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- EMDB-19126: Main density of the DNA bound type IV-A1 CRISPR effector complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-19126
TitleMain density of the DNA bound type IV-A1 CRISPR effector complex with the DinG helicase from P. oleovorans
Map data
Sample
  • Complex: Type IV-A1 CRISPR-Cas effector complex with the DinG helicase from Pseudomonas oleovorans bound to crRNA and target DNA
KeywordsCRISPR / crRNA / DNA binding / type IV CRISPR-Cas / CRISPRi / nuclease deficient / GENE REGULATION
Biological speciesPseudomonas oleovorans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.18 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 12, 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_19126.png

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Map

FileDownload / File: emd_19126.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.334
Minimum - Maximum-0.72544634 - 2.2848141
Average (Standard dev.)-0.0011538164 (±0.06168532)
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: #1

Fileemd_19126_additional_1.map
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Half map: #1

Fileemd_19126_half_map_1.map
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Half map: #2

Fileemd_19126_half_map_2.map
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Sample components

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Entire : Type IV-A1 CRISPR-Cas effector complex with the DinG helicase fro...

EntireName: Type IV-A1 CRISPR-Cas effector complex with the DinG helicase from Pseudomonas oleovorans bound to crRNA and target DNA
Components
  • Complex: Type IV-A1 CRISPR-Cas effector complex with the DinG helicase from Pseudomonas oleovorans bound to crRNA and target DNA

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Supramolecule #1: Type IV-A1 CRISPR-Cas effector complex with the DinG helicase fro...

SupramoleculeName: Type IV-A1 CRISPR-Cas effector complex with the DinG helicase from Pseudomonas oleovorans bound to crRNA and target DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Pseudomonas oleovorans (bacteria)
Molecular weightTheoretical: 332 KDa

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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

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 / Details: 20 mA
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: 2332 / 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: 1022768
Startup modelType of model: NONE / Details: Initial model generated from the data
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2) / Number images used: 55924
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

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