uce inside the quantity necessary for the SPOT assay (200 g) (S2 Table).
Cellulose membrane-bound peptides were automatically ready as outlined by regular SPOT synthesis protocols applying a Spot synthesizer (Abimed) as described in [63]. The software LISA (Jerini) was utilized for the generation from the peptide sequence files and all cysteines have been replaced by 603139-19-1 serines to exclude false-positive spots. A conservative length of 15-mers was chosen to make sure effective coupling steps throughout peptide synthesis within the absence of extensive HPLC and MS analyses of probes. The generated arrays of 15-mer peptides had been synthesized on cellulose-(3-amino-2-hydroxy-propyl)-ether (CAPE) membranes. CAPE membranes were ready from 18 28 cm Whatman 50 paper as described in detail [31]. The SPOT membrane was rinsed for five min with ethanol and washed three instances with TBS (50 mM Tris/HCl, pH 7.6, 150 mM NaCl) for ten min 
prior to blocking with blocking buffer (TBS, 1 x Blocking Buffer (Sigma B-6429), 0.15 M sucrose) for 3 hours. The SH3 domains had been incubated at 10 g/ml using the membrane overnight at 4 in blocking buffer. The membrane was washed three occasions with TBS for ten min. Immuno-detection was carried out by incubating the membrane for 2.5 hours with an anti-GST antibody (Sigma G-1160, 1 g/ml), a secondary anti-mouse antibody HRP conjugate (Sigma A-5906, 1 g/ml) and Luminol remedy (Thermo Scientific # 34080). Pictures have been taken employing a Lumi Imager (Boehringer) and analyzed with all the software Genespotter (Microdiscovery GmbH).
All sequences were aligned with T-coffee (version eight.98) sequence alignment application [64] employing the accurate mode. This mode combines information from Hidden Markov model (HMM) profiles (PSI-coffee) 10205015 and three-dimensional information from structural templates (Expresso) with multiple sequence alignments from other alignment tools (ClustalW2) to create a hugely informed meta-alignment. All multiple sequence alignments were rendered and edited with Jalview [65] to annotate the motifs. The template structures identified by T-coffee and detailed description by Fernandez-Ballester et al. [17] had been used to visually inspect whether crucial interface residues were spatially conserved.
Following manually aligning the top 95% peptides of every SH3 domain, we transformed the alignment into a 15 amino acid-wide position-weighted matrix (PWM), corresponding to the sequence length on the peptide probes, by computing the normalized observed frequency per amino acid for each and every position. Utilizing a sliding window approach we computed a score for every 15-residue partial sequence in a potential binding sequence. A score for a subsequence is obtained by summing the substitution scores, employing the PAM250 substitution matrix, from the observed amino acids to the amino acids inside the PWM per residue position. To account for PWM precise score distributions, we computed for every score the probability of observing such a score offered the PWM against a background distribution of 1000 randomly sampled 15-mers. These p-values have been then corrected for several hypotheses testing by applying the Benjamini-Hochbach correction, which controls the false discovery price (FDR) and converts p-values to q-values. Only subsequences having a q-value 0.0001 have been retained as sequence matches to the PWM.
The coding sequences for the Myosin C-terminal tails had been PCR amplified, cloned by restriction digestion into a pGEX plasmid and transformed into E.coli Rosetta cells (Merck). Cultures were grown at 30 in LB (1% [w/
