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- EMDB-2898: Structures of the CRISPR-Cmr complex reveal mode of RNA target po... -

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Basic information

Entry
Database: EMDB / ID: EMD-2898
TitleStructures of the CRISPR-Cmr complex reveal mode of RNA target positioning
Map dataReconstruction of intact apo CRISPR-Cas complex
Sample
  • Sample: apo CRISPR-Cmr complex
  • 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
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 / cryo EM / Resolution: 4.1 Å
AuthorsTaylor DW / Zhu Y / Staals RHJ / Kornfeld JE / Shinkai A / van der Oost J / Nogales E / Doudna JA
CitationJournal: Science / Year: 2015
Title: Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning.
Authors: David W Taylor / Yifan Zhu / Raymond H J Staals / Jack E Kornfeld / Akeo Shinkai / John van der Oost / Eva Nogales / Jennifer A Doudna /
Abstract: Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR ...Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas surveillance complexes target double-stranded DNA, type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy reconstructions of native type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumblike β hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nucleotide intervals. The Cmr complex is architecturally similar to the type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.
History
DepositionFeb 10, 2015-
Header (metadata) releaseApr 1, 2015-
Map releaseApr 29, 2015-
UpdateMay 13, 2015-
Current statusMay 13, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2898.png

Downloads & links

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Map

FileDownload / File: emd_2898.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of intact apo CRISPR-Cas complex
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.06429065 - 0.12410281
Average (Standard dev.)0.00013883 (±0.00458551)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0640.1240.000

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Supplemental data

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Sample components

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Entire : apo CRISPR-Cmr complex

EntireName: apo CRISPR-Cmr complex
Components
  • Sample: apo CRISPR-Cmr complex
  • 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: apo CRISPR-Cmr complex

SupramoleculeName: apo CRISPR-Cmr complex / type: sample / ID: 1000 / Number unique components: 7
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: 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

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 8 / Details: 20 mM Tris-HCl, pH 8.0, 150 mM NaCl
GridDetails: 4/2 C-flat grids with a thin-layer of carbon over the holes
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK IV
Method: Grids were rapidly plunged into liquid ethane using an FEI Vitrobot MarkIV maintained at 4 degrees C after being blotted for 4-4.5 seconds with a blotting force of 15-20.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.0 mm / Nominal defocus max: -4.5 µm / Nominal defocus min: -2.0 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected at 210,000 times magnification
DetailsData acquired using Leginon. We collected a 6 s exposure fractionated into 20, 300 ms frames with a dose of 8 electrons per square Angstrom per second.
DateJul 31, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 7000 / Average electron dose: 48 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFind3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Relion / Number images used: 250000

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