Computational protocol: Ubiquitination of stalled ribosome triggers ribosome-associated quality control

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Protocol publication

[…] After the gels were extensively washed with Milli-Q water (Millipore), the gel band was excised, diced into 1 mm3 pieces, and destained by 1 ml of 50 mM ammonium bicarbonate (AMBC)/30% acetonitrile (ACN) with agitation for 1 h, then the gels were further washed by 1 ml of 50 mM AMBC/50% ACN for 1 h. Finally, 100% ACN wash was performed to ensure complete gel dehydration. Trypsin solution (Promega, 20 ng/µl in 50 mM AMBC) was subsequently added to gel pieces at approximately equivalent volume and incubated on ice for 30 min. Another small volume of trypsin solution was added to gel samples and incubated at 37 °C for overnight. Digests were extracted by addition of 50 μl of 50% ACN/0.1% trifluoroacetic acid (TFA) for 1 h by shaking. The digested peptides were recovered into fresh eppendorf tubes and additional extraction step was performed with 70% ACN/0.1% TFA for 30 min. The extracted peptides were combined and concentrated by a speed-vac. After centrifugation, the recovered peptides were analyzed by nanoflow HPLC (Easy nLC1000, Thermo Scientific) coupled with a Q Exactive mass spectrometer (Thermo Scientific). The mobile phases were 0.1% formic acid (FA) in water (solvent A) and 0.1% FA in 100% ACN (solvent B). Peptides were directly loaded onto a C18 analytical column (ReproSil-Pur 3 μm, 75 μm inner diameter, and 12 cm length, Nikkyo Technos) and separated using a 80 min two step gradient (0–35% in 70 min, 35–100% in 10 min, and 100% in 10 min of solvent B) at a constant flow rate of 300 nl/min. The Q Exactive was operated in the data-dependent MS/MS mode, using Xcalibur software, with survey scans acquired at a resolution of 140,000 at m/z 200. The top 10 most abundant isotope patterns with charge 2–5 were selected from the survey scans with an isolation window of 2.0 m/z, and fragmented by HCD with normalized collision energies of 28. The maximum ion injection times were 60 ms for both survey and MS/MS scans, and the AGC values were set to 3 × 106 and 5 × 105 for the survey and MS/MS scans, respectively. Ions selected for MS/MS were dynamically excluded for 10 s.Proteome Discoverer software (ver. 1.3, Thermo Scientific) was used to generate peak lists. The MS/MS spectra were searched against a SwissProt database (version 2012_10 of UniProtKB/Swiss-Prot protein database) using the MASCOT search engine. The precursor and fragment mass tolerances were set to 10 ppm and 20 mmu, respectively. Methionine oxidation, protein amino-terminal acetylation, pyroglutamate formation, serine/threonine/tyrosine phosphorylation, and diglycine modification of lysine side chains were set as variable modifications, and cysteine methylthio modification was set as a static modification for database searching. Peptide identification was filtered at a 1% false discovery rate. We also analyzed the MS data by using Paragon search engine. The raw files were converted to MGF files using PD1.3 and were searched by ProteinPilot software (ver. 4.0, AB Sciex) against the SwissProt database. [...] Yeast cell expressing Rqt1-FTP (Flag-TEV-Protein A) were cultured in YPD medium. As a control sample, a yeast strain containing genomically tandem-affinity purification (TAP) tagged uL30 was also expressed in YPD. Purifications were performed as described before. The Rqt1 complex and uL30 control were purified from whole-cell lysates by affinity purification using IgG-conjugated dynabeads, followed by TEV protease cleavage to elute it. Freshly prepared sample was applied to 2 nm pre-coated R3/3 holey carbon-supported grids (Quantifoil) and vitrified using a Vitrobot Mark IV (FEI Company). Data were collected on a Titan Krios TEM (FEI Company) equipped with a Falcon II direct electron detector, operated at 300 keV. The magnification settings resulted in a pixel size of 1.084 Å/pixel. The data set was provided with the semi-automatic software EPU (FEI Company) with an accumulated dose of ~28 e−/Å2 divided on 10 frames. Frames were aligned using Motion Correction software and CTF estimation was performed with CTFFIND4. Particles were picked using Gautomatch ( Data processing was done with RELION. In brief, 2D classification was performed to exclude non-ribosomal particles. The remaining particles were subsequently refined and 3D classified to obtain classes with classical or rotated ribosomes. Images were made with UCSF chimera. […]

Pipeline specifications

Software tools CTFFIND, Gautomatch, RELION, UCSF Chimera
Application cryo-EM
Chemicals Anisomycin