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- EMDB-33198: Structure of the OgeuIscB-omega RNA-target DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-33198
TitleStructure of the OgeuIscB-omega RNA-target DNA complex
Map data
Sample
  • Complex: Structure of the IscB-omegaRNA ribonucleoprotein complex
    • Complex: IscB-omegaRNA
      • Protein or peptide: OgeuIscB
    • Complex: DNA
      • DNA: DNA (5'-D(P*GP*AP*AP*GP*AP*AP*AP*AP*CP*CP*AP*T)-3')
  • RNA: RNA (228-MER)
  • DNA: DNA (49-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: LAURYL DIMETHYLAMINE-N-OXIDE
Biological speciesmetagenome (others) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsKato K / Okazaki O / Isayama Y / Ishikawa J / Nishizawa T / Nishimasu H
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: Nat Commun / Year: 2022
Title: Structure of the IscB-ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9.
Authors: Kazuki Kato / Sae Okazaki / Soumya Kannan / Han Altae-Tran / F Esra Demircioglu / Yukari Isayama / Junichiro Ishikawa / Masahiro Fukuda / Rhiannon K Macrae / Tomohiro Nishizawa / Kira S ...Authors: Kazuki Kato / Sae Okazaki / Soumya Kannan / Han Altae-Tran / F Esra Demircioglu / Yukari Isayama / Junichiro Ishikawa / Masahiro Fukuda / Rhiannon K Macrae / Tomohiro Nishizawa / Kira S Makarova / Eugene V Koonin / Feng Zhang / Hiroshi Nishimasu /
Abstract: Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II ...Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe. Here, we report the cryo-electron microscopy structure of an IscB protein from the human gut metagenome (OgeuIscB) in complex with its cognate ωRNA and a target DNA, at 2.6-Å resolution. This high-resolution structure reveals the detailed architecture of the IscB-ωRNA ribonucleoprotein complex, and shows how the small IscB protein assembles with the ωRNA and mediates RNA-guided DNA cleavage. The large ωRNA scaffold structurally and functionally compensates for the recognition lobe of Cas9, and participates in the recognition of the guide RNA-target DNA heteroduplex. These findings provide insights into the mechanism of the programmable DNA cleavage by the IscB-ωRNA complex and the evolution of the type II CRISPR-Cas9 effector complexes.
History
DepositionApr 10, 2022-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateDec 14, 2022-
Current statusDec 14, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33198.png

Downloads & links

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Map

FileDownload / File: emd_33198.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 1.5
Minimum - Maximum-8.444852 - 11.414227
Average (Standard dev.)-0.0008699077 (±0.17735876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33198_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33198_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 : Structure of the IscB-omegaRNA ribonucleoprotein complex

EntireName: Structure of the IscB-omegaRNA ribonucleoprotein complex
Components
  • Complex: Structure of the IscB-omegaRNA ribonucleoprotein complex
    • Complex: IscB-omegaRNA
      • Protein or peptide: OgeuIscB
    • Complex: DNA
      • DNA: DNA (5'-D(P*GP*AP*AP*GP*AP*AP*AP*AP*CP*CP*AP*T)-3')
  • RNA: RNA (228-MER)
  • DNA: DNA (49-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: LAURYL DIMETHYLAMINE-N-OXIDE

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Supramolecule #1: Structure of the IscB-omegaRNA ribonucleoprotein complex

SupramoleculeName: Structure of the IscB-omegaRNA ribonucleoprotein complex
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #4

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Supramolecule #2: IscB-omegaRNA

SupramoleculeName: IscB-omegaRNA / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: metagenome (others)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #3

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Macromolecule #1: RNA (228-MER)

MacromoleculeName: RNA (228-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 73.640742 KDa
SequenceString: UGUGAGCGGA UAACAAUUCC CCGGCUCUUC CAACUUUAUG GUUGCGACCG UAGGUUGAAA GAGCACAGGC UGAGACAUUC GUAAGGCCG AAAGACCGGA CGCACCCUGG GAUUUCCCCA GUCCCCGGAA CUGCAUAGCG GAUGCCAGUU GAUGGAGCAA U CUAUCAGA ...String:
UGUGAGCGGA UAACAAUUCC CCGGCUCUUC CAACUUUAUG GUUGCGACCG UAGGUUGAAA GAGCACAGGC UGAGACAUUC GUAAGGCCG AAAGACCGGA CGCACCCUGG GAUUUCCCCA GUCCCCGGAA CUGCAUAGCG GAUGCCAGUU GAUGGAGCAA U CUAUCAGA UAAGCCAGGG GGAACAAUCA CCUCUCUGUA UCAGAGAGAG UUUUACAAAA GGAGGAACGG

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Macromolecule #2: DNA (49-MER)

MacromoleculeName: DNA (49-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.092702 KDa
SequenceString:
(DG)(DA)(DA)(DT)(DG)(DG)(DT)(DT)(DT)(DT) (DC)(DT)(DT)(DC)(DG)(DG)(DG)(DG)(DA)(DA) (DT)(DT)(DG)(DT)(DT)(DA)(DT)(DC)(DC) (DG)(DC)(DT)(DC)(DA)(DC)(DA)(DA)(DT)(DT) (DC) (DC)(DT)(DT)(DA)(DG)(DA)(DA)(DA) (DA)

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Macromolecule #3: DNA (5'-D(P*GP*AP*AP*GP*AP*AP*AP*AP*CP*CP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*GP*AP*AP*GP*AP*AP*AP*AP*CP*CP*AP*T)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.281832 KDa
SequenceString:
(DG)(DA)(DA)(DG)(DA)(DA)(DA)(DA)(DC)(DC) (DA)(DT)(DT)(DC)

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Macromolecule #4: OgeuIscB

MacromoleculeName: OgeuIscB / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 56.81968 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MMAVVYVISK SGKPLMPTTR CGHVRILLKE GKARVVERKP FTIQLTYESA EETQPLVLGI DPGRTNIGMS VVTESGESVF NAQIETRNK DVPKLMKDRK QYRMAHRRLK RRCKRRRRAK AAGTAFEEGE KQRLLPGCFK PITCKSIRNK EARFNNRKRP V GWLTPTAN ...String:
MMAVVYVISK SGKPLMPTTR CGHVRILLKE GKARVVERKP FTIQLTYESA EETQPLVLGI DPGRTNIGMS VVTESGESVF NAQIETRNK DVPKLMKDRK QYRMAHRRLK RRCKRRRRAK AAGTAFEEGE KQRLLPGCFK PITCKSIRNK EARFNNRKRP V GWLTPTAN HLLVTHLNVV KKVQKILPVA KVVLELNRFS FMAMNNPKVQ RWQYQRGPLY GKGSVEEAVS MQQDGHCLFC KH GIDHYHH VVPRRKNGSE TLENRVGLCE EHHRLVHTDK EWEANLASKK SGMNKKYHAL SVLNQIIPYL ADQLADMFPG NFC VTSGQD TYLFREEHGI PKDHYLDAYC IACSALTDAK KVSSPKGRPY MVHQFRRHDR QACHKANLNR SYYMGGKLVA TNRH KAMDQ KTDSLEEYRA AHSAADVSKL TVKHPSAQYK DMSRIMPGSI LVSGEGKLFT LSRSEGRNKG QVNYFVSTEG IKYWA RKCQ YLRNNGGLQI YV

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: LAURYL DIMETHYLAMINE-N-OXIDE

MacromoleculeName: LAURYL DIMETHYLAMINE-N-OXIDE / type: ligand / ID: 6 / Number of copies: 1 / Formula: LDA
Molecular weightTheoretical: 229.402 Da
Chemical component information

ChemComp-LDA:
LAURYL DIMETHYLAMINE-N-OXIDE / LDAO, detergent*YM / Lauryldimethylamine oxide

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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

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 792405

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