 4
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.56-2.675.50.6282249141120.8110.2930.6952.594.2
8.85-29.765.10.04248669480.9980.020.04627.296.8

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Processing

Software
NameVersionClassification
XDS20210205data reduction
Aimless0.7.8data scaling
MOLREP11.7.03phasing
REFMAC5.8.0267refinement
Coot0.9.6model building
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7UUX

7uux


Resolution: 2.56→28.86 Å / Cor.coef. Fo:Fc: 0.957 / Cor.coef. Fo:Fc free: 0.936 / SU B: 22.034 / SU ML: 0.223 / Cross valid method: THROUGHOUT / ESU R: 0.683 / ESU R Free: 0.285 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: U VALUES : WITH TLS ADDED HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : RESIDUAL ONLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2329 1768 4.9 %RANDOM
Rwork0.1948 ---
obs0.1967 33970 99.32 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1 Å / Solvent model: MASK
Displacement parametersBiso max: 208.56 Å2 / Biso mean: 65.096 Å2 / Biso min: 26.66 Å2
Baniso -1Baniso -2Baniso -3
1--2.09 Å2-0 Å20 Å2
2--2.94 Å2-0 Å2
3----0.85 Å2
Refinement stepCycle: final / Resolution: 2.56→28.86 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5828 1464 112 59 7463
Biso mean--73.7 42.05 -
Num. residues----776
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0167801
X-RAY DIFFRACTIONr_bond_other_d0.0010.026656
X-RAY DIFFRACTIONr_angle_refined_deg1.3341.71610715
X-RAY DIFFRACTIONr_angle_other_deg1.1062.76815479
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.895699
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.97522.539319
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.107151188
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.4381537
X-RAY DIFFRACTIONr_chiral_restr0.0720.21099
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.027393
X-RAY DIFFRACTIONr_gen_planes_other0.0030.021701
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.56→2.625 Å / Rfactor Rfree error: 0
RfactorNum. reflection% reflection
Rfree0.334 131 -
Rwork0.296 2319 -
obs--93.37 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.6140.40530.10722.08390.04451.18090.01380.201-0.2295-0.09410.03050.17440.1569-0.1427-0.04430.1571-0.0055-0.00190.0671-0.02550.06254.973-26.951-20.633
21.88380.8157-0.2662.4759-0.67151.3845-0.07950.30440.17740.02580.0416-0.3403-0.25070.2220.03780.191-0.0715-0.0490.150.04270.113833.6138.442-18.759
35.14670.9588-1.91182.36042.46464.57440.03860.95750.081-0.49720.2056-0.0641-0.5896-0.1544-0.24410.33230.1512-0.02730.3489-0.05780.177829.051-22.855-31.688
43.99710.4715-1.78484.55430.42134.95130.16170.82860.0868-0.77240.014-0.0869-0.51260.4939-0.17560.3573-0.00460.06920.4012-0.05990.14229.702-23.214-32.268
53.20831.51931.76814.1996-1.32082.7590.086-0.16410.35840.16150.04720.3056-0.1066-0.3379-0.13320.17270.0675-0.00560.1440.06830.2088.198.555-17.783
65.17251.10760.47145.84980.99352.64230.1004-0.55570.2450.23230.16860.3779-0.0631-0.3163-0.2690.22620.0187-0.04680.23490.12960.18668.5576.702-18.349
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A148 - 506
2X-RAY DIFFRACTION2C148 - 506
3X-RAY DIFFRACTION3E1 - 18
4X-RAY DIFFRACTION4F1 - 18
5X-RAY DIFFRACTION5I1 - 18
6X-RAY DIFFRACTION6J1 - 18

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