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- PDB-7xht: Structure of the OgeuIscB-omega RNA-target DNA complex -

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Basic information

Entry
Database: PDB / ID: 7xht
TitleStructure of the OgeuIscB-omega RNA-target DNA complex
Components
  • DNA (49-MER)
  • DNA (5'-D(P*GP*AP*AP*GP*AP*AP*AP*AP*CP*CP*AP*T)-3')
  • OgeuIscB
  • RNA (228-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR-Cas9 / RNP / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesmetagenome (others)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.55 Å
AuthorsKato, K. / Okazaki, O. / Isayama, Y. / Ishikawa, J. / Nishizawa, T. / Nishimasu, H.
Funding support Japan, 1items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: RNA (228-MER)
C: DNA (49-MER)
D: DNA (5'-D(P*GP*AP*AP*GP*AP*AP*AP*AP*CP*CP*AP*T)-3')
A: OgeuIscB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)150,1137
Polymers149,8354
Non-polymers2783
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules CD

#2: DNA chain DNA (49-MER)


Mass: 15092.702 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(P*GP*AP*AP*GP*AP*AP*AP*AP*CP*CP*AP*T)-3')


Mass: 4281.832 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain / Protein , 2 types, 2 molecules BA

#1: RNA chain RNA (228-MER)


Mass: 73640.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)
#4: Protein OgeuIscB


Mass: 56819.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) metagenome (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 3 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-LDA / LAURYL DIMETHYLAMINE-N-OXIDE


Mass: 229.402 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H31NO / Comment: LDAO, detergent*YM

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1Structure of the IscB-omegaRNA ribonucleoprotein complexCOMPLEX#40MULTIPLE SOURCES
2IscB-omegaRNACOMPLEX#41RECOMBINANT
3DNACOMPLEX#31MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: metagenome (others)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 47.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.55 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 792405 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038443
ELECTRON MICROSCOPYf_angle_d0.52912421
ELECTRON MICROSCOPYf_dihedral_angle_d14.8742977
ELECTRON MICROSCOPYf_chiral_restr0.0331529
ELECTRON MICROSCOPYf_plane_restr0.005776

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