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- EMDB-19045: DNA bound type IV-A3 CRISPR effector complex from K. pneumoniae -

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Basic information

Entry
Database: EMDB / ID: EMD-19045
TitleDNA bound type IV-A3 CRISPR effector complex from K. pneumoniae
Map datasharpened map used for model refinement
Sample
  • Complex: Type IV-A3 CRISPR-Cas effector complex from Klebsiella pneumoniae bound to crRNA and target DNA
    • 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
    • DNA: Target Strand (TS)-DNA
    • DNA: Non-Target Strand (NTS)-DNA
  • Ligand: ZINC ION
KeywordsCRISPR / crRNA / DNA binding / type IV CRISPR-Cas / CRISPRi / nuclease deficient / GENE REGULATION
Function / homologyCRISPR type AFERR-associated protein Csf2 / Uncharacterized protein / Uncharacterized protein
Function and homology information
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMiksys A / Cepaite R / Malinauskaite L / Pausch P
Funding supportEuropean Union, 3 items
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 5, 2023-
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_19045.png

Downloads & links

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Map

FileDownload / File: emd_19045.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map used for model refinement
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: 0.44
Minimum - Maximum-2.2865562 - 4.8976874
Average (Standard dev.)0.00013826061 (±0.097829655)
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: unfiltered map

Fileemd_19045_additional_1.map
Annotationunfiltered map
Projections & Slices
AxesZYX

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Additional map: EMReady processed map used for initial model building

Fileemd_19045_additional_2.map
AnnotationEMReady processed map used for initial model building
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19045_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19045_half_map_2.map
Projections & Slices
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Sample components

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

EntireName: Type IV-A3 CRISPR-Cas effector complex from Klebsiella pneumoniae bound to crRNA and target DNA
Components
  • Complex: Type IV-A3 CRISPR-Cas effector complex from Klebsiella pneumoniae bound to crRNA and target DNA
    • 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
    • DNA: Target Strand (TS)-DNA
    • DNA: Non-Target Strand (NTS)-DNA
  • Ligand: ZINC ION

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

SupramoleculeName: Type IV-A3 CRISPR-Cas effector complex from Klebsiella pneumoniae bound to crRNA and target DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Details: The 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
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 332 KDa

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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 #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: Target Strand (TS)-DNA

MacromoleculeName: Target Strand (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: Non-Target Strand (NTS)-DNA

MacromoleculeName: Non-Target Strand (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 #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / 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
20.0 mMHEPES
150.0 mMNaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: 20 mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 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: 1120 / Average exposure time: 48.0 sec. / Average electron dose: 30.0 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: 1082094
Startup modelType of model: NONE / Details: Initial model generated from the data
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2) / Number images used: 104035
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-8rc2:
DNA bound type IV-A3 CRISPR effector complex from K. pneumoniae

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