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- PDB-8fnv: Structure of RdrB from Escherichia coli RADAR defense system -

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Basic information

Entry
Database: PDB / ID: 8fnv
TitleStructure of RdrB from Escherichia coli RADAR defense system
ComponentsAdenosine deaminase
KeywordsIMMUNE SYSTEM / adenosine deaminase / anti-phage defense / RADAR
Function / homologydeaminase activity / Adenosine/adenine deaminase / Metal-dependent hydrolase / Adenosine deaminase
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.11 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
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
Revision 1.2Mar 15, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Adenosine deaminase
B: Adenosine deaminase
C: Adenosine deaminase
D: Adenosine deaminase
E: Adenosine deaminase
F: Adenosine deaminase
G: Adenosine deaminase
H: Adenosine deaminase
I: Adenosine deaminase
J: Adenosine deaminase
K: Adenosine deaminase
L: Adenosine deaminase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,106,34324
Polymers1,105,55912
Non-polymers78512
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "B" and (resid 1 through 122 or resid 126 through 801))
d_2ens_1chain "D"
d_3ens_1(chain "C" and (resid 1 through 122 or resid 126 through 801))
d_4ens_1(chain "A" and (resid 1 through 122 or resid 126 through 801))
d_5ens_1(chain "E" and (resid 1 through 122 or resid 126 through 801))
d_6ens_1(chain "F" and (resid 1 through 122 or resid 126 through 801))
d_7ens_1(chain "G" and (resid 1 through 122 or resid 126 through 801))
d_8ens_1(chain "H" and (resid 1 through 122 or resid 126 through 801))
d_9ens_1(chain "I" and (resid 1 through 122 or resid 126 through 801))
d_10ens_1(chain "J" and (resid 1 through 122 or resid 126 through 801))
d_11ens_1(chain "K" and (resid 1 through 122 or resid 126 through 801))
d_12ens_1(chain "L" and (resid 1 through 122 or resid 126 through 801))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1METALAC1 - 122
d_12ens_1TYRVALC126 - 770
d_21ens_1METVALG1 - 767
d_31ens_1METALAE1 - 122
d_32ens_1TYRVALE126 - 770
d_41ens_1METALAA1 - 122
d_42ens_1TYRVALA126 - 770
d_51ens_1METALAI1 - 122
d_52ens_1TYRVALI126 - 770
d_61ens_1METALAK1 - 122
d_62ens_1TYRVALK126 - 770
d_71ens_1METALAM1 - 122
d_72ens_1TYRVALM126 - 770
d_81ens_1METALAO1 - 122
d_82ens_1TYRVALO126 - 770
d_91ens_1METALAQ1 - 122
d_92ens_1TYRVALQ126 - 770
d_101ens_1METALAS1 - 122
d_102ens_1TYRVALS126 - 770
d_111ens_1METALAU1 - 122
d_112ens_1TYRVALU126 - 770
d_121ens_1METALAW1 - 122
d_122ens_1TYRVALW126 - 770

NCS oper:
IDCodeMatrixVector
1given(0.499868680908, -0.288732628207, -0.816556655264), (-0.866101156906, -0.166964132566, -0.471160019997), (-0.000296402757389, 0.942738801495, -0.333531804031)317.880386878, 495.817199574, 77.4526102089
2given(-0.499993973576, 0.289015067268, 0.816380007888), (-0.288686530528, 0.833132416185, -0.471752545508), (-0.816496242171, -0.471551341843, -0.333126430243)78.1270420197, 183.614094389, 518.988878035
3given(-0.499959454537, 0.866048797366, -0.000156202662962), (-0.866048793045, -0.499959472965, -0.000116004320031), (-0.000178560402902, 7.72816711619E-5, 0.999999981072)125.543999079, 468.500660894, 0.0323612386267
4given(0.000323814028105, -0.57767815486, 0.816264567736), (-0.577672364011, -0.666399319199, -0.471387937084), (0.816268665944, -0.471380840476, -0.333924494804)150.682967775, 537.676562803, 195.869499906
5given(0.500518928699, 0.288313076269, 0.816306542952), (0.865725589538, -0.166769843181, -0.471918449549), (7.50542180495E-5, 0.942901579942, -0.333071471173)-119.826474226, 153.080650043, 77.2377343984
6given(0.500325291531, -0.86583750796, -0.000111637219495), (-0.288428539229, -0.166790362177, 0.942862637314), (-0.816384456253, -0.471705824528, -0.333181684212)270.387466809, 101.43449155, 519.008712321
7given(-0.500316892907, -0.86584235884, 0.00012771056954), (0.865842358139, -0.500316859066, 0.000226685917456), (-0.000132378518464, 0.000223992014578, 0.999999966152)468.477728015, 125.575425728, 0.00504108900986
8given(0.499841985352, 0.866116593133, -0.000191777300034), (0.28838402654, -0.166219656709, 0.942977029922), (0.816696175401, -0.471394816288, -0.332857753795)-72.4132119191, -12.8939813013, 195.517790835
9given(-0.999999966621, -7.36913265437E-5, -0.000247645117532), (-0.000258049420387, 0.333073745187, 0.94290074434), (1.30004801244E-5, 0.942900776772, -0.333073753086)396.070997152, -54.5987852054, 77.2424148397
10given(-4.00167885517E-6, 0.577510156207, -0.816383500238), (0.577209697225, -0.66665413594, -0.471594347361), (-0.816595962158, -0.471226360162, -0.333341794673)245.284720874, 309.095209099, 519.00329005
11given(-0.500591745236, -0.288549405801, -0.816178378183), (0.28811546776, 0.833531751222, -0.471396114687), (0.816331661666, -0.471130618955, -0.33412356703)515.853063143, 69.2676512085, 195.85802158

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Components

#1: Protein
Adenosine deaminase / RdrB


Mass: 92129.875 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: AW119_27895 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8E2SFD7
#2: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: Escherichia coli RdrB dodecamer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
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

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 48.8 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: 2.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233454 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 33.14 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00776775
ELECTRON MICROSCOPYf_angle_d1.363104029
ELECTRON MICROSCOPYf_dihedral_angle_d5.88810100
ELECTRON MICROSCOPYf_chiral_restr0.06811349
ELECTRON MICROSCOPYf_plane_restr0.01813302
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2CELECTRON MICROSCOPYNCS constraints0.000708656857651
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.000706239172885
ens_1d_4CELECTRON MICROSCOPYNCS constraints0.000704112169667
ens_1d_5CELECTRON MICROSCOPYNCS constraints0.000709633471495
ens_1d_6CELECTRON MICROSCOPYNCS constraints0.000704334092889
ens_1d_7CELECTRON MICROSCOPYNCS constraints0.000704355537427
ens_1d_8CELECTRON MICROSCOPYNCS constraints0.000711596053853
ens_1d_9CELECTRON MICROSCOPYNCS constraints0.000713463332595
ens_1d_10CELECTRON MICROSCOPYNCS constraints0.000706767066159
ens_1d_11CELECTRON MICROSCOPYNCS constraints0.000703462677704
ens_1d_12CELECTRON MICROSCOPYNCS constraints0.000709989937069

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