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- PDB-8s36: DNA-bound Type IV-A3 CRISPR effector in complex with DinG helicas... -

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Basic information

Entry
Database: PDB / ID: 8s36
TitleDNA-bound Type IV-A3 CRISPR effector in complex with DinG helicase from K. pneumoniae (state II)
Components
  • (CRISPR type AFERR-associated protein ...) x 3
  • DEAD/DEAH box helicase
  • NTS-NDA
  • TS-DNA
  • crRNA
KeywordsANTIVIRAL PROTEIN / CRISPR / crRNA / DNA binding / type IV CRISPR-Cas / CRISPRi / nuclease deficient / GENE REGULATION
Function / homology
Function and homology information


hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides / DNA 5'-3' helicase / nucleobase-containing compound metabolic process / DNA helicase activity / nucleic acid binding / ATP binding
Similarity search - Function
Helicase superfamily 1/2, DinG/Rad3-like / HELICc2 / ATP-dependent helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain, DinG/Rad3-type / Helicase C-terminal domain / Superfamilies 1 and 2 helicase ATP-binding type-2 domain profile. / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / DEAD-like helicases superfamily / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR type AFERR-associated protein Csf2 / DNA 5'-3' helicase / Uncharacterized protein / Uncharacterized protein
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsSkorupskaite, A. / Ragozius, V. / Cepaite, R. / Klein, N. / Randau, L. / Malinauskaite, L. / Pausch, P.
Funding supportEuropean Union, 4items
OrganizationGrant numberCountry
Other government12-001-01-01-01
European Molecular Biology Organization (EMBO)5342-2023European Union
Other governmentS-MIP-22-10
Other governmentDFG-SPP2141
CitationJournal: Nat Commun / Year: 2024
Title: Structural variation of types IV-A1- and IV-A3-mediated CRISPR interference.
Authors: R Čepaitė / N Klein / A Mikšys / S Camara-Wilpert / V Ragožius / F Benz / A Skorupskaitė / H Becker / G Žvejytė / N Steube / G K A Hochberg / L Randau / R Pinilla-Redondo / L ...Authors: R Čepaitė / N Klein / A Mikšys / S Camara-Wilpert / V Ragožius / F Benz / A Skorupskaitė / H Becker / G Žvejytė / N Steube / G K A Hochberg / L Randau / R Pinilla-Redondo / L Malinauskaitė / P Pausch /
Abstract: CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general ...CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase. The structures reveal how the effector complexes recognize the protospacer adjacent motif and target-strand DNA to form an R-loop structure. Additionally, we reveal differences between types IV-A1 and IV-A3 in DNA interactions and structural motifs that allow for in trans recruitment of DinG. Our study provides a detailed view of type IV-A mediated DNA-interference and presents a structural foundation for engineering type IV-A-based genome editing tools.
History
DepositionFeb 19, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR type AFERR-associated protein Csf2
B: CRISPR type AFERR-associated protein Csf2
C: CRISPR type AFERR-associated protein Csf2
D: CRISPR type AFERR-associated protein Csf2
E: CRISPR type AFERR-associated protein Csf2
F: CRISPR type AFERR-associated protein Csf3
G: CRISPR type AFERR-associated protein Csf1
H: crRNA
I: TS-DNA
J: NTS-NDA
L: CRISPR type AFERR-associated protein Csf2
M: DEAD/DEAH box helicase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)413,95513
Polymers413,89012
Non-polymers651
Water00
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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CRISPR type AFERR-associated protein ... , 3 types, 8 molecules ABCDELFG

#1: Protein
CRISPR type AFERR-associated protein Csf2 / Csf2 (Cas7) / CRISPR-Cas type IV-A3 Cas7 (Csf2)


Mass: 38654.406 Da / Num. of mol.: 6 / Mutation: 6xHis-tag
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: AI2781V1_5179 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Star / References: UniProt: A0A333ESG5
#2: Protein CRISPR type AFERR-associated protein Csf3 / Csf3 (Cas5) / CRISPR-Cas type IV-A3 Cas5 (Csf3)


Mass: 25474.385 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: EAO17_29825 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Star / References: UniProt: A0A8G1XN67
#3: Protein CRISPR type AFERR-associated protein Csf1 / Csf1 (Cas8) / CRISPR-Cas type IV-A3 Cas8 (Csf1)


Mass: 29762.467 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: G4V31_29130 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Star / References: UniProt: A0A7Z7WW72

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain TS-DNA


Mass: 18196.562 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria)
#6: DNA chain NTS-NDA


Mass: 18743.031 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Klebsiella pneumoniae (bacteria)

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RNA chain / Protein / Non-polymers , 3 types, 3 molecules HM

#4: RNA chain crRNA


Mass: 19672.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Star
#7: Protein DEAD/DEAH box helicase / CRISPR-Cas type IV-A3 DinG (Csf4)


Mass: 70114.477 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: BL124_00021750 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Star / References: UniProt: A0A422ZM74
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationN

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Type IV-A3 CRISPR-Cas effector complex with DinG from Klebsiella pneumoniae bound to crRNA and target DNACOMPLEXThe Cas6 subunit found in the type IVA3 system from Klebsiella pneumoniae was present during expression, purification and complex assembly, but was not resolved due to high flexibility#1-#70MULTIPLE SOURCES
2CRISPR effector in complex with a DinG helicase and crRNACOMPLEX#1-#4, #71RECOMBINANT
3Double-stranded DNACOMPLEX#5-#61RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Klebsiella pneumoniae (bacteria)573
33Klebsiella pneumoniae (bacteria)573
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Escherichia coli BL21(DE3) (bacteria)469008Star
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
110 mMHEPES1
2150 mMNaCl1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA current / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 46.33 sec. / Electron dose: 30.31 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4350
Image scansWidth: 4096 / Height: 4096

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4069893
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48197 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1

IDPDB-IDChain-IDSource nameTypeAccession codeInitial refinement model-ID
1MAlphaFoldin silico model
28RC2

8rc2

PDBexperimental model8RC22
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00424957
ELECTRON MICROSCOPYf_angle_d0.72634254
ELECTRON MICROSCOPYf_dihedral_angle_d18.5064365
ELECTRON MICROSCOPYf_chiral_restr0.0433858
ELECTRON MICROSCOPYf_plane_restr0.0064095

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