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- EMDB-30377: Cas1-Cas4 complex from Synechocystis sp. 6803 -

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Basic information

Entry
Database: EMDB / ID: EMD-30377
TitleCas1-Cas4 complex from Synechocystis sp. 6803
Map dataCas1-Cas4 complex
Sample
  • Complex: Cas1-Cas4 from Synechocystis sp. 6803
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 19.2 Å
AuthorsChen Q / Yu Y
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis.
Authors: Chengyong Wu / Dongmei Tang / Jie Cheng / Daojun Hu / Zejing Yang / Xue Ma / Haihuai He / Shaohua Yao / Tian-Min Fu / Yamei Yu / Qiang Chen /
Abstract: CRISPR-Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as ...CRISPR-Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as adaptation in the field. Some CRISPR-Cas systems rely on Cas1 and Cas2 to complete the adaptation process, which has been characterized in a few systems. In contrast, many other CRISPR-Cas systems require an additional factor of Cas4 for efficient adaptation, the mechanism of which remains less understood. Here we present biochemical reconstitution of the Synechocystis sp. PCC6803 type I-D adaptation system, X-ray crystal structures of Cas1-Cas2-prespacer complexes, and negative stained electron microscopy structure of the Cas4-Cas1 complex. Cas4 and Cas2 compete with each other to interact with Cas1. In the absence of prespacer, Cas4 but not Cas2 assembles with Cas1 into a very stable complex for processing the prespacer. Strikingly, the Cas1-prespacer complex develops a higher binding affinity toward Cas2 to form the Cas1-Cas2-prespacer ternary complex for integration. Together, we show a two-step sequential assembly mechanism for the type I-D adaptation module of Synechocystis, in which Cas4-Cas1 and Cas1-Cas2 function as two exclusive complexes for prespacer processing, capture, and integration.
History
DepositionJul 7, 2020-
Header (metadata) releaseMar 31, 2021-
Map releaseMar 31, 2021-
UpdateMar 31, 2021-
Current statusMar 31, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30377.png

Downloads & links

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Map

FileDownload / File: emd_30377.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas1-Cas4 complex
Voxel sizeX=Y=Z: 1.8 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.043577675 - 0.07361306
Average (Standard dev.)0.0006364619 (±0.008108823)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 230.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z230.400230.400230.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0440.0740.001

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Supplemental data

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Sample components

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Entire : Cas1-Cas4 from Synechocystis sp. 6803

EntireName: Cas1-Cas4 from Synechocystis sp. 6803
Components
  • Complex: Cas1-Cas4 from Synechocystis sp. 6803

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Supramolecule #1: Cas1-Cas4 from Synechocystis sp. 6803

SupramoleculeName: Cas1-Cas4 from Synechocystis sp. 6803 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation state3D array

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5
StainingType: NEGATIVE / Material: uranyl acetate

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: OTHER / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 19.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 8866

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