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- PDB-8bd6: Cas12k-sgRNA-dsDNA-TnsC non-productive complex. -

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Basic information

Entry
Database: PDB / ID: 8bd6
TitleCas12k-sgRNA-dsDNA-TnsC non-productive complex.
Components
  • Cas12k
  • DNA
  • DNA non-target strand
  • DNA target strand
  • TnsC
  • sgRNA
KeywordsRNA BINDING PROTEIN / Cas12k / sgRNA / TnsC / CRISPR-Cas / Tn7-like transposons / transposition
Function / homology
Function and homology information


Bacterial TniB / Bacterial TniB protein / : / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / TnsC / Cas12k
Similarity search - Component
Biological speciesScytonema hofmannii (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsSchmitz, M. / Querques, I. / Oberli, S. / Chanez, C. / Jinek, M.
Funding support Switzerland, European Union, United States, 4items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567 Switzerland
European Research Council (ERC)ERC-CoG-820152European Union
Howard Hughes Medical Institute (HHMI)55008735 United States
European Molecular Biology Organization (EMBO)ALTF 296-2020European Union
CitationJournal: Cell / Year: 2022
Title: Structural basis for the assembly of the type V CRISPR-associated transposon complex.
Authors: Michael Schmitz / Irma Querques / Seraina Oberli / Christelle Chanez / Martin Jinek /
Abstract: CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the ...CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.
History
DepositionOct 18, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 4, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2May 24, 2023Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.3Jul 26, 2023Group: Other / Category: pdbx_database_status / Item: _pdbx_database_status.pdb_format_compatible

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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
R: TnsC
S: TnsC
T: TnsC
U: TnsC
V: TnsC
W: TnsC
X: TnsC
Y: TnsC
Z: TnsC
c: DNA
d: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)503,96733
Polymers499,18315
Non-polymers4,78318
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 10 molecules ARSTUVWXYZ

#1: Protein Cas12k


Mass: 79156.773 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
#5: Protein
TnsC


Mass: 31444.617 Da / Num. of mol.: 9
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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RNA chain , 1 types, 1 molecules B

#2: RNA chain sgRNA


Mass: 82376.547 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (bacteria)

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DNA chain , 3 types, 4 molecules CDcd

#3: DNA chain DNA target strand


Mass: 14916.607 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA non-target strand


Mass: 15125.739 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA


Mass: 12303.033 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 18 molecules

#7: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Mg

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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: Target DNA bound Cas12k-sgRNA-TnsC complex in non-productive state
Type: COMPLEX / Details: Cas12k, sgRNA, target DNA, TnsC / Entity ID: #1-#3, #6, #4-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 67.68 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 133000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00931089
ELECTRON MICROSCOPYf_angle_d1.36743433
ELECTRON MICROSCOPYf_dihedral_angle_d21.2112669
ELECTRON MICROSCOPYf_chiral_restr0.0955065
ELECTRON MICROSCOPYf_plane_restr0.0064372

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