+Open data
-Basic information
Entry | Database: PDB / ID: 6nue | |||||||||||||||||||||
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Title | Small conformation of apo CRISPR_Csm complex | |||||||||||||||||||||
Components |
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Keywords | HYDROLASE / TRANSFERASE/RNA / CRISPR / Type III-A / ssRNAase / ssDNase / TRANSFERASE-RNA complex | |||||||||||||||||||||
Function / homology | Function and homology information 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 Similarity search - Function | |||||||||||||||||||||
Biological species | Streptococcus thermophilus (bacteria) | |||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||||||||||||||
Authors | Zhang, K. / Pintilie, G. / Li, S. / Zhu, Y. / Chiu, W. / Huang, Z. | |||||||||||||||||||||
Funding support | United States, China, 6items
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Citation | Journal: 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. | |||||||||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6nue.cif.gz | 446.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6nue.ent.gz | 362.7 KB | Display | PDB format |
PDBx/mmJSON format | 6nue.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6nue_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 6nue_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 6nue_validation.xml.gz | 66.9 KB | Display | |
Data in CIF | 6nue_validation.cif.gz | 102.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/nu/6nue ftp://data.pdbj.org/pub/pdb/validation_reports/nu/6nue | HTTPS FTP |
-Related structure data
Related structure data | 0519MC 0516C 0517C 0518C 6nudC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-CRISPR system ... , 2 types, 4 molecules JABM
#1: Protein | Mass: 86930.672 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: cas10, csm1 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A0A7HFE1, Hydrolases; Acting on ester bonds, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases |
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#2: Protein | Mass: 14238.391 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm2 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A0A7HIX1 |
-CRISPR type III-associated RAMP protein ... , 2 types, 6 molecules CENOPI
#3: Protein | Mass: 24600.918 Da / Num. of mol.: 5 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm3, CDA68_00842 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A0A7HIF0 #5: Protein | | Mass: 33786.949 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus (bacteria) / Gene: csm4, CDA68_00841 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: A0A0A7HGA1 |
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-RNA chain / Non-polymers , 2 types, 2 molecules H
#4: RNA chain | Mass: 22935.553 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Streptococcus thermophilus (bacteria) Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
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#6: Chemical | ChemComp-ATP / |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Small conformation of apo CRISPR_Csm complex / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT |
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Molecular weight | Units: MEGADALTONS / Experimental value: NO |
Source (natural) | Organism: Streptococcus thermophilus (bacteria) |
Source (recombinant) | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Buffer solution | pH: 7.6 |
Specimen | Conc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: unspecified |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 7 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||
3D reconstruction | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81540 / Symmetry type: POINT |