Structural basis of AAUAAA polyadenylation signal recognition by the human CPSF complex.

Protein expression and purification Human CPSF subunit constructs were cloned into MacroLab vectors developed by Scott Gradia (University of California, Berkeley) 21 . His6-tagged full-length CPSF160 (Uniprot Q10570 ) was inserted into the 438B vector (Addgene #55219); His 6 -(StrepII) 2 -WDR33 (Uniprot Q9C0J8-1, residues 1-410) was inserted into a modified version of the 438B vector; untagged CPSF30 (Uniprot O95639-3, residues 1-178), was inserted into the 438A vector (Addgene #55218); StrepII-GFP-tagged Fip1 (Uniprot Q6UN15-4, residues 130-195) was inserted into the 438Rgfp vector (Addgene #55211) by ligation-independent cloning. The four constructs were combined in a single plasmid using the MacroBac ligation-independent cloning system 21 . Recombinant baculovirus was generated using the Bac-to-Bac system (Invitrogen) according to standard protocols and used to infect Sf9 cells at a density of 1.0 × 10 6 ml −1 . Cells were harvested 72 hr post infection, resuspended in 20 mM Tris-Cl pH 7.5, 200 mM NaCl, 10% glycerol and 0.05% Tween20, supplemented with Protease Inhibitor Cocktail (GE Healthcare), and lysed by sonication. The complex was purified on Ni-NTA resin (Qiagen), followed by purification on Streptactin superflow resin (IBA). Expression tags were removed by overnight incubation with TEV protease. Synthetic 10-mer PAS RNA (5’-ACAAUAAAGG-3’) was added in 1.2-fold molar excess and the resulting protein-RNA complex was further purified by size exclusion chromatography on a Superdex-200 column (GE Healthcare) in 20 mM HEPES pH 7.5, 140 mM KCl and 1 mM DTT. Peak fractions (as indicated in Supplementary figure 1A ) were collected and flash-frozen in liquid nitrogen.

Cryo-EM sample preparationand data collection The CPSF-PAS

RNA complex was vitrified on Quantifoil Cu 400 mesh R1.2/1.3 grids previously glow-discharged for 15 seconds on each side. 4 μl of sample at 0.3 mg/ml was deposited on the EM grid, incubated for 15 seconds, blotted for 7 seconds and plunge-frozen in liquid ethane-propane mixture using a FEI Vitrobot Mark IV at 22 °C and 100% humidity. Data collection was performed on a FEI Titan Krios microscope operated at 300 kV and equipped with a Gatan K2 Summit detector and a Gatan Quantum GIF LS energy filter. Image acquisition and initial processing was managed by Focus 22 . Micrographs with a pixel size of 1.058 Å were collected in super-resolution mode and consisted of 50 frames for a total exposure time of 10 seconds and a total dose of 80 e - / Å 2 . The defocus ranged between -2.2 and -1 μm.

Image processing

Motion correction and averaging were performed with MotionCor2 23 . Final micrographs are dose-weighted sums of the full stack removing the first frame. Contrast transfer function (CTF) parameters were determined with CTFFIND4 24 . Subsequent processing was performed in RELION-2 25 . A dataset of 1070 images was selected with a CTF resolution estimate between 2.6 and 3.0 Å. 1600 particles were manually picked from a random subset of micrographs and the resulting 2D class averages used as input for the RELION-2 Autopick function on binned micrographs, obtaining a total 265,000 particles. After selection of 263,000 particles with best Autopick figure of merit, the particles were subjected to two rounds of 2D classification, selecting particles from 72 and 27 classes, respectively. The final dataset contained 137,000 particles representing 51% of the picked particle dataset. Aligned particles were then extracted from unbinned micrographs and an initial reference-free model was generated using RELION-2 (Initial model function). 3D classification of the dataset into 3 classes yielded models with similar features, number of particles and final resolution, suggesting structural homogeneity. Therefore, the whole dataset was directly subjected to 3D refine alignment. After post-processing in RELION-2 the final map reached a resolution of 3.1 Å. The resolution was measured by Fourier shell correlation between reconstructions from two independently refined half-datasets (gold-standard FSC in RELION-2). Local resolution was calculated within RELION-2 with ResMap 26 from two data-independent reconstructions. Model building Model building was performed in Coot 27 using the CPSF160-WDR33 heterodimer as starting model (PDB 6F9N) and refined using phenix.real_space_refine 28 . Figures were prepared with Chimera 29 and PyMol ( https://www.pymol.org )

Supplementary Material Supplementary Figures 1-4 Supplementary Table 1