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- EMDB-5719: Electron microscopy of the negatively-stained Cmr complex from Th... -

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Basic information

Entry
Database: EMDB / ID: EMD-5719
TitleElectron microscopy of the negatively-stained Cmr complex from Thermus thermophilus HB8.
Map data3D structure by EM single particle analysis of negatively-stained images
Sample
  • Sample: Cmr complex from Thermus thermophilus
  • Protein or peptide: Cmr2
  • Protein or peptide: Cmr3
  • Protein or peptide: Cmr1TRPM8
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5
  • Protein or peptide: Cmr6
  • RNA: crRNA
Keywordsadaptive immune system / cmr complex / CRISPR-Cas / Thermus thermophilus
Function / homology
Function and homology information


defense response to virus / cytoplasm
Similarity search - Function
CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1791 / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / CRISPR-associated protein Cmr2 / CRISPR-associated protein Cmr2, N-terminal ...CRISPR-associated protein (Cas_Cmr5) / CRISPR-associated protein, TM1791 / CRISPR-associated protein TM1795 / CRISPR-associated protein, Cmr3 / CRISPR-associated protein (Cas_Cmr3) / CRISPR-associated protein, Cmr5 / CRISPR-associated RAMP Cmr4 / AF1862-like domain superfamily / 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: 26.0 Å
AuthorsAgari Y / Maki-Yonekura S / Staals RH / van Duijin E / 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 13, 2013-
Header (metadata) releaseAug 28, 2013-
Map releaseOct 23, 2013-
UpdateOct 23, 2013-
Current statusOct 23, 2013Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.62
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.62
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5719_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5719.map.gz / Format: CCP4 / Size: 309.6 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D structure by EM single particle analysis of negatively-stained images
Voxel sizeX=Y=Z: 4.6 Å
Density
Contour LevelBy AUTHOR: 0.62 / Movie #1: 0.62
Minimum - Maximum-1.27373731 - 2.00673509
Average (Standard dev.)0.01052072 (±0.40630186)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin424242
Dimensions414148
Spacing414148
CellA: 188.59999 Å / B: 188.59999 Å / C: 220.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.64.64.6
M x/y/z414148
origin x/y/z0.0000.0000.000
length x/y/z188.600188.600220.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS424242
NC/NR/NS414148
D min/max/mean-1.2742.0070.011

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Supplemental data

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Sample components

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Entire : Cmr complex from Thermus thermophilus

EntireName: Cmr complex from Thermus thermophilus
Components
  • Sample: Cmr complex from Thermus thermophilus
  • Protein or peptide: Cmr2
  • Protein or peptide: Cmr3
  • Protein or peptide: Cmr1TRPM8
  • Protein or peptide: Cmr4
  • Protein or peptide: Cmr5
  • Protein or peptide: Cmr6
  • RNA: crRNA

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Supramolecule #1000: Cmr complex from Thermus thermophilus

SupramoleculeName: Cmr complex from Thermus thermophilus / type: sample / ID: 1000 / Details: sample was monodisperse / Oligomeric state: hetero multimer / Number unique components: 7
Molecular weightExperimental: 400 KDa / Theoretical: 360 KDa / Method: mass spectorometry

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Macromolecule #1: Cmr2

MacromoleculeName: Cmr2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8 / Location in cell: cytoplasm
Molecular weightExperimental: 65 KDa / Theoretical: 65 KDa
SequenceUniProtKB: GGDEF domain-containing protein

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Macromolecule #2: Cmr3

MacromoleculeName: Cmr3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightExperimental: 40 KDa / Theoretical: 40 KDa
SequenceUniProtKB: CRISPR-associated protein Cmr3

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Macromolecule #3: Cmr1

MacromoleculeName: Cmr1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightExperimental: 40 KDa / Theoretical: 40 KDa
SequenceUniProtKB: Type III-B CRISPR module RAMP protein Cmr1

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Macromolecule #4: Cmr4

MacromoleculeName: Cmr4 / type: protein_or_peptide / ID: 4 / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightExperimental: 30 KDa / Theoretical: 30 KDa
SequenceUniProtKB: Type III-B CRISPR module RAMP protein Cmr4

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Macromolecule #5: Cmr5

MacromoleculeName: Cmr5 / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Oligomeric state: trimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightExperimental: 10 KDa / Theoretical: 10 KDa
SequenceUniProtKB: CRISPR system Cmr subunit Cmr5

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Macromolecule #6: Cmr6

MacromoleculeName: Cmr6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightExperimental: 40 KDa / Theoretical: 40 KDa
SequenceUniProtKB: Type III-B CRISPR module RAMP protein Cmr6

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Macromolecule #7: crRNA

MacromoleculeName: crRNA / type: rna / ID: 7 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightTheoretical: 20 KDa

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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

Concentration0.1 mg/mL
BufferpH: 8 / Details: 20mM Tris-HCl, 150mM NaCl
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl acetate for 30 seconds.
GridDetails: 200 mesh gold grid with thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: JEOL
DateMar 11, 2011
Image recordingNumber real images: 105

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 8976
DetailsThe particles were selected interactively at the computer terminal.

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