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- EMDB-26066: Structure of Enterobacter cloacae Cap2-CdnD02 2:1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-26066
TitleStructure of Enterobacter cloacae Cap2-CdnD02 2:1 complex
Map data
Sample
  • Complex: Ternary complex of Cap2 and CdnD02 in a 2:1 stoichiometry.
    • Protein or peptide: Cap2
    • Protein or peptide: Cyclic AMP-AMP-GMP synthase
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


nucleotide metabolic process / nucleotidyltransferase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / defense response to virus / GTP binding / ATP binding / metal ion binding
Similarity search - Function
Second Messenger Oligonucleotide or Dinucleotide Synthetase domain / Nucleotidyltransferase superfamily
Similarity search - Domain/homology
Cyclic AMP-AMP-GMP synthase
Similarity search - Component
Biological speciesEnterobacter cloacae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGu Y / Ye Q / Ledvina HE / Quan Y / Lau RK / Zhou H / Whiteley AT / Corbett KD
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM104141 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21 AI148814 United States
CitationJournal: Nature / Year: 2023
Title: An E1-E2 fusion protein primes antiviral immune signalling in bacteria.
Authors: Hannah E Ledvina / Qiaozhen Ye / Yajie Gu / Ashley E Sullivan / Yun Quan / Rebecca K Lau / Huilin Zhou / Kevin D Corbett / Aaron T Whiteley /
Abstract: In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor ...In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor cyclic GMP-AMP synthase (cGAS) detects viral infection to produce the nucleotide second messenger cyclic GMP-AMP (cGAMP), which initiates stimulator of interferon genes (STING)-dependent antiviral signalling. Bacteria encode evolutionary predecessors of cGAS called cGAS/DncV-like nucleotidyltransferases (CD-NTases), which detect bacteriophage infection and produce diverse nucleotide second messengers. How bacterial CD-NTase activation is controlled remains unknown. Here we show that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryo-electron microscopy structure of the Cap2-CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling eukaryotic E1 and E2 proteins. The structure captures a reactive-intermediate state with the CD-NTase C terminus positioned in the Cap2 E1 active site and conjugated to AMP. Cap2 conjugates the CD-NTase C terminus to a target molecule that primes the CD-NTase for increased cGAMP production. We further demonstrate that a specific endopeptidase, Cap3, balances Cap2 activity by cleaving CD-NTase-target conjugates. Our data demonstrate that bacteria control immune signalling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of the E1 and E2 machinery across domains of life.
History
DepositionJan 26, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateApr 26, 2023-
Current statusApr 26, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26066.png

Downloads & links

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Map

FileDownload / File: emd_26066.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.29307374 - 0.61477655
Average (Standard dev.)-0.0004174199 (±0.009071684)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 369.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_26066_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26066_half_map_2.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 : Ternary complex of Cap2 and CdnD02 in a 2:1 stoichiometry.

EntireName: Ternary complex of Cap2 and CdnD02 in a 2:1 stoichiometry.
Components
  • Complex: Ternary complex of Cap2 and CdnD02 in a 2:1 stoichiometry.
    • Protein or peptide: Cap2
    • Protein or peptide: Cyclic AMP-AMP-GMP synthase
  • Ligand: ADENOSINE MONOPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Ternary complex of Cap2 and CdnD02 in a 2:1 stoichiometry.

SupramoleculeName: Ternary complex of Cap2 and CdnD02 in a 2:1 stoichiometry.
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Enterobacter cloacae (bacteria)
Molecular weightTheoretical: 179.4 KDa

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Macromolecule #1: Cap2

MacromoleculeName: Cap2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Enterobacter cloacae (bacteria)
Molecular weightTheoretical: 67.024953 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSTVVQQVPA ELQAALTLIN NDPRMRTNNA WALSADKRWS LKFTAELSVP CSSFMPDNSV WHLVLWQEET LIRIEVYPDK SEGISATFQ HQNYNFSDAS TREWTSGNPA LENTPTVFGR NLWGLEPEAL LDRISWRLSR LLLWIDAAAQ EKLATTGDAV E LPAFPDQS ...String:
MSTVVQQVPA ELQAALTLIN NDPRMRTNNA WALSADKRWS LKFTAELSVP CSSFMPDNSV WHLVLWQEET LIRIEVYPDK SEGISATFQ HQNYNFSDAS TREWTSGNPA LENTPTVFGR NLWGLEPEAL LDRISWRLSR LLLWIDAAAQ EKLATTGDAV E LPAFPDQS PFTVIGFSEQ IDDLPFWASK TGEWGFASST GLPGAHGTLF LREFLDNKGK LIRTTKWSPF MRKGARTTNA VW SVLPTLP VLAPWQAPRT WQELSHCFAQ CGLSLPDLFS DIGRSVRALR KQRAPGLLLL GFPLENKIGD EPARIHWLAL RLA GLSNTM TKRPGFRPTE RNRRTWDREQ PLSQEPIRWV RTQNWAADQL RTRGEAANDI RSKKVLIIGA GSLGSMIAEN LMRI GVVSQ GILDADLLQT GNLSRHALTM TSVGHNKAAA LVEHLNRILP DASARSFSCA FPPESEVAKN SLRQYDVIID CTGDD GVLK SLAAFDWKSE KIFISLAMTW RAEGLFAFAA SETSFPVTDA SSRFNASASP EIDMDEARIE GIGAWHPVFP ARADDV QLW AAVGTKFICR VVSAPGRIYE YFKQMPDGTV EKEPHEY

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Macromolecule #2: Cyclic AMP-AMP-GMP synthase

MacromoleculeName: Cyclic AMP-AMP-GMP synthase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Enterobacter cloacae (bacteria)
Molecular weightTheoretical: 45.506828 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKSSHHHHHH ENLYFQSNAE LQPQFNEFLA NIRPTDTQKE DWKSGARTLR ERLKNFEPLK EIVVSTFLQG SIRRSTAIRP LGDKRPDVD IVVVTNLDHT RMSPTDAMDL FIPFLEKYYP GKWETQGRSF GITLSYVELD LVITAIPESG AEKSHLEQLY K SESVLTVN ...String:
MKSSHHHHHH ENLYFQSNAE LQPQFNEFLA NIRPTDTQKE DWKSGARTLR ERLKNFEPLK EIVVSTFLQG SIRRSTAIRP LGDKRPDVD IVVVTNLDHT RMSPTDAMDL FIPFLEKYYP GKWETQGRSF GITLSYVELD LVITAIPESG AEKSHLEQLY K SESVLTVN SLEEQTDWRL NKSWTPNTGW LSESNSAQVE DAPASEWKAH PLVLPDREKN EWGRTHPLAQ IRWTAEKNRL CN GHYINLV RAVKWWRQQN SEDLPKYPKG YPLEHLIGNA LDNGTTSMAQ GLVQLMDTFL SRWAAIYNQK SKPWLSDHGV AEH DVMARL TAEDFCSFYE GIASAAEIAR NALASEEPQE SAQLWRQLFG SKFPLPGPQG GDRNGGFTTP SKPAEPQKTG RFA

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Macromolecule #3: ADENOSINE MONOPHOSPHATE

MacromoleculeName: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: AMP
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-AMP:
ADENOSINE MONOPHOSPHATE / AMP*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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

Concentration0.9 mg/mL
BufferpH: 8.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average exposure time: 10.0 sec. / Average electron dose: 64.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 147709
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7ljl


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7tqd:
Structure of Enterobacter cloacae Cap2-CdnD02 2:1 complex

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