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- EMDB-75710: IscB-TID truncated and wRNA bound to Target ssRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-75710
TitleIscB-TID truncated and wRNA bound to Target ssRNA
Map data
Sample
  • Complex: IscB-TID truncated and wRNA bound to Target ssRNA
    • Protein or peptide: IscB
    • RNA: wRNA (233-MER)
    • RNA: Target ssRNA
  • Ligand: MAGNESIUM ION
KeywordsIscB / Cas9 / HNH / CRISPR-Cas / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsXu C / Yang Q / Niu X / Ke A
Funding support United States, 1 items
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, 2026-
Header (metadata) releaseJun 3, 2026-
Map releaseJun 3, 2026-
UpdateJul 1, 2026-
Current statusJul 1, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_75710.png

Downloads & links

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Map

FileDownload / File: emd_75710.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 324 pix.
= 267.3 Å		0.83 Å/pix.
x 324 pix.
= 267.3 Å		0.83 Å/pix.
x 324 pix.
= 267.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.31508362 - 0.7891042
Average (Standard dev.)-0.00031111177 (±0.013136429)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 267.3 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_75710_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_75710_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : IscB-TID truncated and wRNA bound to Target ssRNA

EntireName: IscB-TID truncated and wRNA bound to Target ssRNA
Components
  • Complex: IscB-TID truncated and wRNA bound to Target ssRNA
    • Protein or peptide: IscB
    • RNA: wRNA (233-MER)
    • RNA: Target ssRNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: IscB-TID truncated and wRNA bound to Target ssRNA

SupramoleculeName: IscB-TID truncated and wRNA bound to Target ssRNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Macromolecule #1: IscB

MacromoleculeName: IscB / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 50.433199 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MAVVYVISKS GKPLMPTTRC GHVRILLKEG KARVVERKPF TIQLTYESAE ETQPLVLGID PGRTNIGMSV VTESGESVFN AQIETRNKD VPKLMKDRKQ YRMAHRRLKR RCKRRRRAKA AGTAFEEGEK QRLLPGCFKP ITCKSIRNKE ARFNNRKRPV G WLTPTANH ...String:
MAVVYVISKS GKPLMPTTRC GHVRILLKEG KARVVERKPF TIQLTYESAE ETQPLVLGID PGRTNIGMSV VTESGESVFN AQIETRNKD VPKLMKDRKQ YRMAHRRLKR RCKRRRRAKA AGTAFEEGEK QRLLPGCFKP ITCKSIRNKE ARFNNRKRPV G WLTPTANH LLVTHLNVVK KVQKILPVAK VVLELNRFSF MAMNNPKVQR WQYQRGPLYG KGSVEEAVSM QQDGHCLFCK HG IDEYHHV VPRRKNGSET LENRVGLCEE HNQLVHTDKE WEANLASKKS GMNKKYHALS VLNQIIPYLA DQLADMFPGN FCV TSGQDT YLFREEHGIP KDHYLDAYCI ACSALTDAKK VSSPKGRPYM VHQFRRHDRQ ACHKANLNRS YYMGGKLVAT NRHK AMDQK TDSLEEYRAA HSAADVSKLT VKHPSAQYNG GLQIYV

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Macromolecule #2: wRNA (233-MER)

MacromoleculeName: wRNA (233-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 75.414812 KDa
SequenceString: AAAAGAGUGA ACGAGAGGCU CUUUCAACUU GAAAAGGUUG AAAGAGCACA GGCUGAGACA UUCGUAAGGC CGAAAGGCCG GACGCACCC UGGGAUUUCC CCAGUCCCCG GAACUGCAUA GCGGAUGUCA GUUGAUCGGC CGAGUAAUUU ACGUCGACGU U GACGUCGA ...String:
AAAAGAGUGA ACGAGAGGCU CUUUCAACUU GAAAAGGUUG AAAGAGCACA GGCUGAGACA UUCGUAAGGC CGAAAGGCCG GACGCACCC UGGGAUUUCC CCAGUCCCCG GAACUGCAUA GCGGAUGUCA GUUGAUCGGC CGAGUAAUUU ACGUCGACGU U GACGUCGA UGGUUGCGGC CGAUCAGAUA AGCCAGGGGG AACAAUCACC UCUCUGGAAA CAGAGAGAGU UUUAC

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Macromolecule #3: Target ssRNA

MacromoleculeName: Target ssRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.642989 KDa
SequenceString:
UCUUCUAGUC UCGUUCACUC UUUUCUCU

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5357082
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 328824
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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