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- EMDB-6122: RNA-targeting by the Type III-A CRISPR-Cas Csm complex of Thermus... -

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Basic information

Entry
Database: EMDB / ID: EMD-6122
TitleRNA-targeting by the Type III-A CRISPR-Cas Csm complex of Thermus thermophilus
Map dataReconstruction of Csm complex
Sample
  • Sample: Type III-A CRISPR-Cas Csm complex of Thermus thermophilus
  • Protein or peptide: Csm1
  • Protein or peptide: Csm2
  • Protein or peptide: Csm3
  • Protein or peptide: Csm4
  • Protein or peptide: Csm5
  • RNA: CRISPR RNA
KeywordsCRISPR RNA-guided targeting complex
Function / homology
Function and homology information


phosphoric diester hydrolase activity / exonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / ATP binding / metal ion binding
Similarity search - Function
CRISPR-associated protein Csm5 / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR-associated RAMP Csm3 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 ...CRISPR-associated protein Csm5 / CRISPR-associated protein Csm5 / CRISPR-associated protein, Csm2 Type III-A / CRISPR-associated protein, Csm2 Type III-A / Csm2 Type III-A / : / CRISPR-associated RAMP Csm3 / CRISPR-associated RAMP Csm3 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / Csm1, subunit domain B / Csm1 subunit domain B / Cas10/Cmr2, second palm domain / : / CRISPR type III-associated protein / CRISPR type III-associated protein / RAMP superfamily / HD domain / GGDEF domain profile. / GGDEF domain / HD domain / Reverse transcriptase/Diguanylate cyclase domain
Similarity search - Domain/homology
CRISPR system Cms protein Csm5 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A) / CRISPR system Cms protein Csm4 / CRISPR system Cms endoribonuclease Csm3 / CRISPR system Cms protein Csm2
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsStaals RHJ / Zhu Y / Taylor DW / Kornfeld JE / Sharma K / Barendregt A / Koehorst JJ / Vlot M / Neupane N / Varossieau K ...Staals RHJ / Zhu Y / Taylor DW / Kornfeld JE / Sharma K / Barendregt A / Koehorst JJ / Vlot M / Neupane N / Varossieau K / Sakamoto K / Suzuki T / Dohmae N / Yokoyama S / Schaap PJ / Urlaub H / Heck AJR / Nogales E / Doudna JA / Shinkai A / van der Oost J
CitationJournal: Mol Cell / Year: 2014
Title: RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus.
Authors: Raymond H J Staals / Yifan Zhu / David W Taylor / Jack E Kornfeld / Kundan Sharma / Arjan Barendregt / Jasper J Koehorst / Marnix Vlot / Nirajan Neupane / Koen Varossieau / Keiko Sakamoto / ...Authors: Raymond H J Staals / Yifan Zhu / David W Taylor / Jack E Kornfeld / Kundan Sharma / Arjan Barendregt / Jasper J Koehorst / Marnix Vlot / Nirajan Neupane / Koen Varossieau / Keiko Sakamoto / Takehiro Suzuki / Naoshi Dohmae / Shigeyuki Yokoyama / Peter J Schaap / Henning Urlaub / Albert J R Heck / Eva Nogales / Jennifer A Doudna / Akeo Shinkai / John van der Oost /
Abstract: CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type ...CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.
History
DepositionOct 6, 2014-
Header (metadata) releaseNov 5, 2014-
Map releaseDec 3, 2014-
UpdateDec 10, 2014-
Current statusDec 10, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.65
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.65
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6122.png

Downloads & links

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Map

FileDownload / File: emd_6122.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Csm complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 144 pix.
= 403.2 Å		2.8 Å/pix.
x 144 pix.
= 403.2 Å		2.8 Å/pix.
x 144 pix.
= 403.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.65 / Movie #1: 4.65
Minimum - Maximum-12.448145869999999 - 13.10042
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/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-12.44813.1000.000

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Supplemental data

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Sample components

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Entire : Type III-A CRISPR-Cas Csm complex of Thermus thermophilus

EntireName: Type III-A CRISPR-Cas Csm complex of Thermus thermophilus
Components
  • Sample: Type III-A CRISPR-Cas Csm complex of Thermus thermophilus
  • Protein or peptide: Csm1
  • Protein or peptide: Csm2
  • Protein or peptide: Csm3
  • Protein or peptide: Csm4
  • Protein or peptide: Csm5
  • RNA: CRISPR RNA

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

SupramoleculeName: Type III-A CRISPR-Cas Csm complex of Thermus thermophilus
type: sample / ID: 1000 / Details: The sample was monodisperse.
Oligomeric state: 1 Csm1: 3 Csm2: 6 Csm3: 2 Csm4: 1 Csm5: 1 crRNA
Number unique components: 6
Molecular weightExperimental: 426 KDa / Method: Native mass spectrometry

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Macromolecule #1: Csm1

MacromoleculeName: Csm1 / type: protein_or_peptide / ID: 1 / Name.synonym: Cas10 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightTheoretical: 90 KDa
SequenceUniProtKB: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
GO: metal ion binding, phosphoric diester hydrolase activity
InterPro: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1

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Macromolecule #2: Csm2

MacromoleculeName: Csm2 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightTheoretical: 16 KDa
SequenceUniProtKB: CRISPR system Cms protein Csm2 / InterPro: CRISPR-associated protein, Csm2 Type III-A

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Macromolecule #3: Csm3

MacromoleculeName: Csm3 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightTheoretical: 27 KDa
SequenceUniProtKB: CRISPR system Cms endoribonuclease Csm3
InterPro: CRISPR-associated RAMP Csm3, CRISPR type III-associated protein

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Macromolecule #4: Csm4

MacromoleculeName: Csm4 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightTheoretical: 32 KDa
SequenceUniProtKB: CRISPR system Cms protein Csm4

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Macromolecule #5: Csm5

MacromoleculeName: Csm5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightTheoretical: 45 KDa
SequenceUniProtKB: CRISPR system Cms protein Csm5
InterPro: CRISPR-associated protein Csm5, CRISPR type III-associated protein

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Macromolecule #6: CRISPR RNA

MacromoleculeName: CRISPR RNA / type: rna / ID: 6 / Name.synonym: crRNA / Details: endogenous crRNA of variable lengths / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Thermus thermophilus (bacteria) / Strain: HB8
Molecular weightTheoretical: 14 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

Concentration0.03 mg/mL
BufferpH: 7.4
Details: 25 mM HEPES, pH 7.5, 100 mM KCl, 1 mM TCEP, 5% glycerol
StainingType: NEGATIVE
Details: Sample was negatively stained with four consecutive droplets of 2% uranyl acetate.
GridDetails: 200 mesh Cu grid with thin carbon, glow discharged with a sputter coater
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification.
Legacy - Electron beam tilt params: 0
DetailsData acquired using Leginon.
DateOct 18, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 420 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 1.4 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

DetailsWe used a low-pass-filtered E. coli Cascade structure as an initial model for three-dimensional reconstruction using iterative projection matching refinement with libraries from the EMAN2 and SPARX software packages.
CTF correctionDetails: each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 60000
Final two d classificationNumber classes: 200

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