SUPPLMTARY IFORMATIO a doi:10.108/nature10402 b 100 nm 100 nm c SAXS Model d ulers assigned to reference- Back-projected free class averages class averages Refinement against single particles Reconstructed density 1.0 fourier shell correlation 0.9 0.8 Unbound Target-bound 0.7 0.6 0. fsc = 0. 0.4 0. 0.2 fsc=0.14 0.1 0.0 20.0 Figure10.0 Supplementary legends resolution (Angstroms) 6.66 Supplementary Figure 1 ryo electron microscopy and three dimensional reconstruction. Images of the unbound (a) and RA bound ascade complex (b) collected at 100,000X magnification. c, Initial models for three dimensional reconstruction were determined using a low resolution SAXS reconstruction14. The SAXS reconstruction was low pass filtered to 60Å resolution, forward projected at an angular increment of 1 degrees, and a multi reference alignment was performed using,000 reference free class averages of each of the ascade complexes. The WWW. AT U R. O M / AT U R aligned class averages were back projected to generate a new density, which was 1
RSARH SUPPLMTARY IFORMATIO using,000 reference free class averages of each of the ascade complexes. The aligned class averages were back projected to generate a new density, which was then used for another iteration of projection matching. The unbound and targetbound datasets were processed separately, each using their corresponding initial model. Three dimensional refinements of the starting densities were performed using an iterative projection matching and Fourier reconstruction approach using libraries from the MA2 and SPARX software packages 24,2. d, The resolution of each structure was estimated using Fourier shell correlations 2. Resolutions reported in the paper were made using a conservative 0. Fourier shell correlation criterion. 2 WWW.ATUR.OM/ATUR
Supplementary Figure legends SUPPLMTARY IFORMATIO RSARH microscopy and b a Supplementary Figure 1 ryo electron c three dimensional crra crra reconstruction. Images of the unbound (a) and RA bound ascade complex (b) r s eq u e n c, Initial models for three dimensional collected at 100,000X magnification. e c stem-loop reconstruction were determined using a low resolution SAXS reconstruction14. The ace sp A SAXS reconstruction was low pass filtered to 60Å resolution, forward projected at an angular increment of 1 degrees, and a multi reference alignment was performed Seed cr R using,000 reference free class averages of each of the ascade complexes. The -handle aligned class averages were back projected to generate a new density, which was d then used for another iteration of projection matching. The unbound and target e as bound datasets were processed separately, each using their corresponding initial sa2 model. Three dimensional refinements of the starting densities were performed using an iterative projection matching and Fourier reconstruction approach using 24,2 libraries from the MA2 and SPARX software 90 packages 90. d, The resolution of 90 each structure was estimated using Fourier shell correlations2. Resolutions reported in the paper were made using a conservative 0. Fourier shell correlation criterion. Supplementary Figure 2 Three dimensional docking of crystal structures into ascade. a, Atomic coordinates for homologous structures of as, the crra stem loop and asb from T. thermophilus HB8 were docked in the ~8 Å cryo M map. b, rystal structure of a RISPR specific endoribonuclease (magenta) and the crra stem loop (red) docked in the segmented density for as (gray mesh) and the crra (green surface) (PDB: 2Y8W) 11. c, crra sequence (61nt) modeled into the crra density. d, rystal structure of a asb homolog (se2) form T. thermophilus (yellow) docked in the segmented density for asb (gray mesh) (PDB: 2ZA). Surface charge representation of the. coli asb sequence threaded onto the se2 crystal structure. e, rystal structure of a distant as homolog from the P2 strain of Sulfolobus solfataricus (PDB: PS0)4. The sa2 structure is significantly different form the as density and the two structures do not superimpose with high fidelity. However, shared secondary structure elements are detectable (blue, yellow, magenta and orange) and slight modifications to the pitch or angle of these elements reveal a similar architecture. W W W. A T U R. O M / A T U R
RSARH SUPPLMTARY IFORMATIO a b ascade ascade spacer 1 2 6 1 1 1 12 as asa asb as asd as as1 as2 se1 se2 se4 ase se crra ascade ascade ascade as c DA target 148 98 64 0 6 22 16 6 RA target 148 98 64 0 6 22 16 0 1 20 Spacer 0 1 10 20 Spacer Self PAM 0 1 10 20 0 1 10 20 min A Self PAM 0 1 20 0 1 10 20 0 1 10 20 0 1 10 20 min D B A D B 6 Supplementary Figure Structural interrogation of ascade by limited proteolysis. a, Schematic of the RISPR/as locus in the. coli K12 genome. ascade assembles from an unequal number of as proteins and a crra (asa 1B 2 6D 1 1crRA 1). b c) Limited trypsin digestion of ascade bound to a nucleic acid substrate complementary to the spacer, the spacer plus the handle (self) or to an oligo that contains a protospacer adjacent motif (PAM). A proteolytic product appears at the first time point (green arrow) in all samples. The asb subunits are sensitive to proteolysis 20 minutes after binding to DA or RA targets (red arrow). 4 WWW.ATUR.OM/ATUR
SUPPLMTARY IFORMATIO RSARH 2 1 90 90 4 6 Supplementary Figure 4 The as subunits are superimposable between the target bound and unbound structures. The as hexamer from the target bound (surface) and unbound structures (mesh) of ascade. The red arrow indicates additional density for 6 in the unbound structure. WWW.ATUR.OM/ATUR
RSARH SUPPLMTARY IFORMATIO 8-nt -Handle 2-nt Spacer 21-nt -Stem asa as6 asd 8 7 6 4 2 2 1 0 29 28 27 26 2 24 2 22 21 20 19 18 17 16 1 14 1 12 11 10 9-8 -7-6 - -4 - -2-1 A U A A A A U A U U U U A A U U U U A A A 1 Seed T A T T T T A T A A as as4 T A T A A A A T T A as as as2 as as1 A A A T T A A A A T T T T A A A A A T T T A U U as ascade ascade ascade ascade ascade target -8 to 8 1 to 16 9 to 24 17 to 2 2 to 40 Supplementary Figure Molecular recognition of foreign nucleic acids by ascade. Top, Schematic of the ascade architecture highlighting the relative position of each as protein and the three distinct segments of the mature crra (8 nt handle / 2 nt spacer / 21 nt stem loop). The spacer sequence is shown in red and the protein subunits are colored according to Fig 1. Bottom, Target binding affinities at discrete regions along the crra were determined by electrophoretic mobility shift assays with increasing concentrations of ascade (0 1000 nm) in each panel. Sixteen nucleotide ssda oligos that tile along the length of the crra were end labeled with 2P. The last lane in each panel includes 00 nm ascade and a ssda oligo that is not complementary to the crra (). ascade makes low affinity, non sequence specific interactions with DA 14. Supplemental references 2 van Heel, M. & Schatz, M. Fourier shell correlation threshold criteria. J Struct Biol 11, 20 262 (200). Agari, Y., Yokoyama, S., Kuramitsu, S. & Shinkai, A. X ray crystal structure of a RISPR associated protein, se2, from Thermus thermophilus HB8. Proteins Structure Function and Bioinformatics, 106 1067 (2008). 4 Lintner,.. et al. Structural and functional characterization of an archaeal ASAD complex for RISPR mediated viral defense. J Biol hem (2011). 6 WWW.ATUR.OM/ATUR