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- EMDB-19688: DNA-bound Type IV-A3 CRISPR effector in complex with DinG helicas... -

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Basic information

Entry
Database: EMDB / ID: EMD-19688
TitleDNA-bound Type IV-A3 CRISPR effector in complex with DinG helicase from K. pneumoniae (state I)
Map dataSharp map
Sample
  • Complex: Type IV-A3 CRISPR-Cas effector complex with DinG from Klebsiella pneumoniae bound to crRNA and target DNA
    • Complex: CRISPR effector in complex with a DinG helicase and crRNA
      • Protein or peptide: CRISPR type AFERR-associated protein Csf2
      • Protein or peptide: CRISPR type AFERR-associated protein Csf3
      • Protein or peptide: CRISPR type AFERR-associated protein Csf1
      • RNA: crRNA
      • Protein or peptide: DEAD/DEAH box helicase
    • Complex: Double-stranded DNA
      • DNA: TS-DNA
      • DNA: NTS-DNA
  • Ligand: ZINC ION
KeywordsCRISPR / crRNA / DNA binding / type IV CRISPR-Cas / CRISPRi / nuclease deficient / GENE REGULATION / ANTIVIRAL PROTEIN
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
CRISPR type AFERR-associated protein Csf2 / DNA 5'-3' helicase / Uncharacterized protein / Uncharacterized protein
Similarity search - Component
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle 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, 4 items
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, 2024-
Header (metadata) releaseNov 6, 2024-
Map releaseNov 6, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19688.png

Downloads & links

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Map

FileDownload / File: emd_19688.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharp map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 360 pix.
= 396. Å		1.1 Å/pix.
x 360 pix.
= 396. Å		1.1 Å/pix.
x 360 pix.
= 396. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.18997891 - 0.55085224
Average (Standard dev.)-0.002633956 (±0.015972637)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Volume map

Fileemd_19688_additional_1.map
AnnotationVolume map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19688_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19688_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Type IV-A3 CRISPR-Cas effector complex with DinG from Klebsiella ...

EntireName: Type IV-A3 CRISPR-Cas effector complex with DinG from Klebsiella pneumoniae bound to crRNA and target DNA
Components
  • Complex: Type IV-A3 CRISPR-Cas effector complex with DinG from Klebsiella pneumoniae bound to crRNA and target DNA
    • Complex: CRISPR effector in complex with a DinG helicase and crRNA
      • Protein or peptide: CRISPR type AFERR-associated protein Csf2
      • Protein or peptide: CRISPR type AFERR-associated protein Csf3
      • Protein or peptide: CRISPR type AFERR-associated protein Csf1
      • RNA: crRNA
      • Protein or peptide: DEAD/DEAH box helicase
    • Complex: Double-stranded DNA
      • DNA: TS-DNA
      • DNA: NTS-DNA
  • Ligand: ZINC ION

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Supramolecule #1: Type IV-A3 CRISPR-Cas effector complex with DinG from Klebsiella ...

SupramoleculeName: Type IV-A3 CRISPR-Cas effector complex with DinG from Klebsiella pneumoniae bound to crRNA and target DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Details: The Cas6 subunit found in the type IV-A3 system from K. pneumoniae was present during expression, purification and complex assembly, but was not resolved due to high flexibility
Molecular weightTheoretical: 428 KDa

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Supramolecule #2: CRISPR effector in complex with a DinG helicase and crRNA

SupramoleculeName: CRISPR effector in complex with a DinG helicase and crRNA
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4, #7
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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Supramolecule #3: Double-stranded DNA

SupramoleculeName: Double-stranded DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5-#6
Source (natural)Organism: Klebsiella pneumoniae (bacteria)

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Macromolecule #1: CRISPR type AFERR-associated protein Csf2

MacromoleculeName: CRISPR type AFERR-associated protein Csf2 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 38.654406 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRTLNFNGKI STLEPLTVTV KNAVSTSGHR LPRNGGFNAA PYFPGTSIRG TLRHAAHKVI VDRVGLNADG KSPFDLAEHF MLAQGVDIN GEAETFAPGE INAGAELRSK NPLISLFGRW GLSGKVGIGN AIPDGDNQWG MFGGGARSIM FQRDESLMEF L ETDQVDRL ...String:
MRTLNFNGKI STLEPLTVTV KNAVSTSGHR LPRNGGFNAA PYFPGTSIRG TLRHAAHKVI VDRVGLNADG KSPFDLAEHF MLAQGVDIN GEAETFAPGE INAGAELRSK NPLISLFGRW GLSGKVGIGN AIPDGDNQWG MFGGGARSIM FQRDESLMEF L ETDQVDRL ERLLEEQAEA SVDISQIKTE QDALKKAMKS ADKDTKAELQ IKVRELDEKI QARKDQKQES RESIRRPIDP YE AFITGAE LSHRMSIKNA TDEEAGLFIS ALIRFAAEPR FGGHANHNCG LVEAHWTVTT WKPGELVPVT LGEIVITPNG VEI TGDELF AMVKAFNENQ SFDFTARGHH HHHH

UniProtKB: CRISPR type AFERR-associated protein Csf2

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Macromolecule #2: CRISPR type AFERR-associated protein Csf3

MacromoleculeName: CRISPR type AFERR-associated protein Csf3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 25.474385 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLNFKPYRVI MSSLTPVVIS GIAPSLDGIL YEALSQAIPS NEPGVVLARL KEILLFNDEL GVFHASSLRF GITPEQGIGA TTSMRCDYL SPEKLSTAMF SPRTRRGLFT RVLLTGGPTK RRMTTRPAYS APYLTFDFVG SSEAVEILLN HAHVGVGYDY F SAANGEFN ...String:
MLNFKPYRVI MSSLTPVVIS GIAPSLDGIL YEALSQAIPS NEPGVVLARL KEILLFNDEL GVFHASSLRF GITPEQGIGA TTSMRCDYL SPEKLSTAMF SPRTRRGLFT RVLLTGGPTK RRMTTRPAYS APYLTFDFVG SSEAVEILLN HAHVGVGYDY F SAANGEFN NVTILPLDID TSISNEGMAL RPVPVNSGLN GIKGVSPLIP PYFVGEKLNI VHPAPVRTQL ISSLLRG

UniProtKB: Uncharacterized protein

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Macromolecule #3: CRISPR type AFERR-associated protein Csf1

MacromoleculeName: CRISPR type AFERR-associated protein Csf1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 29.762467 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNHPVESVYS ALTSILLPYM GEPVPVQRNC SCCGRAPSEF DGVGFELVNA YRERVVHCRP CQTFFVSAPE LMGVENPKKP TTGQKFGMW SGVGAVINVE DNSSVLLAPQ GVVNKLPEHF FDHVEVITAT SGQHLEYLFN TELKFPLIYI QNFGVKTYEL V RSLRVSLS ...String:
MNHPVESVYS ALTSILLPYM GEPVPVQRNC SCCGRAPSEF DGVGFELVNA YRERVVHCRP CQTFFVSAPE LMGVENPKKP TTGQKFGMW SGVGAVINVE DNSSVLLAPQ GVVNKLPEHF FDHVEVITAT SGQHLEYLFN TELKFPLIYI QNFGVKTYEL V RSLRVSLS ADAIYTCADQ LLTRQNEVLY MLDLKKAKEL HQEIKNYSKK EMDIFIRTVT LLAYSRITPE AASNEFKKNN LI PLLLLLP TDPHQRLSIL HLLKKV

UniProtKB: Uncharacterized protein

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Macromolecule #7: DEAD/DEAH box helicase

MacromoleculeName: DEAD/DEAH box helicase / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 70.114477 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNTSAVFESA GLSLRKVQQD YIEAAAGALT QDHKVALISA ETGVGKTLGY LVPALLILLK NPEAKFVIAT NSHALMHQIF RSDRPLLEQ IAEQCGIKVT FSRLMGKANY VSLEKVRGLL LMDEFTDLDT VKVLEKLANW SKPLVEFEEE YGELPAQITP E MVTYSIWD ...String:
MNTSAVFESA GLSLRKVQQD YIEAAAGALT QDHKVALISA ETGVGKTLGY LVPALLILLK NPEAKFVIAT NSHALMHQIF RSDRPLLEQ IAEQCGIKVT FSRLMGKANY VSLEKVRGLL LMDEFTDLDT VKVLEKLANW SKPLVEFEEE YGELPAQITP E MVTYSIWD DIQDIDDIRL NALSANFIVT THAMVMVDCM CNHRILGDKE NMYLIIDEAD IFVDMLEVWK QRRFNLRELT SA FNEHIPR NGVHVIDQLM NDVTSIAGDL HFCSTPAAVA LFDNSFNALS KVGREIKNEA ARKAFFDCIY SWEMLGLSGG QKG VGVSNK RREPALIAVN PFIGMNVGRY CTQWRSALLT SATLSITSTP ETGMEWLCKA LGLTSDTISI RKIFSPDVYG SMKL TIAGA DFPKVFNDPK EQIFSGQWLK AVVEQLSCIQ GPALVLTASH YETRMIANQL GEVSQPVYIQ KAGQALSEII KQYQE KPGI LISAGASVGV SPRGENGEQI FQDLIITRIP FLPPDRMKAE SLYGYLKERG YSRTFEAVNR NIYLENLRKV IRKAKQ SVG RGIRSENDTV RIIILDPRFP EPTDLSSKHR SLEHIIPVRF RREYRSCEIL SPAYFEEDIQ C

UniProtKB: DNA 5'-3' helicase

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Macromolecule #4: crRNA

MacromoleculeName: crRNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 19.672641 KDa
SequenceString:
UUAUCGGCGA GACCGGGAUG CACCUCCCGA AGGGUCUCGG UGUUUCCCCU GCGUGCGGGG G

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Macromolecule #5: TS-DNA

MacromoleculeName: TS-DNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 18.196562 KDa
SequenceString: (DC)(DC)(DC)(DT)(DC)(DC)(DC)(DT)(DC)(DC) (DA)(DG)(DC)(DT)(DT)(DC)(DC)(DG)(DA)(DG) (DA)(DC)(DC)(DC)(DT)(DT)(DC)(DG)(DG) (DG)(DA)(DG)(DG)(DT)(DG)(DC)(DA)(DT)(DC) (DC) (DC)(DG)(DG)(DT)(DC)(DT) ...String:
(DC)(DC)(DC)(DT)(DC)(DC)(DC)(DT)(DC)(DC) (DA)(DG)(DC)(DT)(DT)(DC)(DC)(DG)(DA)(DG) (DA)(DC)(DC)(DC)(DT)(DT)(DC)(DG)(DG) (DG)(DA)(DG)(DG)(DT)(DG)(DC)(DA)(DT)(DC) (DC) (DC)(DG)(DG)(DT)(DC)(DT)(DC)(DG) (DC)(DT)(DT)(DG)(DG)(DC)(DC)(DT)(DC)(DC) (DT)(DC)

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Macromolecule #6: NTS-DNA

MacromoleculeName: NTS-DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 18.743031 KDa
SequenceString: (DG)(DA)(DG)(DG)(DA)(DG)(DG)(DC)(DC)(DA) (DA)(DG)(DA)(DT)(DC)(DT)(DC)(DA)(DA)(DT) (DT)(DT)(DC)(DG)(DT)(DA)(DC)(DA)(DA) (DG)(DA)(DA)(DA)(DT)(DC)(DC)(DT)(DT)(DT) (DG) (DA)(DG)(DA)(DT)(DG)(DA) ...String:
(DG)(DA)(DG)(DG)(DA)(DG)(DG)(DC)(DC)(DA) (DA)(DG)(DA)(DT)(DC)(DT)(DC)(DA)(DA)(DT) (DT)(DT)(DC)(DG)(DT)(DA)(DC)(DA)(DA) (DG)(DA)(DA)(DA)(DT)(DC)(DC)(DT)(DT)(DT) (DG) (DA)(DG)(DA)(DT)(DG)(DA)(DA)(DG) (DC)(DT)(DG)(DG)(DA)(DG)(DG)(DG)(DA)(DG) (DG)(DG)

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5 / Component:
ConcentrationName
10.0 mMHEPES
150.0 mMNaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: AIR / Details: 20 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 4350 / Average exposure time: 46.33 sec. / Average electron dose: 30.31 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 92000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 4069893
Startup modelType of model: INSILICO MODEL / In silico model: AlphaFold
Details: AlphaFold model for chain M (DinG) was fitted using ChimeraX and Coot. PDB deposition 8RC2 was used for the remaining chains.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 45309
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainPDB ID
chain_id: M, source_name: AlphaFold, initial_model_type: in silico model
source_name: PDB, initial_model_type: experimental model

8rc2

Output model

PDB-8s35:
DNA-bound Type IV-A3 CRISPR effector in complex with DinG helicase from K. pneumoniae (state I)

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