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- PDB-8ctl: IscB and wRNA bound to Target DNA (locked state) -

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Basic information

Entry
Database: PDB / ID: 8ctl
TitleIscB and wRNA bound to Target DNA (locked state)
Components
  • DNA non-target strand
  • DNA target strand
  • IscB
  • RNA (222-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR / IscB / HEARO RNA / omega RNA / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
unidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSchuler, G.A. / Hu, C. / Ke, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118174 United States
CitationJournal: Science / Year: 2022
Title: Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9.
Authors: Gabriel Schuler / Chunyi Hu / Ailong Ke /
Abstract: Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ...Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.
History
DepositionMay 16, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: IscB
C: RNA (222-MER)
A: DNA target strand
B: DNA non-target strand


Theoretical massNumber of molelcules
Total (without water)165,5614
Polymers165,5614
Non-polymers00
Water00
1


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

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Components

#1: Protein IscB


Mass: 56819.680 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain (production host): T7 Express (NEB)
#2: RNA chain RNA (222-MER)


Mass: 71877.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain (production host): T7 Express (NEB)
#3: DNA chain DNA target strand


Mass: 18305.646 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA non-target strand


Mass: 18557.873 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) unidentified (others)

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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: Cryo-EM structure of IscB in complex with RNA and target DNA
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 190 kDa/nm / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Strain: T7 Express (NEB)
Buffer solutionpH: 7.25
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaCl1
250 mMHEPESC8H18N2O4S1
35 mMMagnesium chlorideC9H15O6P1
42 mM2-MercaptoethanolHOCH2CH2SH1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 6.5 seconds before plunging

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 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: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71831 / Symmetry type: POINT

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