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- PDB-8axa: Cryo-EM structure of shCas12k-sgRNA-dsDNA ternary complex (type V... -

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Basic information

Entry
Database: PDB / ID: 8axa
TitleCryo-EM structure of shCas12k-sgRNA-dsDNA ternary complex (type V-K CRISPR-associated transposon)
Components
  • Cas12k
  • DNA non-target strand
  • DNA target strand
  • sgRNASubgenomic mRNA
KeywordsDNA BINDING PROTEIN / CRISPR / complex / transposition
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / Cas12k
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsTenjo-Castano, F. / Sofos, N. / Stella, S. / Fuglsang, A. / Pape, T. / Mesa, P. / Stutzke, L.S. / Temperini, P. / Montoya, G.
Funding support Denmark, 4items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Novo Nordisk FoundationNNF18OC0055061 Denmark
CitationJournal: To Be Published
Title: Conformational Landscape of the Type V-K CRISPR-associated Transposon Integration Assembly
Authors: Tenjo-Castano, F. / Sofos, N. / Stella, S. / Fuglsang, A. / Pape, T. / Mesa, P. / Stutzke, L.S. / Temperini, P. / Montoya, G.
History
DepositionAug 31, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas12k
B: sgRNA
C: DNA target strand
D: DNA non-target strand


Theoretical massNumber of molelcules
Total (without water)192,6324
Polymers192,6324
Non-polymers00
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis, homology
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Cas12k


Mass: 74738.258 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8M0FGU0
#2: RNA chain sgRNA / Subgenomic mRNA


Mass: 84006.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli)
#3: DNA chain DNA target strand


Mass: 16858.855 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli)
#4: DNA chain DNA non-target strand


Mass: 17027.963 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli)
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O

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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: Ternary complex of ShCas12k bound to sgRNA and dsDNA. / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.193 MDa / Experimental value: YES
Source (natural)Organism: Scytonema hofmannii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium chlorideKCl1
230 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
35 mMmagnesium chlorideMgCl21
41 mMTris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 9.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3 s blotting, 4 degrees celcius

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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 FIELDBright-field microscopy / Nominal magnification: 96000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 45 sec. / Electron dose: 45 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2231

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4cryoSPARC3.2.0CTF correction
7UCSF ChimeraXmodel fitting
8Coot0.9.6model fitting
10PHENIXmodel refinement
11cryoSPARC3.2.0initial Euler assignment
12cryoSPARC3.3.1final Euler assignment
13cryoSPARC3.2.0classification
14cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 858078
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 136646 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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