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- EMDB-8660: Spacer capture and integration by a type I-F Cas1:Cas2-3 CRISPR a... -

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Basic information

Entry
Database: EMDB / ID: EMD-8660
TitleSpacer capture and integration by a type I-F Cas1:Cas2-3 CRISPR adaptation complex
Map dataSpacer capture and integration by a type I-F Cas1:Cas2-3 CRISPR adaptation complex
Sample
  • Complex: type I-F Cas1:Cas2-3 CRISPR adaptation complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / metal ion binding
Similarity search - Function
CRISPR-associated protein Cas1, YPEST subtype / CRISPR-associated endonuclease Cas1, N-terminal domain / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1
Similarity search - Domain/homology
CRISPR-associated endonuclease Cas1
Similarity search - Component
Biological speciesPectobacterium atrosepticum (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 25.0 Å
AuthorsFagerlund RD / Wilkinson ME / Bostina M / Fineran PC
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.
Authors: Robert D Fagerlund / Max E Wilkinson / Oleg Klykov / Arjan Barendregt / F Grant Pearce / Sebastian N Kieper / Howard W R Maxwell / Angela Capolupo / Albert J R Heck / Kurt L Krause / Mihnea ...Authors: Robert D Fagerlund / Max E Wilkinson / Oleg Klykov / Arjan Barendregt / F Grant Pearce / Sebastian N Kieper / Howard W R Maxwell / Angela Capolupo / Albert J R Heck / Kurt L Krause / Mihnea Bostina / Richard A Scheltema / Raymond H J Staals / Peter C Fineran /
Abstract: CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, ...CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas1-Cas2-3 complex from with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.
History
DepositionApr 1, 2017-
Header (metadata) releaseApr 26, 2017-
Map releaseJun 28, 2017-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8660.png

Downloads & links

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Map

FileDownload / File: emd_8660.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSpacer capture and integration by a type I-F Cas1:Cas2-3 CRISPR adaptation complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.12 Å/pix.
x 80 pix.
= 249.6 Å		3.12 Å/pix.
x 80 pix.
= 249.6 Å		3.12 Å/pix.
x 80 pix.
= 249.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.17194505 - 0.43533912
Average (Standard dev.)0.012038743 (±0.06389361)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 249.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.123.123.12
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z249.600249.600249.600
α/β/γ90.00090.00090.000
start NX/NY/NZ00-41
NX/NY/NZ737384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.1720.4350.012

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Supplemental data

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Sample components

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Entire : type I-F Cas1:Cas2-3 CRISPR adaptation complex

EntireName: type I-F Cas1:Cas2-3 CRISPR adaptation complex
Components
  • Complex: type I-F Cas1:Cas2-3 CRISPR adaptation complex

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Supramolecule #1: type I-F Cas1:Cas2-3 CRISPR adaptation complex

SupramoleculeName: type I-F Cas1:Cas2-3 CRISPR adaptation complex / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Pectobacterium atrosepticum (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
StainingType: NEGATIVE / Material: Uranyl formate
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL 2200FS
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Startup modelType of model: OTHER / Details: tomography
Final reconstructionResolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 7565
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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