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- PDB-8rfj: DNA bound type IV-A1 CRISPR effector complex with the DinG helica... -

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Basic information

Entry
Database: PDB / ID: 8rfj
TitleDNA bound type IV-A1 CRISPR effector complex with the DinG helicase from P. oleovorans
Components
  • (CRISPR type AFERR-associated protein ...) x 4
  • ATP-dependent DNA helicase DinG
  • Non-target strand (NTS-) DNA
  • Target strand (TS-)DNA
  • crRNA
KeywordsGENE REGULATION / CRISPR / crRNA / DNA binding / type IV CRISPR-Cas / CRISPRi / nuclease deficient
Function / homology
Function and homology information


hydrolase activity, acting on acid anhydrides, in phosphorus-containing anhydrides / nucleobase-containing compound metabolic process / helicase activity / nucleic acid binding / ATP binding
Similarity search - Function
CRISPR-associated protein, Csf1 family / CRISPR type IV/AFERR-associated protein Csf2 / 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. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR type AFERR-associated protein Csf1 / CRISPR type AFERR-associated protein Csf2 / CRISPR type AFERR-associated protein Csf3 / ATP-dependent DNA helicase DinG / Uncharacterized protein
Similarity search - Component
Biological speciesPseudomonas oleovorans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsMiksys, A. / Cepaite, R. / Malinauskaite, L. / Pausch, P.
Funding supportEuropean Union, 3items
OrganizationGrant numberCountry
Other government01.2.2-CPVA-V-716-01-0001
European Molecular Biology Organization (EMBO)5342-2023European Union
Other governmentS-MIP-22-10
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
DepositionDec 12, 2023Deposition 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: Target strand (TS-)DNA
J: Non-target strand (NTS-) DNA
K: ATP-dependent DNA helicase DinG
M: CRISPR type AFERR-associated protein Csf5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)398,58913
Polymers398,52412
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area64010 Å2
ΔGint-298 kcal/mol
Surface area121060 Å2
MethodPISA

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Components

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CRISPR type AFERR-associated protein ... , 4 types, 8 molecules ABCDEFGM

#1: Protein
CRISPR type AFERR-associated protein Csf2


Mass: 37193.965 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas oleovorans (bacteria) / Gene: NCTC10692_04846 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A379PIR9
#2: Protein CRISPR type AFERR-associated protein Csf3


Mass: 24451.830 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas oleovorans (bacteria) / Gene: NCTC10692_04845 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A379PL53
#3: Protein CRISPR type AFERR-associated protein Csf1


Mass: 27239.332 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas oleovorans (bacteria) / Gene: NCTC10692_04847 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A379PIR4
#8: Protein CRISPR type AFERR-associated protein Csf5


Mass: 25635.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas oleovorans (bacteria) / Gene: NCTC10692_04848 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A379PNK2

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DNA chain , 2 types, 2 molecules IJ

#5: DNA chain Target strand (TS-)DNA


Mass: 18882.029 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas oleovorans (bacteria)
#6: DNA chain Non-target strand (NTS-) DNA


Mass: 18663.979 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas oleovorans (bacteria)

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

#4: RNA chain crRNA


Mass: 19711.727 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas oleovorans (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#7: Protein ATP-dependent DNA helicase DinG


Mass: 77969.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas oleovorans (bacteria) / Gene: NCTC10692_04849 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A379PL92
#9: 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
IDNameTypeEntity IDParent-IDSource
1Type IV-A1 CRISPR-Cas effector complex with the DinG helicase from Pseudomonas oleovorans bound to crRNA and target DNACOMPLEX#8, #1-#70MULTIPLE SOURCES
2Type IV-A1 CRISPR-Cas effector complex with the DinG helicase from Pseudomonas oleovorans bound to crRNACOMPLEX#8, #1-#4, #71RECOMBINANT
3Target DNACOMPLEX#5-#61RECOMBINANT
Molecular weightValue: 0.332 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pseudomonas oleovorans (bacteria)301
33Pseudomonas oleovorans (bacteria)301
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
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA / Film material: CARBON / 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 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: 48 sec. / Electron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2332
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2particle selection
2EPUimage acquisition
4cryoSPARC4.2CTF correction
7Coot0.9.8model fitting
8ISOLDE1.4model fitting
10PHENIX1.19.2-4158model refinement
11cryoSPARC4.2initial Euler assignment
12cryoSPARC4.2final Euler assignment
14cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1022768
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55924 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325458
ELECTRON MICROSCOPYf_angle_d0.53535142
ELECTRON MICROSCOPYf_dihedral_angle_d16.6154507
ELECTRON MICROSCOPYf_chiral_restr0.0414013
ELECTRON MICROSCOPYf_plane_restr0.0044107

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