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- PDB-7pla: Cryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA... -

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Basic information

Entry
Database: PDB / ID: 7pla
TitleCryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA target
Components
  • DNA non-target strand
  • DNA target strand
  • ShCas12k
  • sgRNASubgenomic mRNA
KeywordsDNA BINDING PROTEIN / Cas12k / sgRNA / target DNA / Protein-RNA-DNA complex / Tn7 / Transposition / CRISPR
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesScytonema hofmannii (Cyanobacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsSchmitz, M. / 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 30, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: ShCas12k
B: sgRNA
C: DNA target strand
D: DNA non-target strand


Theoretical massNumber of molelcules
Total (without water)186,9204
Polymers186,9204
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12180 Å2
ΔGint-85 kcal/mol
Surface area54130 Å2
MethodPISA

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Components

#1: Protein ShCas12k


Mass: 73744.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Scytonema hofmannii (Cyanobacteria) / Production host: Escherichia coli (E. coli)
#2: RNA chain sgRNA / Subgenomic mRNA


Mass: 82376.547 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Scytonema hofmannii (Cyanobacteria)
#3: DNA chain DNA target strand


Mass: 15301.837 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA non-target strand


Mass: 15497.987 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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 with sgRNA and target dsDNA
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.187 MDa / Experimental value: NO
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 FIELDBright-field microscopy
Specimen holderModel: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51.81 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.1_4122+SVNrefinement
PHENIX1.19.1_4122+SVNrefinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138000 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 185.47 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00429393
ELECTRON MICROSCOPYf_angle_d0.679413857
ELECTRON MICROSCOPYf_chiral_restr0.03671727
ELECTRON MICROSCOPYf_plane_restr0.0044868
ELECTRON MICROSCOPYf_dihedral_angle_d21.02293337

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