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- EMDB-2418: Structure and activity of an RNA-targeting Type III-B CRISPER-Cas... -

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Basic information

Entry
Database: EMDB / ID: EMD-2418
TitleStructure and activity of an RNA-targeting Type III-B CRISPER-Cas complex
Map dataReconstruction of Type III-B CRISPR-Cas complex from Thermus thermophilus
Sample
  • Sample: Type III-B CRISPR-Cas complex of Thermus thermophilus
  • Protein or peptide: CRISPR-associated protein TM1791
  • Protein or peptide: CRISPR system Cmr subunit Cmr5
  • Protein or peptide: CRISPR-associated protein Cmr3
  • Protein or peptide: CRISPR-associated protein Crm2
  • Protein or peptide: CRISPR-associated RAMP Cmr4
  • Protein or peptide: CRISPR-associated protein TM1795
  • RNA: CRISPR RNA
KeywordsCRISPR-Cas / RNA-targeting / Cmr complex
Function / homology
Function and homology information


regulation of defense response to virus / defense response to virus / cytoplasm
Similarity search - Function
CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1791 / CRISPR-associated protein, TM1791 / CRISPR-associated protein TM1795 / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 ...CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1791 / CRISPR-associated protein, TM1791 / CRISPR-associated protein TM1795 / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / CRISPR-associated protein Cmr2 / CRISPR-associated protein Cmr2 / CRISPR-associated protein Cmr2, N-terminal / CRISPR-Cas system, Cmr2 subunit, D1 domain, cysteine cluster / CRISPR-associated protein Cmr2, N-terminal / CRISPR type III-associated protein / RAMP superfamily / GGDEF domain profile. / GGDEF domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
Type III-B CRISPR module RAMP protein Cmr6 / CRISPR system Cmr subunit Cmr5 / Type III-B CRISPR module RAMP protein Cmr4 / Type III-B CRISPR module RAMP protein Cmr1 / CRISPR-associated protein Cmr3 / GGDEF domain-containing protein
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsStaals RHJ / Agari Y / Maki-Yonekura S / Zhu Y / Taylor DW / van Duijn E / Barendregt A / Vlot M / Koehorst JJ / Sakamoto K ...Staals RHJ / Agari Y / Maki-Yonekura S / Zhu Y / Taylor DW / van Duijn E / Barendregt A / Vlot M / Koehorst JJ / Sakamoto K / Masuda A / Dohmae N / Schaap PJ / Doudna JA / Heck AJ / Yonekura K / van der Oost J / Shinkai A
CitationJournal: Mol Cell / Year: 2013
Title: Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus.
Authors: Raymond H J Staals / Yoshihiro Agari / Saori Maki-Yonekura / Yifan Zhu / David W Taylor / Esther van Duijn / Arjan Barendregt / Marnix Vlot / Jasper J Koehorst / Keiko Sakamoto / Akiko ...Authors: Raymond H J Staals / Yoshihiro Agari / Saori Maki-Yonekura / Yifan Zhu / David W Taylor / Esther van Duijn / Arjan Barendregt / Marnix Vlot / Jasper J Koehorst / Keiko Sakamoto / Akiko Masuda / Naoshi Dohmae / Peter J Schaap / Jennifer A Doudna / Albert J R Heck / Koji Yonekura / John van der Oost / Akeo Shinkai /
Abstract: The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six ...The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.
History
DepositionJul 18, 2013-
Header (metadata) releaseAug 14, 2013-
Map releaseOct 16, 2013-
UpdateNov 6, 2013-
Current statusNov 6, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.22
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3.22
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2418.tif

Downloads & links

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Map

FileDownload / File: emd_2418.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Type III-B CRISPR-Cas complex from Thermus thermophilus
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 3.22 / Movie #1: 3.22
Minimum - Maximum-9.27186775 - 17.75046158
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-72-72-72
Dimensions144144144
Spacing144144144
CellA=B=C: 403.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z403.200403.200403.200
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-9.27217.750-0.000

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Supplemental data

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Sample components

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Entire : Type III-B CRISPR-Cas complex of Thermus thermophilus

EntireName: Type III-B CRISPR-Cas complex of Thermus thermophilus
Components
  • Sample: Type III-B CRISPR-Cas complex of Thermus thermophilus
  • Protein or peptide: CRISPR-associated protein TM1791
  • Protein or peptide: CRISPR system Cmr subunit Cmr5
  • Protein or peptide: CRISPR-associated protein Cmr3
  • Protein or peptide: CRISPR-associated protein Crm2
  • Protein or peptide: CRISPR-associated RAMP Cmr4
  • Protein or peptide: CRISPR-associated protein TM1795
  • RNA: CRISPR RNA

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Supramolecule #1000: Type III-B CRISPR-Cas complex of Thermus thermophilus

SupramoleculeName: Type III-B CRISPR-Cas complex of Thermus thermophilus / type: sample / ID: 1000 / Number unique components: 12
Molecular weightExperimental: 365 KDa / Method: MS/MS

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Macromolecule #1: CRISPR-associated protein TM1791

MacromoleculeName: CRISPR-associated protein TM1791 / type: protein_or_peptide / ID: 1 / Name.synonym: Cmr6, TTHB165 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 38 KDa / Theoretical: 38 KDa
SequenceUniProtKB: Type III-B CRISPR module RAMP protein Cmr6 / InterPro: CRISPR-associated protein, TM1791

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Macromolecule #3: CRISPR system Cmr subunit Cmr5

MacromoleculeName: CRISPR system Cmr subunit Cmr5 / type: protein_or_peptide / ID: 3
Name.synonym: Cmr5, CRISPR type III-B/RAMP module-associated protein Cmr5
Number of copies: 3 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 12 KDa / Theoretical: 12 KDa
SequenceUniProtKB: CRISPR system Cmr subunit Cmr5 / GO: regulation of defense response to virus / InterPro: CRISPR-associated protein, Cmr5

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Macromolecule #4: CRISPR-associated protein Cmr3

MacromoleculeName: CRISPR-associated protein Cmr3 / type: protein_or_peptide / ID: 4 / Name.synonym: Cmr3, TTHB160 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 39 KDa / Theoretical: 39 KDa
SequenceUniProtKB: CRISPR-associated protein Cmr3 / InterPro: CRISPR-associated protein, Cmr3

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Macromolecule #5: CRISPR-associated protein Crm2

MacromoleculeName: CRISPR-associated protein Crm2 / type: protein_or_peptide / ID: 5 / Name.synonym: Cmr2, TTHB160 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 65 KDa / Theoretical: 65 KDa
SequenceUniProtKB: GGDEF domain-containing protein / InterPro: CRISPR-associated protein Cmr2

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Macromolecule #6: CRISPR-associated RAMP Cmr4

MacromoleculeName: CRISPR-associated RAMP Cmr4 / type: protein_or_peptide / ID: 6 / Name.synonym: Cmr4, TTHB163 / Number of copies: 4 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 37 KDa / Theoretical: 37 KDa
SequenceUniProtKB: Type III-B CRISPR module RAMP protein Cmr4 / InterPro: CRISPR-associated RAMP Cmr4

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Macromolecule #7: CRISPR-associated protein TM1795

MacromoleculeName: CRISPR-associated protein TM1795 / type: protein_or_peptide / ID: 7 / Name.synonym: Cmr1, TTHB162 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 44 KDa
SequenceUniProtKB: Type III-B CRISPR module RAMP protein Cmr1 / InterPro: CRISPR-associated protein TM1795

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Macromolecule #2: CRISPR RNA

MacromoleculeName: CRISPR RNA / type: rna / ID: 2 / Name.synonym: crRNA / Details: 40-46 nucleotide endogenous crRNA bound to complex / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Thermus thermophilus (bacteria)

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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

Concentration0.03 mg/mL
BufferpH: 7.4
Details: 25 mM Hepes, pH 7.5, 100 mM KCl, 1 mM TCEP, 5% glycerol
StainingType: NEGATIVE
Details: Sample applied to glow-discharged continuous carbon grids. After 1 min, grids were stained consecutively in 6 droplets of 2% (w/v) uranyl acetate solution and excess stain removed by gentle ...Details: Sample applied to glow-discharged continuous carbon grids. After 1 min, grids were stained consecutively in 6 droplets of 2% (w/v) uranyl acetate solution and excess stain removed by gentle blotting with filter paper.
GridDetails: 200 mesh Cu grid
VitrificationCryogen name: NONE / Instrument: OTHER

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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: -1.4 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected at 210,000 times magnification
DetailsData acquired using Leginon
DateJan 29, 2013
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 300 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: ACE2 micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, SPARX / Number images used: 45000

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