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- PDB-8fnu: Structure of RdrA from Streptococcus suis RADAR defense system -

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Basic information

Entry
Database: PDB / ID: 8fnu
TitleStructure of RdrA from Streptococcus suis RADAR defense system
ComponentsKAP NTPase domain-containing protein
KeywordsIMMUNE SYSTEM / RADAR / ATPase / RdrA / antiphage system
Function / homologyKAP family P-loop domain / KAP family P-loop domain / P-loop containing nucleoside triphosphate hydrolase / KAP NTPase domain-containing protein
Function and homology information
Biological speciesStreptococcus suis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsDuncan-Lowey, B. / Johnson, A.G. / Rawson, S. / Mayer, M.L. / Kranzusch, P.J.
Funding support United States, 4items
OrganizationGrant numberCountry
The Pew Charitable Trusts United States
Burroughs Wellcome Fund United States
The G. Harold and Leila Y. Mathers Foundation United States
Parker Institute for Cancer Immunotherapy United States
CitationJournal: Cell / Year: 2023
Title: Cryo-EM structure of the RADAR supramolecular anti-phage defense complex.
Authors: Brianna Duncan-Lowey / Nitzan Tal / Alex G Johnson / Shaun Rawson / Megan L Mayer / Shany Doron / Adi Millman / Sarah Melamed / Taya Fedorenko / Assaf Kacen / Alexander Brandis / Tevie ...Authors: Brianna Duncan-Lowey / Nitzan Tal / Alex G Johnson / Shaun Rawson / Megan L Mayer / Shany Doron / Adi Millman / Sarah Melamed / Taya Fedorenko / Assaf Kacen / Alexander Brandis / Tevie Mehlman / Gil Amitai / Rotem Sorek / Philip J Kranzusch /
Abstract: RADAR is a two-protein bacterial defense system that was reported to defend against phage by "editing" messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing ...RADAR is a two-protein bacterial defense system that was reported to defend against phage by "editing" messenger RNA. Here, we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal an RdrB active site that targets mononucleotides. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the massive accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a mechanism of anti-phage defense.
History
DepositionDec 28, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Mar 15, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: KAP NTPase domain-containing protein
B: KAP NTPase domain-containing protein
E: KAP NTPase domain-containing protein
F: KAP NTPase domain-containing protein
G: KAP NTPase domain-containing protein
D: KAP NTPase domain-containing protein
C: KAP NTPase domain-containing protein


Theoretical massNumber of molelcules
Total (without water)744,8937
Polymers744,8937
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "D"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "A"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "G"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1ASNLEUF1 - 796
d_21ens_1ASNLEUB1 - 796
d_31ens_1ASNLEUG1 - 796
d_41ens_1ASNLEUA1 - 796
d_51ens_1ASNLEUC1 - 796
d_61ens_1ASNLEUD1 - 796
d_71ens_1ASNLEUE1 - 796

NCS oper:
IDCodeMatrixVector
1given(0.606280677855, -0.795244154617, 0.00323638799079), (0.795139856196, 0.606259080286, 0.0142315375201), (-0.0132796366308, -0.00605492513301, 0.999893488894)261.441780899, -92.5354385287, 4.76346903221
2given(-0.900725060193, -0.434387980389, 0.00120350864707), (0.434387289599, -0.900725842631, -0.00079940712476), (0.00143128418668, -0.000197257171358, 0.999998956257)513.429916406, 322.68495457, -0.270659097152
3given(-0.244244788318, -0.969664674838, -0.00974175295298), (0.969651039827, -0.244330823956, 0.00890558400334), (-0.0110156407426, -0.00727095840085, 0.999912890617)490.380284179, 58.4614866459, 4.43721280157
4given(0.614772247022, 0.788672073779, -0.00717247050756), (-0.788696202525, 0.614782395067, -0.000952280604244), (0.00365847147822, 0.00624233593891, 0.999973824072)-86.572641743, 258.851451269, -2.27214015433
5given(-0.23865667176, 0.971084747204, -0.00611610759775), (-0.971095641041, -0.23862373932, 0.00565393532209), (0.0040310018879, 0.00728867481463, 0.99996531252)61.0683883743, 484.49284314, -2.712594564
6given(-0.903715391213, 0.428107280924, 0.00475896036052), (-0.428086118428, -0.903724834224, 0.0048681840836), (0.00638489571396, 0.00236220801512, 0.999976826271)323.546615839, 511.445042379, -2.11290123447

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Components

#1: Protein
KAP NTPase domain-containing protein / RdrA


Mass: 106413.305 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus suis (bacteria) / Gene: ERS132531_01777 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0Z8XTP6

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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

ComponentName: RdrA heptameric complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Streptococcus suis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium chlorideKCl1
250 mMHEPES1
31 mMTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 42.02 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193305 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 31.05 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00647145
ELECTRON MICROSCOPYf_angle_d1.27463574
ELECTRON MICROSCOPYf_dihedral_angle_d7.6226090
ELECTRON MICROSCOPYf_chiral_restr0.0587056
ELECTRON MICROSCOPYf_plane_restr0.0198008
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2FELECTRON MICROSCOPYNCS constraints0.0394164517977
ens_1d_3FELECTRON MICROSCOPYNCS constraints0.0394087377055
ens_1d_4FELECTRON MICROSCOPYNCS constraints0.0394072184331
ens_1d_5FELECTRON MICROSCOPYNCS constraints0.000703813061842
ens_1d_6FELECTRON MICROSCOPYNCS constraints0.000701915893833
ens_1d_7FELECTRON MICROSCOPYNCS constraints0.0394088013792

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