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- EMDB-62001: Cryo-EM structure of the HfmIscB-omega RNA-target DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-62001
TitleCryo-EM structure of the HfmIscB-omega RNA-target DNA complex
Map data
Sample
  • Complex: Cryo-EM structure of the OavvIscB-omega RNA-target DNA complex
    • Protein or peptide: HfmIscB
    • RNA: Omega RNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsRNA-guided DNA nuclease / RNA BINDING PROTEIN
Biological speciesmetagenome (others) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.78 Å
AuthorsNagahata N / Yamada S / Yamashita K / Nishimasu H
Funding support Japan, 3 items
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, 2024-
Header (metadata) releaseFeb 4, 2026-
Map releaseFeb 4, 2026-
UpdateMar 4, 2026-
Current statusMar 4, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_62001.png

Downloads & links

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Map

FileDownload / File: emd_62001.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 288 pix.
= 298.8 Å		1.04 Å/pix.
x 288 pix.
= 298.8 Å		1.04 Å/pix.
x 288 pix.
= 298.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0375 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.3383789 - 3.6397984
Average (Standard dev.)-0.0066023516 (±0.063411005)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 298.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_62001_msk_1.map
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Half map: #2

Fileemd_62001_half_map_1.map
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Half map: #1

Fileemd_62001_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of the OavvIscB-omega RNA-target DNA complex

EntireName: Cryo-EM structure of the OavvIscB-omega RNA-target DNA complex
Components
  • Complex: Cryo-EM structure of the OavvIscB-omega RNA-target DNA complex
    • Protein or peptide: HfmIscB
    • RNA: Omega RNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Cryo-EM structure of the OavvIscB-omega RNA-target DNA complex

SupramoleculeName: Cryo-EM structure of the OavvIscB-omega RNA-target DNA complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: metagenome (others)

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Macromolecule #1: HfmIscB

MacromoleculeName: HfmIscB / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 62.381379 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MADLSKHHHH SGHHHTGHHH HSGSHHHTGS AAGGEEDKKP AGGEGGGAHI NLKVKGQDGN EVFFRIKRST QLKKLMNAYC DRQSVDMTA IAFLFDGRRL RAEQTPDELE MEDGDEIDAM LHQTGGSSGS MAYCFVVDKN NRPLAPTKVN KGWYLVRKGR A KIKSRYPM ...String:
MADLSKHHHH SGHHHTGHHH HSGSHHHTGS AAGGEEDKKP AGGEGGGAHI NLKVKGQDGN EVFFRIKRST QLKKLMNAYC DRQSVDMTA IAFLFDGRRL RAEQTPDELE MEDGDEIDAM LHQTGGSSGS MAYCFVVDKN NRPLAPTKVN KGWYLVRKGR A KIKSRYPM VIQLEKEVEP DKYDESRVVV GIDDGSAHVG LAIVQKCPTK NKVVFKGTIE QRQDVKHLMD VRRGYRRYHR YH KRYRQAR FNNRHSSKRS GRLAPSIKQK KDAVLRVLYQ LNRWINIQEY YLEDVCMDIR AMTDGYKPYR WQYQKSSRLD ENL RKAAII RDGCKCQECG RSNCVLEVAH IKARKYGGAD TIGNLITLCS GCHQKTEGRE REFEDRYFKI IGSKPKRFDY AMHV MQGKN YLRERISELG PLHLTNGGET ANKRIEWNIV KSHSNDAICI TDCISDTCDV KEWIIKPMRR KSKAKTDNVL GIRHR DLVS YTYKNGEIHT GYVTALYPEL LALNFQSKTK HCKKVNAQKC RLLWKFDKIY WLEQCV

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Macromolecule #2: Omega RNA

MacromoleculeName: Omega RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: metagenome (others)
Molecular weightTheoretical: 115.337195 KDa
SequenceString: GGAAUUGUGA GCGGAUAACA AUUCCCCAGG ACUAAUUAUU AAGCAGUCUA AUAUAUAGAC AAAAGUGUAA ACAAGUGGAC AAGUGUAUA AUUACAUUUC UUGUUUACAC UUGCUUAACA AUAGGUUUCA AGCCUUAGUG ACUGCUACUA UCGAAAGAUA U GUUGCAGA ...String:
GGAAUUGUGA GCGGAUAACA AUUCCCCAGG ACUAAUUAUU AAGCAGUCUA AUAUAUAGAC AAAAGUGUAA ACAAGUGGAC AAGUGUAUA AUUACAUUUC UUGUUUACAC UUGCUUAACA AUAGGUUUCA AGCCUUAGUG ACUGCUACUA UCGAAAGAUA U GUUGCAGA UACGAACUAC GUUAGGGAAA AGGUUAAAGA CACACCUUUA GAUGUACUCG UCAGUCUGAA GCUCUGUGAG UG CCAAUCA AGAAACUGUG CUAAUGUCCU GUACAGAUAA CAGAGAAACA CAUAUACCUU CCCCGACAUU GGCAAGACGA AGA UUACUC CGAAAGGAAG GUAUCCAGAG AUGGAAAAUG AA

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Macromolecule #3: Target DNA strand

MacromoleculeName: Target DNA strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.047669 KDa
SequenceString:
(DG)(DA)(DA)(DT)(DG)(DG)(DT)(DT)(DC)(DC) (DT)(DC)(DC)(DC)(DG)(DG)(DG)(DG)(DA)(DA) (DT)(DT)(DG)(DT)(DT)(DA)(DT)(DC)(DC) (DG)(DC)(DT)(DC)(DA)(DC)(DA)(DA)(DT)(DT) (DC) (DC)(DT)(DT)(DA)(DG)(DA)(DA)(DA) (DA)

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Macromolecule #4: Non-target DNA strand

MacromoleculeName: Non-target DNA strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 4.329829 KDa
SequenceString:
(DG)(DG)(DG)(DA)(DG)(DG)(DA)(DA)(DC)(DC) (DA)(DT)(DT)(DC)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 17 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.78 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 689275
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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