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- EMDB-62004: Cryo-EM structure of the NbaCas9-guide RNA-target DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-62004
TitleCryo-EM structure of the NbaCas9-guide RNA-target DNA complex
Map data
Sample
  • Complex: Cryo-EM structure of the NbCas9-guide RNA-target DNA complex
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: Guide RNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: water
KeywordsRNA-guided DNA nuclease / RNA BINDING PROTEIN
Function / homology:
Function and homology information
Biological speciesNitrospirota bacterium (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 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_62004.png

Downloads & links

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Map

FileDownload / File: emd_62004.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 210 pix.
= 232.4 Å		1.11 Å/pix.
x 210 pix.
= 232.4 Å		1.11 Å/pix.
x 210 pix.
= 232.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.10667 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.0077889 - 2.7732046
Average (Standard dev.)0.00048221843 (±0.075331904)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 232.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_62004_msk_1.map
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Half map: #2

Fileemd_62004_half_map_1.map
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Half map: #1

Fileemd_62004_half_map_2.map
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Sample components

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Entire : Cryo-EM structure of the NbCas9-guide RNA-target DNA complex

EntireName: Cryo-EM structure of the NbCas9-guide RNA-target DNA complex
Components
  • Complex: Cryo-EM structure of the NbCas9-guide RNA-target DNA complex
    • Protein or peptide: HNH nuclease domain-containing protein
    • RNA: Guide RNA
    • DNA: Target DNA strand
    • DNA: Non-target DNA strand
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: Cryo-EM structure of the NbCas9-guide RNA-target DNA complex

SupramoleculeName: Cryo-EM structure of the NbCas9-guide RNA-target DNA complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Nitrospirota bacterium (bacteria)

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Macromolecule #1: HNH nuclease domain-containing protein

MacromoleculeName: HNH nuclease domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Nitrospirota bacterium (bacteria)
Molecular weightTheoretical: 84.610656 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSEKELVLGI AYGGKYTGLG VVDRRNNQVL YARTIKMRDD VADILKGRRE QRGIRRTQQT RKKRLRELKK YLNSIGYDNS TELFKSIYS LAHKRGYDYA DMPTPEEIEE MDEKEKKEWK DTQRNSRYRK EVLADVRKVM SNGGASDEQI KRVESIFNKQ Y RPKRFNNR ...String:
GSEKELVLGI AYGGKYTGLG VVDRRNNQVL YARTIKMRDD VADILKGRRE QRGIRRTQQT RKKRLRELKK YLNSIGYDNS TELFKSIYS LAHKRGYDYA DMPTPEEIEE MDEKEKKEWK DTQRNSRYRK EVLADVRKVM SNGGASDEQI KRVESIFNKQ Y RPKRFNNR ILTKCKVCGK NTPLRRNVRE LLLENIVRFL PLESELKETL KRTILEGQQG NINKLFRKLK FNQKDWPGKN LT DIAKNKL PGRLPFCKEH FAENEKFTTI EKSTFRLAPS LKTKIENVLT VIKDEVMPNF ALDRVVMESN NFDIAAKTKG KKR LAKEEY SKGHKENRET LMESLLRETD GRCVYCGKTI TLADANKDAI YPKKAGGSNI FANLVACCRS CNENKGGRTP SESG IMPNP EVVATIKNDL KKKILDDARS IKQLDFNKYM SHASIGWRHM RDRLKELTGN DKLPVERLSG IVTAYFRRWW GFKKE RAND KHHALDAVIL ASRKDYTDEG LVAMTLKPAN SDGREFDPEK HIKESEEFKR NKGSRGSALY DKNPLSIKNG KIARRY MVT EIERGKEDAV ISEEWREKLK EAFDRFGVSN GKCLTDLQTK EVGLYGQKNP MSLKCAVRGA GKGQIVLIGN NAFKTNV HN VGVAVYLDEK GKKRACELKN QRLAKHFVEP QDEIKGKILF TLRKGDTVKA EDGNIYRILE LGERPVVDIK WVPTSDGK K KRVKTAIHAT KLTKL

UniProtKB: UNIPROTKB: A0A3C0V2Q6

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Macromolecule #2: Guide RNA

MacromoleculeName: Guide RNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Nitrospirota bacterium (bacteria)
Molecular weightTheoretical: 62.928344 KDa
SequenceString: GGGGAAAUUA GGUGCGCUUG GCGUUACAGU UAAGGCUCUU UGAAAAUGAU GAUUGAAACG AAAAAAGAGC CUUAAUUGUA AAACGCCUA UAUGAUGAAG GAAUGUACGC UUGGGUUUGU CCAGCCUAAG CCAAACUAAA ACUACAGUCG AUAAUGACAC C UUCAUUGU ...String:
GGGGAAAUUA GGUGCGCUUG GCGUUACAGU UAAGGCUCUU UGAAAAUGAU GAUUGAAACG AAAAAAGAGC CUUAAUUGUA AAACGCCUA UAUGAUGAAG GAAUGUACGC UUGGGUUUGU CCAGCCUAAG CCAAACUAAA ACUACAGUCG AUAAUGACAC C UUCAUUGU GGGUUAGGAC AUUAAAAAAA UUUUCAG

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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: 12.973302 KDa
SequenceString:
(DA)(DC)(DC)(DC)(DG)(DT)(DT)(DC)(DC)(DC) (DC)(DG)(DC)(DC)(DA)(DA)(DG)(DC)(DG)(DC) (DA)(DC)(DC)(DT)(DA)(DA)(DT)(DT)(DT) (DC)(DC)(DC)(DC)(DG)(DG)(DG)(DA)(DT)(DC) (DC) (DT)(DC)(DT)

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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: 13.498658 KDa
SequenceString:
(DA)(DG)(DA)(DG)(DG)(DA)(DT)(DC)(DC)(DC) (DG)(DG)(DG)(DG)(DA)(DA)(DA)(DT)(DT)(DA) (DG)(DG)(DA)(DC)(DG)(DC)(DG)(DA)(DA) (DC)(DC)(DG)(DG)(DG)(DG)(DG)(DA)(DA)(DC) (DG) (DG)(DG)(DT)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 9 / 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: 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: 2.0 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: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 453280
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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