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- PDB-8rkt: Conformational Landscape of the Type V-K CRISPR-associated Transp... -

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Basic information

Entry
Database: PDB / ID: 8rkt
TitleConformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K Cas12k domain local-refinement map
Components
  • Non-target strand - LE
  • ShCas12k
  • Small ribosomal subunit protein uS15
  • Target strand - LE
  • TniQ
  • sgRNA
KeywordsDNA BINDING PROTEIN / CRISPR-associated Transposon genome editing transposition
Function / homology
Function and homology information


cytosolic small ribosomal subunit / rRNA binding / structural constituent of ribosome / translation
Similarity search - Function
: / TniQ / TniQ / Ribosomal protein S15, bacterial-type / Ribosomal protein S15 signature. / Ribosomal protein S15 / Ribosomal_S15 / Ribosomal protein S15 / S15/NS1, RNA-binding
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / RNA / RNA (> 10) / RNA (> 100) / Small ribosomal subunit protein uS15 / TniQ (Homology model) / ShCas12k
Similarity search - Component
Biological speciesScytonema hofmannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.35 Å
AuthorsTenjo-Castano, F. / Mesa, P. / Montoya, G.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk Foundation Denmark
CitationJournal: Mol Cell / Year: 2024
Title: Conformational landscape of the type V-K CRISPR-associated transposon integration assembly.
Authors: Francisco Tenjo-Castaño / Nicholas Sofos / Luisa S Stutzke / Piero Temperini / Anders Fuglsang / Tillmann Pape / Pablo Mesa / Guillermo Montoya /
Abstract: CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site ...CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted ∼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.
History
DepositionDec 30, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: sgRNA
2: Non-target strand - LE
3: Target strand - LE
A: ShCas12k
B: Small ribosomal subunit protein uS15
C: TniQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)256,2699
Polymers256,1146
Non-polymers1553
Water5,819323
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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RNA chain , 1 types, 1 molecules 1

#1: RNA chain sgRNA


Mass: 84006.516 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)

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DNA chain , 2 types, 2 molecules 23

#2: DNA chain Non-target strand - LE


Mass: 20961.504 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)
#3: DNA chain Target strand - LE


Mass: 40822.160 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)

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Protein , 3 types, 3 molecules ABC

#4: Protein ShCas12k


Mass: 79479.312 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: A0A8X6EH11
#5: Protein Small ribosomal subunit protein uS15


Mass: 10376.003 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Gene: rpsO, rps15, WA1_22700 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A139X9A4
#6: Protein TniQ


Mass: 20468.783 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: A0A8J0PCL5

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Non-polymers , 3 types, 326 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: Zn
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 323 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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: recognition complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0./1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN 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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 19936

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Processing

EM software
IDNameVersionCategory
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
13cryoSPARC4.43D reconstruction
20ISOLDEmodel refinement
21Cootmodel refinement
22PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144000 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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