Professional Documents
Culture Documents
the RNA-target cleavage activity in Cmr4. Mol. Cell 56, 4354 (2014). Medline
doi:10.1016/j.molcel.2014.09.002
15. F. DiMaio, M. D. Tyka, M. L. Baker, W. Chiu, D. Baker, Refinement of protein
structures into low-resolution density maps using rosetta. J. Mol. Biol. 392, 181
190 (2009). Medline doi:10.1016/j.jmb.2009.07.008
16. B. Wiedenheft, G. C. Lander, K. Zhou, M. M. Jore, S. J. Brouns, J. van der Oost, J.
A. Doudna, E. Nogales, Structures of the RNA-guided surveillance complex from
a bacterial immune system. Nature 477, 486489 (2011). Medline
doi:10.1038/nature10402
17. M. L. Hochstrasser, D. W. Taylor, P. Bhat, C. K. Guegler, S. H. Sternberg, E.
Nogales, J. A. Doudna, CasA mediates Cas3-catalyzed target degradation during
CRISPR RNA-guided interference. Proc. Natl. Acad. Sci. U.S.A. 111, 66186623
(2014). Medline doi:10.1073/pnas.1405079111
18. Z. Chen, H. Yang, N. P. Pavletich, Mechanism of homologous recombination from
the RecA-ssDNA/dsDNA structures. Nature 453, 4894 (2008). Medline
doi:10.1038/nature06971
19. C. Anders, O. Niewoehner, A. Duerst, M. Jinek, Structural basis of PAMdependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569
573 (2014). Medline doi:10.1038/nature13579
20. H. Nishimasu, F. A. Ran, P. D. Hsu, S. Konermann, S. I. Shehata, N. Dohmae, R.
Ishitani, F. Zhang, O. Nureki, Crystal structure of Cas9 in complex with guide
RNA and target DNA. Cell 156, 935949 (2014). Medline
doi:10.1016/j.cell.2014.02.001
21. O. W. Ryan, J. M. Skerker, M. J. Maurer, X. Li, J. C. Tsai, S. Poddar, M. E. Lee, W.
DeLoache, J. E. Dueber, A. P. Arkin, J. H. Cate, Selection of chromosomal DNA
libraries using a multiplex CRISPR system. eLife 3, e03703 (2014). Medline
22. G. W. Goldberg, W. Jiang, D. Bikard, L. A. Marraffini, Conditional tolerance of
temperate phages via transcription-dependent CRISPR-Cas targeting. Nature
514, 633637 (2014). Medline doi:10.1038/nature13637
23. J. A. Mindell, N. Grigorieff, Accurate determination of local defocus and specimen
tilt in electron microscopy. J. Struct. Biol. 142, 334347 (2003). Medline
doi:10.1016/S1047-8477(03)00069-8
24. X. C. Bai, I. S. Fernandez, G. McMullan, S. H. Scheres, Ribosome structures to
near-atomic resolution from thirty thousand cryo-EM particles. eLife 2, e00461
(2013). Medline doi:10.7554/eLife.00461
25. S. H. Scheres, Beam-induced motion correction for sub-megadalton cryo-EM
particles. eLife 3, e03665 (2014). Medline
26. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C.
Meng, T. E. Ferrin, UCSF Chimeraa visualization system for exploratory
research and analysis. J. Comput. Chem. 25, 16051612 (2004). Medline
doi:10.1002/jcc.20084
27. A. I. Cocozaki, N. F. Ramia, Y. Shao, C. R. Hale, R. M. Terns, M. P. Terns, H. Li,
Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex.
Structure 20, 545553 (2012). Medline doi:10.1016/j.str.2012.01.018
28. Y. Shao, A. I. Cocozaki, N. F. Ramia, R. M. Terns, M. P. Terns, H. Li, Structure of
the Cmr2-Cmr3 subcomplex of the Cmr RNA silencing complex. Structure 21,
376384 (2013). Medline doi:10.1016/j.str.2013.01.002
29. K. Sakamoto, Y. Agari, K. Agari, S. Yokoyama, S. Kuramitsu, A. Shinkai, X-ray
crystal structure of a CRISPR-associated RAMP module Cmr5 protein from
Thermus thermophilus HB8. Proteins 75, 528532 (2009). Medline
doi:10.1002/prot.22358
ACKNOWLEDGMENTS
The structures of intact apo-Cmr, smaller apo-Cmr, and target-bound Cmr have
been deposited into the EMDataBank with accession codes EMD-2898, EMD2899, and EMD-2900, respectively. We thank R. Louder, S. Howes, E. Kellogg, R.
Zhang, P. Grob, Y. He, T. Houweling, Z. Yu and M.J. de la Cruz for expert electron
microscopy assistance. D.W.T is a Damon Runyon Fellow supported by the
Damon Runyon Cancer Research Foundation (DRG-2218-15). R.H.J.S. was
supported by the University of Otagos Division of Health Sciences Career
Development postdoctoral fellowship. Y.Z. and J.O. received financial support
from the Netherlands Organisation for Scientific Research (NWO), via a
Gravitation grant to the Soehngen Institute for Anaerobic Microbiology
(024.002.002) and an ALW-TOP project (854.10.003), respectively. This work
was supported in part by JSPS KAKENHI Grant Number 25440013 (to A.S.).
J.A.D and E.N. are Howard Hughes Medical Institute Investigators. The T.
thermophilus Cmr complex and T. thermophilus strain producing Cmr6-His
protein are available from A.S. under a material transfer agreement with RIKEN.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/cgi/content/full/science.aaa4535/DC1
Materials and methods
Figs. S1 to S3
Table S1
Movies S1 to S3
References (2329)
9 December 2014; accepted 11 March 2015
Published online 2 April 2015
10.1126/science.aaa4535
Fig. 1. Fig. 1. Architecture of the native crRNA-bound (apo) and ssRNA target-bound
Cmr. (AC) Cryo-EM reconstructions of intact apo-Cmr (crRNA-bound) (A), a smaller apoCmr (B), and ssRNA target-bound Cmr (C), at 4.1-, 4.5- and 4.4- resolution (using the
0.143 gold standard Fourier Shell Correlation criterion), respectively. Subunits are
segmented and colored as indicated. (D) Difference map between intact apo-Cmr and
target-bound Cmr at 10- (solid, blue) superimposed on the apo-Cmr structure
(transparent). (E) Aligning the smaller (surface) and intact (mesh) apo-Cmr complexes
based on Cmr2Cmr3 (left) or Cmr1Cmr6 (right), shows that the helical geometry is
preserved. (F) Alignment of apo-Cmr (surface) and target-bound Cmr complexes
(transparent mesh) based on the Cmr4 backbone (removed for clarity) (see also movies
S1, S2)..
/ sciencemag.org/content/early/recent / 2 April 2015 / Page 5 / 10.1126/science.aaa4535
Fig. 3. The Cmr4 backbone and Cmr5 subunits position target ssRNA for segmented cleavage.
(A) The channel formed between Cmr4 and Cmr5 backbones creates the binding cleft for target RNA.
(B) Cmr4 -hairpins (thumbs) intercalate between every fifth base pair of duplex crRNA:target RNA,
disrupting the helix. (C) Expanded view of (B) shows how the thumb-like domain of the lower Cmr4
positions the kinked target near the catalytic loop. (D) Pseudo-atomic model created by docking
available crystal structures of Cmr subunits and our homology model of Cmr4 into the target-bound
Cmr structure. (E) Model of target recognition and cleavage shows that the loop (red) of the homology
model (presented here) containing catalytic residues H16 and D27 previously identified (14) is
positioned near the target. (F, G) The Cmr6 subunit (gold) also contains a long thumb-like extension
(F), which disrupts base-paring between the crRNA and target RNA (G) for the 5-most cleavage event.
(H, I) The thumb of the Cmr3 subunit (H) engages the crRNA 5-handle and bottom target:crRNA
segment (I). (See also movie S3 and Supplementary Materials and Methods.)
/ sciencemag.org/content/early/recent / 2 April 2015 / Page 7 / 10.1126/science.aaa4535