PDBsum entry 3ps0

RNA binding protein

PDB id: 3ps0

Contents

Protein chains

293 a.a.

272 a.a.

Waters ×545

PDB id:

3ps0

Name:

RNA binding protein

Title:

The structure of the crispr-associated protein, csa2, from sulfolobus solfataricus

Structure:

Crispr-associated protein, csa2. Chain: a, b, c, d. Engineered: yes

Source:

Sulfolobus solfataricus. Organism_taxid: 273057. Strain: p2. Gene: sso1442. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.00Å R-factor: 0.185 R-free: 0.221

Authors:

N.G.Lintner,M.Sdano,M.J.Young,C.M.Lawrence

Key ref:

N.G.Lintner et al. (2011). Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE). J Biol Chem, 286, 21643-21656. PubMed id: 21507944 DOI: 10.1074/jbc.M111.238485

Date:

30-Nov-10 Release date: 20-Apr-11

Protein chains

Q97Y91  (CSA2B_SULSO) -  CRISPR-associated aCascade subunit Cas7/Csa2 2 from Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)

Seq: 321 a.a.

Struc: 293 a.a.

Protein chains

Q97Y91  (CSA2B_SULSO) -  CRISPR-associated aCascade subunit Cas7/Csa2 2 from Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)

Seq: 321 a.a.

Struc: 272 a.a.

Enzyme reactions

• Enzyme class: Chains A, B, C, D: E.C.?

DOI no: 10.1074/jbc.M111.238485

J Biol Chem 286:21643-21656 (2011)

PubMed id: 21507944

Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE).

N.G.Lintner, M.Kerou, S.K.Brumfield, S.Graham, H.Liu, J.H.Naismith, M.Sdano, N.Peng, Q.She, V.Copié, M.J.Young, M.F.White, C.M.Lawrence.

ABSTRACT

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

Selected figure(s)

Figure 2.
S. solfataricus Cas6 generates crRNA. A, a two-repeat spacer unit CRISPR transcript was cleaved by Cas6 at a single site in each repeat, yielding fragments of 109 and 43 nt for cleavage at repeat 1, 106 and 46 nt for cleavage at repeat 2, and the 63-nt mature crRNA for cleavage at both repeats. B, a synthetic RNA corresponding to a single CRISPR repeat with a 15-unit 5′ extension is cleaved by Cas6 at a single site, generating an 8-nt repeat-derived 5′ extension (“psi-tag”). C, schematic illustrating the 2-repeat transcript and the expected cleavage products. D, schematic illustrating the synthetic substrate.

Figure 5.
The structural similarity between Csa2 and Cas6 is limited to the RNA-recognition motif. A, Csa2 and Cas6 are shown in equivalent orientations based on an SSM Structural alignment. The RRM-like subdomains are colored violet in both structures. The Csa2 1–3 domain is colored red, the 2–4 domain is colored orange, and the C-terminal subdomain is colored yellow. The portions of the Cas6 N-terminal domain that do not exhibit similarity to the Csa2 RRM subdomain are depicted in green and the C-terminal domain in light cyan. The conserved clusters on Csa2 and the putative active site residues on Cas6 are shown as “sticks” colored in dark cyan.

The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2011, 286, 21643-21656) copyright 2011.

Figures were selected by an automated process.