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- EMDB-27769: Type I-C Cas4-Cas1-Cas2 complex bound to a 24 bp duplex PAM/PAM p... -

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Basic information

Entry
Database: EMDB / ID: EMD-27769
TitleType I-C Cas4-Cas1-Cas2 complex bound to a 24 bp duplex PAM/PAM prespacer
Map data
Sample
  • Complex: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate
KeywordsCRISPR Cas adaptation type I-C / Protein-DNA complex / DNA BINDING PROTEIN
Biological speciesAlkalihalobacillus halodurans C-125 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsDhingra Y / Suresh SK / Juneja P / Sashital DG
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115874 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM140876 United States
CitationJournal: Mol Cell / Year: 2022
Title: PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation.
Authors: Yukti Dhingra / Shravanti K Suresh / Puneet Juneja / Dipali G Sashital /
Abstract: Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of ...Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.
History
DepositionAug 2, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27769.png

Downloads & links

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Map

FileDownload / File: emd_27769.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 384 pix.
= 348.019 Å		0.91 Å/pix.
x 384 pix.
= 348.019 Å		0.91 Å/pix.
x 384 pix.
= 348.019 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9063 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.734962 - 1.1551456
Average (Standard dev.)-0.00017064366 (±0.018766712)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 348.0192 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27769_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27769_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate

EntireName: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate
Components
  • Complex: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate

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Supramolecule #1: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate

SupramoleculeName: Type I-C Cas4-Cas1-Cas2 complex with a PAM/PAM substrate
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Alkalihalobacillus halodurans C-125 (bacteria)

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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

Concentration0.75 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol, 2 mM DTT, and 2 mM MnCl2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 160000
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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