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- PDB-7plh: Scytonema hofmannii TnsC bound to AMPPNP and DNA -

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Basic information

Entry
Database: PDB / ID: 7plh
TitleScytonema hofmannii TnsC bound to AMPPNP and DNA
Components
  • DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*T)-3')
  • ShTnsC
KeywordsDNA BINDING PROTEIN / TnsC / Transposition protein / AAA+ ATPase / Tn7 / CRISPR
Function / homologyPHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10)
Function and homology information
Biological speciesScytonema hofmannii (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsQuerques, I. / Jinek, M.
Funding supportEuropean Union, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567European Union
European Research Council (ERC)ERC-CoG-820152European Union
CitationJournal: Nature / Year: 2021
Title: Target site selection and remodelling by type V CRISPR-transposon systems.
Authors: Irma Querques / Michael Schmitz / Seraina Oberli / Christelle Chanez / Martin Jinek /
Abstract: Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided ...Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.
History
DepositionAug 31, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: ShTnsC
B: ShTnsC
C: ShTnsC
D: ShTnsC
E: ShTnsC
F: ShTnsC
G: ShTnsC
H: DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*T)-3')
I: DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)237,31923
Polymers233,6059
Non-polymers3,71414
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25350 Å2
ΔGint-81 kcal/mol
Surface area86330 Å2
MethodPISA

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Components

#1: Protein
ShTnsC


Mass: 31444.617 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (bacteria) / Production host: Escherichia coli (E. coli)
#2: DNA chain DNA (5'-D(P*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*TP*AP*T)-3')


Mass: 6746.438 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Complex of ShTnsC with AMPPNP and dsDNA / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
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: OTHER
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 66.39 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 59.72 ° / Axial rise/subunit: 6.78 Å / Axial symmetry: C1
3D reconstructionResolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77544 / Symmetry type: HELICAL

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