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- EMDB-5929: CasA mediates Cas3-catalyzed target degradation during CRISPR RNA... -

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Basic information

Entry
Database: EMDB / ID: EMD-5929
TitleCasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference
Map dataCryo-EM reconstruction of Cascade bound to a 72 bp target dsDNA
Sample
  • Sample: Cascade bound to a 72 bp target dsDNA
  • Protein or peptide: CRISPR system Cascade subunit CasA
  • Protein or peptide: CRISPR system Cascade subunit CasB
  • Protein or peptide: CRISPR system Cascade subunit CasC
  • Protein or peptide: CRISPR system Cascade subunit CasD
  • Protein or peptide: CRISPR system Cascade subunit CasE
  • RNA: R44 CRISPR RNA
  • DNA: 72 bp dsDNA target with R44 protospacer
KeywordsCascade / CRISPR RNA / Cas3 / bacterial immunity
Function / homology
Function and homology information


protein-containing complex => GO:0032991 / CRISPR-cas system / protein binding / DNA/RNA hybrid binding / : / maintenance of CRISPR repeat elements / RNA processing / RNA endonuclease activity / defense response to virus / endonuclease activity ...protein-containing complex => GO:0032991 / CRISPR-cas system / protein binding / DNA/RNA hybrid binding / : / maintenance of CRISPR repeat elements / RNA processing / RNA endonuclease activity / defense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / nucleic acid binding / protein-containing complex / DNA binding / RNA binding / zinc ion binding
Similarity search - Function
CRISPR-associated protein, CT1975 / CRISPR-associated protein, CT1975 / CT1975-like protein / CRISPR-associated protein, CasD / CRISPR-associated protein, CasD / CRISPR-associated protein Cse2 / CRISPR-associated protein Cse2 / Cse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein Cse1 ...CRISPR-associated protein, CT1975 / CRISPR-associated protein, CT1975 / CT1975-like protein / CRISPR-associated protein, CasD / CRISPR-associated protein, CasD / CRISPR-associated protein Cse2 / CRISPR-associated protein Cse2 / Cse2 superfamily / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR-associated protein Cse3 / CRISPR associated protein / CRISPR_assoc / CRISPR-associated protein, Cas5 / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
CRISPR system Cascade subunit CasB / CRISPR system Cascade subunit CasE / CRISPR system Cascade subunit CasD / CRISPR system Cascade subunit CasC / CRISPR system Cascade subunit CasA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Enterobacteria phage P7 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsHochstrasser ML / Taylor DW / Bhat P / Guegler CK / Sternberg SH / Nogales E / Doudna JA
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.
Authors: Megan L Hochstrasser / David W Taylor / Prashant Bhat / Chantal K Guegler / Samuel H Sternberg / Eva Nogales / Jennifer A Doudna /
Abstract: In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA ...In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.
History
DepositionMar 19, 2014-
Header (metadata) releaseApr 16, 2014-
Map releaseApr 16, 2014-
UpdateMay 14, 2014-
Current statusMay 14, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.11
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5929.png

Downloads & links

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Map

FileDownload / File: emd_5929.map.gz / Format: CCP4 / Size: 2.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of Cascade bound to a 72 bp target dsDNA
Voxel sizeX=Y=Z: 2.18 Å
Density
Contour LevelBy AUTHOR: 3.11 / Movie #1: 3.11
Minimum - Maximum0.0 - 14.437739369999999
Average (Standard dev.)0.33693373 (±1.27328527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-34-39-47
Dimensions857590
Spacing857590
CellA: 163.5 Å / B: 185.3 Å / C: 196.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.182.182.18
M x/y/z758590
origin x/y/z0.0000.0000.000
length x/y/z163.500185.300196.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-39-34-47
NC/NR/NS758590
D min/max/mean0.00014.4380.337

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Supplemental data

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Sample components

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Entire : Cascade bound to a 72 bp target dsDNA

EntireName: Cascade bound to a 72 bp target dsDNA
Components
  • Sample: Cascade bound to a 72 bp target dsDNA
  • Protein or peptide: CRISPR system Cascade subunit CasA
  • Protein or peptide: CRISPR system Cascade subunit CasB
  • Protein or peptide: CRISPR system Cascade subunit CasC
  • Protein or peptide: CRISPR system Cascade subunit CasD
  • Protein or peptide: CRISPR system Cascade subunit CasE
  • RNA: R44 CRISPR RNA
  • DNA: 72 bp dsDNA target with R44 protospacer

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Supramolecule #1000: Cascade bound to a 72 bp target dsDNA

SupramoleculeName: Cascade bound to a 72 bp target dsDNA / type: sample / ID: 1000
Oligomeric state: 1 CasA: 2 CasB: 6 CasC: 1 CasD: 1 CasE: 1 crRNA: 1 target dsDNA
Number unique components: 7
Molecular weightTheoretical: 443.8 KDa

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Macromolecule #1: CRISPR system Cascade subunit CasA

MacromoleculeName: CRISPR system Cascade subunit CasA / type: protein_or_peptide / ID: 1
Name.synonym: CRISPR type I-E/Ecoli-associated protein CasA/Cse1, CRISPR-associated protein CasA/Cse1, CasA, Cse1
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli
Molecular weightTheoretical: 55.9 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
SequenceUniProtKB: CRISPR system Cascade subunit CasA
GO: defense response to virus, DNA binding, RNA binding, protein-containing complex => GO:0032991
InterPro: CRISPR-associated protein Cse1

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Macromolecule #2: CRISPR system Cascade subunit CasB

MacromoleculeName: CRISPR system Cascade subunit CasB / type: protein_or_peptide / ID: 2 / Name.synonym: CasB, Cse2 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli
Molecular weightTheoretical: 18.7 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
SequenceUniProtKB: CRISPR system Cascade subunit CasB
GO: defense response to virus, RNA binding, protein-containing complex => GO:0032991
InterPro: CRISPR-associated protein Cse2

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Macromolecule #3: CRISPR system Cascade subunit CasC

MacromoleculeName: CRISPR system Cascade subunit CasC / type: protein_or_peptide / ID: 3 / Name.synonym: CasC, Cas4, Cse4
Details: The 6 CasC subunits make a helical stack forming a groove in which the crRNA lies.
Number of copies: 6 / Oligomeric state: helical / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
SequenceUniProtKB: CRISPR system Cascade subunit CasC
GO: defense response to virus, RNA binding, protein-containing complex => GO:0032991, protein binding
InterPro: CRISPR-associated protein, CT1975

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Macromolecule #4: CRISPR system Cascade subunit CasD

MacromoleculeName: CRISPR system Cascade subunit CasD / type: protein_or_peptide / ID: 4 / Name.synonym: CasD, Cas5 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli
Molecular weightTheoretical: 25.2 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
SequenceUniProtKB: CRISPR system Cascade subunit CasD
GO: defense response to virus, RNA binding, protein-containing complex => GO:0032991
InterPro: CRISPR-associated protein, Cas5, CRISPR-associated protein Cas5, N-terminal, CRISPR-associated protein, CasD

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Macromolecule #5: CRISPR system Cascade subunit CasE

MacromoleculeName: CRISPR system Cascade subunit CasE / type: protein_or_peptide / ID: 5
Name.synonym: CasE, Cas6e, CasE endoRNase, crRNA endonuclease
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli
Molecular weightTheoretical: 22.3 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)
SequenceUniProtKB: CRISPR system Cascade subunit CasE
GO: defense response to virus, RNA binding, protein-containing complex => GO:0032991, RNA processing, GO: 0090305, endonuclease activity
InterPro: CRISPR-associated protein Cse3

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Macromolecule #6: R44 CRISPR RNA

MacromoleculeName: R44 CRISPR RNA / type: rna / ID: 6 / Name.synonym: crRNA / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12 / synonym: E. coli
Molecular weightTheoretical: 18.6 KDa
SequenceString:
AUAAACCGAC GGUAUUGUUC AGAUCCUGGC UUGCCAACAG GAGUUCCCCG CGCCAGCGGG G

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Macromolecule #7: 72 bp dsDNA target with R44 protospacer

MacromoleculeName: 72 bp dsDNA target with R44 protospacer / type: dna / ID: 7 / Name.synonym: dsDNA target
Details: This strand is annealed to the complementary non-target strand to form the dsDNA target.
Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Enterobacteria phage P7 (virus)
Molecular weightTheoretical: 44.4 KDa
SequenceString:
CATGAGGTCC CTCGTTTAGT CTGTTGGCAA GCCAGGATCT GAACAATACC GTCATCGGAG GTACGATCAA GG

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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

Concentration2.3 mg/mL
BufferpH: 7.5
Details: 50 mM HEPES, pH 7.5, 100 mM KCl, 5% glycerol, 0.1 mM EDTA, 1 mM TCEP
GridDetails: C-flat 2/2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 78 K / Instrument: FEI VITROBOT MARK I
Method: A 4 uL drop of purified sample was placed onto C-flat grids that had been glow-discharged in a nitrogen atmosphere for 60 seconds using an Edwards Carbon Evaporator. The grids were blotted ...Method: A 4 uL drop of purified sample was placed onto C-flat grids that had been glow-discharged in a nitrogen atmosphere for 60 seconds using an Edwards Carbon Evaporator. The grids were blotted for 4 seconds using a blotting offset of -1 mm.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: -2.8 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 100000
Sample stageSpecimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 78 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
Legacy - Electron beam tilt params: 0
DetailsData acquired using Leginon.
DateDec 26, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 3580 / Average electron dose: 20 e/Å2
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Whole micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 280000
DetailsImage pre-processing performed in Appion. Particles were selected using template-based picking in Appion.

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