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- EMDB-26097: Cas3-Cas8 complex from type I-A CRISPR-Cas system -

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Basic information

Entry
Database: EMDB / ID: EMD-26097
TitleCas3-Cas8 complex from type I-A CRISPR-Cas system
Map data
Sample
  • Complex: full R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
Biological speciesGeobacter sulfurreducens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsHu C / Ni D
Funding support United States, Switzerland, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118117 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118160 United States
Swiss National Science FoundationNCCR Switzerland
CitationJournal: Mol Cell / Year: 2022
Title: Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.
Authors: Chunyi Hu / Dongchun Ni / Ki Hyun Nam / Sonali Majumdar / Justin McLean / Henning Stahlberg / Michael P Terns / Ailong Ke /
Abstract: Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme ...Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.
History
DepositionJan 28, 2022-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateAug 17, 2022-
Current statusAug 17, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26097.png

Downloads & links

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Map

FileDownload / File: emd_26097.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 350 pix.
= 255.85 Å		0.73 Å/pix.
x 350 pix.
= 255.85 Å		0.73 Å/pix.
x 350 pix.
= 255.85 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.731 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.225
Minimum - Maximum-0.6113941 - 0.7299612
Average (Standard dev.)-0.00011194189 (±0.031726103)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 255.85 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : full R-loop state of Cas3-Cascade complex from Pyrococcus furiosu...

EntireName: full R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
Components
  • Complex: full R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system

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Supramolecule #1: full R-loop state of Cas3-Cascade complex from Pyrococcus furiosu...

SupramoleculeName: full R-loop state of Cas3-Cascade complex from Pyrococcus furiosus type I-A CRISPR/Cas system
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#15 / Details: full R-loop state
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightExperimental: 450 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

Concentration1 mg/mL
BufferpH: 7.5 / Component - Concentration: 150.0 mM / Component - Formula: NaClSodium chloride / Component - Name: sodium chloride
GridModel: Quantifoil R1.2/1.3 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 6 seconds.

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 1209 / Average exposure time: 2.5 sec. / Average electron dose: 50.0 e/Å2

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Image processing

CTF correctionSoftware - Name: EMAN
Initial angle assignmentType: OTHER
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 32211
DetailsCas3-Cas8 complex

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Atomic model buiding 1

RefinementProtocol: OTHER

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