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- PDB-11hr: Engineered IscB and wRNA bound to Target ssDNA -

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Basic information

Entry
Database: PDB / ID: 11hr
TitleEngineered IscB and wRNA bound to Target ssDNA
Components
  • IscB
  • RNA (233-MER)
  • Target ssDNA
KeywordsRNA BINDING PROTEIN/RNA/DNA / IscB / Cas9 / HNH / CRISPR-Cas / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsXu, C. / Yang, Q. / Niu, X. / Ke, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35118174 United States
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Structure basis for single-strand nucleic acid targeting by IscB and variants.
Authors: Chengtao Xu / Qi Yang / Xiaolin Niu / Ailong Ke /
Abstract: Transposon-encoded IscB was defined as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome-editing applications. We previously ...Transposon-encoded IscB was defined as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome-editing applications. We previously repurposed IscB and related Cas9s as efficient RNA editors by removing their double-stranded DNA (dsDNA) recognition module, the target-adjacent motif (TAM)/protospacer adjacent motif-interacting domain. Here, we report four cryo-electron microscopy structures of IscB, with or without TAM-interaction domain (TID), in complex with single-stranded nucleic acid (ssNA) targets. Structures reveal that, regardless of TID presence, IscB engages ssNA using the same mechanism. IscB initially facilitates formation of a 10-nt seed duplex with ssNA; further base-pairing is blocked by an alternatively positioned HNH nuclease that acts as a roadblock. In this intermediate state, neither HNH nor RuvC is competent for target cleavage. Only upon full duplex formation is the HNH roadblock dislodged by the duplex extension between guide RNA and ssNA. HNH and RuvC nuclease active sites become exposed as the result. A similar set of conformational rearrangements likely governs IscB activity during dsDNA target interrogation. Guided by the structural and mechanistic insights, we introduced mutations to either improve ssNA binding or ease HNH dislodging. Both approaches improved the RNA-targeting efficiency of IscB in vitro and in human cells.
History
DepositionFeb 25, 2026Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Jun 3, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Jul 1, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: IscB
B: RNA (233-MER)
D: Target ssDNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,7146
Polymers140,6003
Non-polymers1143
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein IscB


Mass: 56779.602 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)
#2: RNA chain RNA (233-MER)


Mass: 75414.812 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)
#3: DNA chain Target ssDNA


Mass: 8405.396 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestY
Has protein modificationN

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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: Engineered IscB and wRNA bound to Target ssDNA / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Buffer solutionpH: 7.5
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: 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

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6173103
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 293636 / Symmetry type: POINT
RefinementHighest resolution: 2.8 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048666
ELECTRON MICROSCOPYf_angle_d0.53612655
ELECTRON MICROSCOPYf_dihedral_angle_d22.0263377
ELECTRON MICROSCOPYf_chiral_restr0.0331546
ELECTRON MICROSCOPYf_plane_restr0.003873

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