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- EMDB-0516: Small conformation of ssRNA-bound CRISPR_Csm complex -

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Basic information

Entry
Database: EMDB / ID: EMD-0516
TitleSmall conformation of ssRNA-bound CRISPR_Csm complex
Map data
SampleSmall conformation of ssRNA-bound CRISPR_Csm complex
  • (CRISPR system ...) x 2
  • (nucleic-acidNucleic acid) x 2
  • (CRISPR type III-associated RAMP protein ...) x 2
  • ligand
Function / homology
Function and homology information


exonuclease activity / defense response to virus / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding / ATP binding
CRISPR type III-associated protein / HD domain / Csm1, subunit domain B / Csm4, C-terminal / CRISPR-associated RAMP Csm3 / CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 / GGDEF domain / CRISPR type III-associated RAMP protein Csm4 / CRISPR-associated protein, Csm2 Type III-A
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
Biological speciesStreptococcus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZhang K / Pintilie G / Li S / Zhu Y / Chiu W / Huang Z
Funding support United States, China, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P41GM103832 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01GM079429 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)U54GM103297 United States
National Natural Science Foundation of China (NSFC)31825008 China
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD021600 United States
National Natural Science Foundation of China (NSFC)31422014 China
CitationJournal: Cell Res. / Year: 2019
Title: Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry.
Authors: Minghui Guo / Kaiming Zhang / Yuwei Zhu / Grigore D Pintilie / Xiaoyu Guan / Shanshan Li / Michael F Schmid / Zhuo Ma / Wah Chiu / Zhiwei Huang /
Abstract: The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading ...The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5'-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3' flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.
Validation ReportPDB-ID: 6nud

SummaryFull reportAbout validation report
History
DepositionJan 31, 2019-
Header (metadata) releaseFeb 27, 2019-
Map releaseMar 13, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nud
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0516.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 384 pix.
= 407.04 Å
1.06 Å/pix.
x 384 pix.
= 407.04 Å
1.06 Å/pix.
x 384 pix.
= 407.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.9540308 - 3.347922
Average (Standard dev.)0.00083021907 (±0.0987355)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 407.03998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z407.040407.040407.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-1.9543.3480.001

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Supplemental data

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Sample components

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Entire Small conformation of ssRNA-bound CRISPR_Csm complex

EntireName: Small conformation of ssRNA-bound CRISPR_Csm complex / Number of components: 8

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Component #1: protein, Small conformation of ssRNA-bound CRISPR_Csm complex

ProteinName: Small conformation of ssRNA-bound CRISPR_Csm complex / Recombinant expression: No
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #2: protein, CRISPR system Cms protein Csm2

ProteinName: CRISPR system Cms protein Csm2 / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 14.238391 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #3: nucleic-acid, crRNA

nucleic acidName: crRNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
ACGGAAACUU UCGUAACUGU UUAAUUCUGU UCACUUAUUC CACCGAUAUA AACCUAAUUA CCUCGAGAGG GG
MassTheoretical: 22.935553 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)

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Component #4: protein, CRISPR type III-associated RAMP protein Csm4

ProteinName: CRISPR type III-associated RAMP protein Csm4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 33.786949 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #5: protein, CRISPR system single-strand-specific deoxyribonuclease C...

ProteinName: CRISPR system single-strand-specific deoxyribonuclease Cas10/Csm1 (subtype III-A)
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 86.930672 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #6: nucleic-acid, target ssRNA

nucleic acidName: target ssRNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GGGAAUAAGU GAACAGAAUU AAACAGUUAC GAAAAAAAAA AAGGGUACC
MassTheoretical: 15.980722 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)

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Component #7: protein, CRISPR type III-associated RAMP protein Csm3

ProteinName: CRISPR type III-associated RAMP protein Csm3 / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 24.556908 kDa
SourceSpecies: Streptococcus thermophilus (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Component #8: ligand, ADENOSINE-5'-TRIPHOSPHATE

LigandName: ADENOSINE-5'-TRIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.507181 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.4 mg/mL / pH: 7.6
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 7 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 50092
3D reconstructionSoftware: cryoSPARC / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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