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 species
Thermus thermophilus (bacteria)
Method
single particle reconstruction / cryo EM / Resolution: 4.1 Å
Journal: 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
Deposition
Feb 10, 2015
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Header (metadata) release
Apr 1, 2015
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Map release
Apr 29, 2015
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Update
May 13, 2015
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Current status
May 13, 2015
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_2898.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Reconstruction of intact apo CRISPR-Cas complex
Voxel size
X=Y=Z: 1 Å
Density
Contour Level
By AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum
-0.06429065 - 0.12410281
Average (Standard dev.)
0.00013883 (±0.00458551)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
320
320
320
Spacing
320
320
320
Cell
A=B=C: 320.0 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1
1
1
M x/y/z
320
320
320
origin x/y/z
0.000
0.000
0.000
length x/y/z
320.000
320.000
320.000
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
320
320
320
D min/max/mean
-0.064
0.124
0.000
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Supplemental data
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Sample components
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Entire : apo CRISPR-Cmr complex
Entire
Name: 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
Supramolecule
Name: apo CRISPR-Cmr complex / type: sample / ID: 1000 / Number unique components: 7
Molecular weight
Experimental: 365 KDa / Method: MS/MS
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Macromolecule #1: CRISPR-associated protein TM1791
Macromolecule
Name: 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 weight
Experimental: 38 KDa / Theoretical: 38 KDa
Sequence
UniProtKB: Type III-B CRISPR module RAMP protein Cmr6 / InterPro: CRISPR-associated protein, TM1791
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Macromolecule #3: CRISPR system Cmr subunit Cmr5
Macromolecule
Name: 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 weight
Experimental: 12 KDa / Theoretical: 12 KDa
Sequence
UniProtKB: 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
Macromolecule
Name: 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 weight
Experimental: 39 KDa / Theoretical: 39 KDa
Sequence
UniProtKB: CRISPR-associated protein Cmr3 / InterPro: CRISPR-associated protein, Cmr3
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Macromolecule #5: CRISPR-associated protein Crm2
Macromolecule
Name: 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 weight
Experimental: 65 KDa / Theoretical: 65 KDa
Sequence
UniProtKB: GGDEF domain-containing protein / InterPro: CRISPR-associated protein Cmr2
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Macromolecule #6: CRISPR-associated RAMP Cmr4
Macromolecule
Name: 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 weight
Experimental: 37 KDa / Theoretical: 37 KDa
Sequence
UniProtKB: Type III-B CRISPR module RAMP protein Cmr4 / InterPro: CRISPR-associated RAMP Cmr4
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Macromolecule #7: CRISPR-associated protein TM1795
Macromolecule
Name: 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 weight
Experimental: 44 KDa
Sequence
UniProtKB: Type III-B CRISPR module RAMP protein Cmr1 / InterPro: CRISPR-associated protein TM1795
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Macromolecule #2: CRISPR RNA
Macromolecule
Name: 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
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
1.0 mg/mL
Buffer
pH: 8 / Details: 20 mM Tris-HCl, pH 8.0, 150 mM NaCl
Grid
Details: 4/2 C-flat grids with a thin-layer of carbon over the holes
Vitrification
Cryogen 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
Microscope
FEI TITAN KRIOS
Alignment procedure
Legacy - Astigmatism: Objective astigmatism was corrected at 210,000 times magnification
Details
Data 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.
Date
Jul 31, 2014
Image recording
Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 7000 / Average electron dose: 48 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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