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- EMDB-7485: Cas4-dependent prespacer processing ensures high-fidelity program... -

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Basic information

Entry
Database: EMDB / ID: EMD-7485
TitleCas4-dependent prespacer processing ensures high-fidelity programming of CRISPR arrays
Map dataCas1-Cas4 complex
Sample
  • Complex: Cas1-Cas4 complex
    • Protein or peptide: Cas1
    • Protein or peptide: Cas4
Biological speciesBacillus halodurans (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsLee H / Zhou Y / Taylor DW / Sashital DG
Funding support United States, 3 items
OrganizationGrant numberCountry
Cancer Prevention and Research Institute of TexasRR160088 United States
Welch FoundationF-1938 United States
National Institutes of Health/National Human Genome Research InstituteR01 GM115874 United States
CitationJournal: Mol Cell / Year: 2018
Title: Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.
Authors: Hayun Lee / Yi Zhou / David W Taylor / Dipali G Sashital /
Abstract: CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems ...CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.
History
DepositionFeb 27, 2018-
Header (metadata) releaseApr 11, 2018-
Map releaseApr 11, 2018-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7485.png

Downloads & links

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Map

FileDownload / File: emd_7485.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCas1-Cas4 complex
Voxel sizeX=Y=Z: 3.6 Å
Density
Contour LevelBy EMDB: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.12762192 - 0.13523985
Average (Standard dev.)0.0009198695 (±0.01490172)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 230.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.63.63.6
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z230.400230.400230.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-128-128-128
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-0.1280.1350.001

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Supplemental data

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Sample components

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Entire : Cas1-Cas4 complex

EntireName: Cas1-Cas4 complex
Components
  • Complex: Cas1-Cas4 complex
    • Protein or peptide: Cas1
    • Protein or peptide: Cas4

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Supramolecule #1: Cas1-Cas4 complex

SupramoleculeName: Cas1-Cas4 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus halodurans (bacteria)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: Cas1

MacromoleculeName: Cas1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus halodurans (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITFL TKNGRFLARV VGESRGNVVL RKTQYRISEN DQESTKIARN FITGKVYNSK WMLERMTREH PLRVNVEQFK ATSQLLSVMM QEIRNCDSLE SLRGWEGQAA ...String:
MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITFL TKNGRFLARV VGESRGNVVL RKTQYRISEN DQESTKIARN FITGKVYNSK WMLERMTREH PLRVNVEQFK ATSQLLSVMM QEIRNCDSLE SLRGWEGQAA INYNKVFDQM ILQQKEEFAF HGRSRRPPKD NVNAMLSFAY TLLANDVAAA LETVGLDAYV GFMHQDRPGR ASLALDLMEE LRGLYADRFV LSLINRKEMT ADGFYKKENG AVLMTDEARK TFLKAWQTKK QEKITHPYLG EKMSWGLVPY VQALLLARFL RGDLDEYPPF LWK

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Macromolecule #2: Cas4

MacromoleculeName: Cas4 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus halodurans (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MASNEEDRYL MLSGLQHFQF CKRQWALIHI EQQWEENVRT IEGQHLHKKA DQPFMKEKRG SKLTVRAMPI QSKNLQISGI CDVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA LRDKVVQMAK ...String:
MASNEEDRYL MLSGLQHFQF CKRQWALIHI EQQWEENVRT IEGQHLHKKA DQPFMKEKRG SKLTVRAMPI QSKNLQISGI CDVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA LRDKVVQMAK EMHHYYENRH TPKVKTGPFC NNCSLQSICL PKLMNKRSVK RYIEGRLSE

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
StainingType: NEGATIVE / Material: uranyl acetate
GridSupport film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: unspecified

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Electron microscopy

MicroscopeJEOL 2010
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: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 30.0 e/Å2

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Image processing

Startup modelType of model: OTHER / Details: ab initio
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 4000

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