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- PDB-9k32: Cryo-EM structure of the NbaCas9-guide RNA-target DNA complex -

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Basic information

Entry
Database: PDB / ID: 9k32
TitleCryo-EM structure of the NbaCas9-guide RNA-target DNA complex
Components
  • Guide RNA
  • HNH nuclease domain-containing protein
  • Non-target DNA strand
  • Target DNA strand
KeywordsRNA BINDING PROTEIN / RNA-guided DNA nuclease
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / :
Function and homology information
Biological speciesNitrospirota bacterium (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 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: To Be Published
Title: Cryo-EM structure of the NbCas9-guide RNA-target DNA complex
Authors: Nagahata, N.
History
DepositionOct 18, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 4, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 4, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: HNH nuclease domain-containing protein
B: Guide RNA
C: Target DNA strand
D: Non-target DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)174,2159
Polymers174,0114
Non-polymers2045
Water1629
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: 12973.302 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Non-target DNA strand


Mass: 13498.658 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 HNH nuclease domain-containing protein


Mass: 84610.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nitrospirota bacterium (bacteria) / Gene: DCP24_02765 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3C0V2Q6
#2: RNA chain Guide RNA


Mass: 62928.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nitrospirota bacterium (bacteria) / Production host: Expression vector pET-mod (others)

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Non-polymers , 3 types, 14 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O

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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 NbCas9-guide RNA-target DNA complex
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Nitrospirota bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6
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: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 453280 / Symmetry type: POINT

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