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- PDB-9k30: Cryo-EM structure of the TbaIscB-omega RNA-target DNA complex -

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Basic information

Entry
Database: PDB / ID: 9k30
TitleCryo-EM structure of the TbaIscB-omega RNA-target DNA complex
Components
  • Non-target DNA strand
  • Omega RNA
  • Target DNA strand
  • TbaIscB
KeywordsRNA BINDING PROTEIN / RNA-guided DNA nuclease
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTissierellia bacterium (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsNagahata, N. / Yamada, S. / Yamashita, K. / Nishimasu, H.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR23B6 Japan
Japan Society for the Promotion of Science (JSPS)JP21H05281 Japan
Japan Society for the Promotion of Science (JSPS)JP22H00403 Japan
CitationJournal: Nat Struct Mol Biol / Year: 2026
Title: Structural visualization of the molecular evolution of CRISPR-Cas9.
Authors: Naoto Nagahata / Kazuki Kato / Sota Yamada / Soumya Kannan / Sae Okazaki / Yukari Isayama / Masahiro Hiraizumi / Keitaro Yamashita / Eugene V Koonin / Feng Zhang / Hiroshi Nishimasu /
Abstract: RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR-Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element- ...RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR-Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element-guided activity) systems, respectively. Sequence and structural comparisons indicate that IscB (~500 residues) evolved into Cas9 (~700-1,600 residues) through protein expansion coupled with guide RNA miniaturization. However, the specific sequence of events in this evolutionary transition remains unknown. Here, we report cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases-two IscBs and two Cas9s-each in complex with its cognate guide RNA and target DNA. Comparisons of these four complex structures to previously reported IscB and Cas9 structures indicate that evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the acquisition of the zinc-finger-containing REC3 domain, followed by bridge helix extension and REC1 domain acquisition. These structural changes led to expansion of the REC lobe, increasing the target DNA cleavage specificity. Additionally, the structural conservation of the RNA scaffolds indicates that the dual CRISPR RNA (crRNA) and trans-activating crRNA guides of CRISPR-Cas9 evolved from the single ωRNA guides of OMEGA systems. Our findings provide insights into the succession of structural changes involved in the exaptation of transposon-associated RNA-guided nucleases for the role of effector nucleases in adaptive immune systems.
History
DepositionOct 18, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 4, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TbaIscB
B: Omega RNA
C: Target DNA strand
D: Non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)288,0106
Polymers287,9204
Non-polymers902
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain Target DNA strand


Mass: 13028.337 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Non-target DNA strand


Mass: 13433.622 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules AB

#1: Protein TbaIscB


Mass: 119250.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tissierellia bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#2: RNA chain Omega RNA


Mass: 142207.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tissierellia bacterium (bacteria)
Production host: Cloning vector pCOLA-AraC-pBAD-lacI-LVA-TetR-LVA (others)

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN
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: Cryo-EM structure of the TbIscB-omega RNA-target DNA complex
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Tissierellia bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
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: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
9Servalcatmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94390 / Symmetry type: POINT

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