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

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Basic information

Entry
Database: PDB / ID: 8rku
TitleConformational Landscape of the Type V-K CRISPR-associated TransposonIntegration Assembly CAST V-K TnsC domain local-refinement map
Components
  • Non-target strand - LE
  • ShTnsC
  • Target strand - LE
KeywordsDNA BINDING PROTEIN / CRISPR-associated Transposon genome editing transposition
Function / homologyBacterial TniB / Bacterial TniB protein / P-loop containing nucleoside triphosphate hydrolase / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA (> 100) / TnsC
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsTenjo-Castano, F. / Mesa, P. / Montoya, G.
Funding support Denmark, European Union, 2items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
European Research Council (ERC)101096548European Union
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
2: Non-target strand - LE
3: Target strand - LE
D: ShTnsC
E: ShTnsC
F: ShTnsC
G: ShTnsC
H: ShTnsC
I: ShTnsC
J: ShTnsC
K: ShTnsC
L: ShTnsC
M: ShTnsC
N: ShTnsC
O: ShTnsC
P: ShTnsC
Q: ShTnsC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)508,83144
Polymers501,39116
Non-polymers7,44128
Water9,692538
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 23

#1: DNA chain Non-target strand - LE


Mass: 20961.504 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)
#2: 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 , 1 types, 14 molecules DEFGHIJKLMNOPQ

#3: Protein
ShTnsC


Mass: 31400.496 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8J0PCL3

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Non-polymers , 3 types, 566 molecules

#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 538 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: Type V-K CRISPR-associated transposon post-transposition state after transesterification
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 1 MDa / Experimental 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 holderSpecimen 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
EM imaging opticsEnergyfilter name: TFS Selectris X

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
7Cootmodel fitting
8UCSF ChimeraXmodel fitting
14cryoSPARC4.43D reconstruction
15PHENIXmodel refinement
16ISOLDEmodel refinement
17Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 258000 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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