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Structures of the RNA-guided surveillance complex from a bacterial immune system

Nature volume 477, pages 486–489 (2011)Cite this article

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Abstract

Bacteria and archaea acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPRs). These repetitive loci maintain a genetic record of all prior encounters with foreign transgressors1,2,3,4,5,6. CRISPRs are transcribed and the long primary transcript is processed into a library of short CRISPR-derived RNAs (crRNAs) that contain a unique sequence complementary to a foreign nucleic-acid challenger7,8,9,10,11,12. In Escherichia coli, crRNAs are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defence), which is required for protection against bacteriophages13,14. Here we use cryo-electron microscopy to determine the subnanometre structures of Cascade before and after binding to a target sequence. These structures reveal a sea-horse-shaped architecture in which the crRNA is displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing. Cascade engages invading nucleic acids through high-affinity base-pairing interactions near the 5′ end of the crRNA. Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) for destruction of invading nucleic-acid sequences.

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Figure 1: Structure of the Cascade complex from E. coli.
Figure 2: Programmed capping of the CasC helix.
Figure 3: Target binding triggers a concerted conformational change.
Figure 4: A model for pathogen surveillance and signalling by Cascade.

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Accession codes

Data deposits

The cryo-electron microscopy density maps for Cascade and Cascade bound to a 32-nucleotide target have been deposited at the Electron Microscopy Data Bank under accession numbers 5314 and 5315, respectively.

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Acknowledgements

We are grateful to the Doudna and Nogales lab members for their reading of this manuscript, and to P. Grob, S. Hill, R. Hall and T. Houweling for technical support. This project was funded by a National Science Foundation grant to J.A.D., a Veni grant to S.J.J.B. (863.08.014) and a NWO Vici grant to J.v.d.O. (865.05.001). G.C.L. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation. B.W. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. E.N. and J.A.D. are Howard Hughes Medical Institute investigators.

Author information

Author notes

  1. Blake Wiedenheft and Gabriel C. Lander: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA

    Blake Wiedenheft, Kaihong Zhou, Jennifer A. Doudna & Eva Nogales

  2. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Blake Wiedenheft, Kaihong Zhou, Jennifer A. Doudna & Eva Nogales

  3. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Gabriel C. Lander & Eva Nogales

  4. Department of Agrotechnology and Food Sciences, Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands

    Matthijs M. Jore, Stan J. J. Brouns & John van der Oost

  5. Department of Chemistry, University of California, Berkeley, 94720, California, USA

    Jennifer A. Doudna

  6. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Jennifer A. Doudna

Authors
  1. Blake Wiedenheft
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Contributions

M.M.J., S.J.J.B. and J.v.d.O. designed expression constructs. B.W. and K.Z. purified samples. B.W., K.Z., M.M.J. and S.J.J.B. performed assays. B.W. and G.C.L. carried out the cryo-electron microscopy. G.C.L. performed the electron microscopy processing and segmentation analysis. All authors contributed to experimental design, data analysis and manuscript preparation.

Corresponding authors

Correspondence to Jennifer A. Doudna or Eva Nogales.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-5 with legends and additional references. (PDF 4650 kb)

Supplementary Movie 1

This movie shows a 360° rotation of the ˜8 Å resolution structure Cascade. (MOV 10186 kb)

Supplementary Movie 2

This movie shows a 360° rotation of the segmented Cascade structure. (MOV 12490 kb)

Supplementary Movie 3

This movie shows the conformational rearrangement of the CasA (purple), B (yellow) and E (magenta) subunits, while the helical backbone of the complex remains relatively undisturbed. (MOV 2790 kb)

Supplementary Movie 4

This movie shows the structural transition of Cascade under the CasC carapace. Target binding results in duplex formation along the length of the spacer sequence. Duplex formation (green) induces a rotation in CasE. The motion of CasE (magenta) is directly coupled with movement of the CasB dimer (yellow), which forms a protein bridge between the head (CasE) and tail (CasA, purple) of the complex. CasD (brown) is a hinge for the rotation of CasA. (MOV 2213 kb)

Supplementary Movie 5

This movie shows how base pairing in the crRNA spacer results in a concerted conformational change that disrupts the hook-like structure at the 5' end of the crRNA and results in a decrease in resolvable density for the distal domain of C6. (MOV 2346 kb)

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Wiedenheft, B., Lander, G., Zhou, K. et al. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477, 486–489 (2011). https://doi.org/10.1038/nature10402

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