Computational protocol: Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome

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Protocol publication

[…] Ribosome profiling experiments were performed as described previously (), but with some modifications. Approximately five A260 units of the Ski-3-TAP tagged pullouts were used for ribosome profiling. The pullout fraction was digested with RNase I (Ambion) at 25°C for 45 min in a shaker at 500 rpm followed by 5 min incubation on ice with SUPERase-In (Ambion). Following nuclease digestion, ribosomes were pelleted through a sucrose cushion (20 mM HEPES pH 7.5, 100 mM KOAc, 10 mM MgCl2, 750 mM sucrose, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml cycloheximide, Protease Inhibitor Cocktail (Roche)) by centrifugation at 312 000 × g for 45 min at 4°C. Ribosomal pellets were resuspended in ribosome-splitting buffer (20 mM Tris, 400 mM KCl, 2 mM MgCl2, 1 mM DTT and 1 mM puromycin) and incubated on ice for 30 min. Ribosomal subunits were pelleted again by centrifugation at 250 000 × g for 90 min at 4°C. The supernatant from this centrifugation step was used as the source for ribosome protected fragments (RPFs).RPFs were further purified and size selected in a 15% denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel for fragments between 26–62 nt using markers. Gel extracted fragments were precipitated and processed as given in the protocol (ARTseq™ Ribosome Profiling Kit, Epicentre, WI, USA) for library preparation and high-throughput sequencing. Sequencing was performed on an Illumina HiSeq 1500. Reads were adapter-trimmed using the software Cutadapt (version 1.2.1, EMBnet.journal, 2011). Reads mapping to ribosomal RNA, tRNA, small nuclear and nucleolar RNA were removed. Remaining reads were mapped to the yeast genome (R64–1–1, 19 July 2014) using Tophat (v2.0.8b) () with the following parameters: -a 4 –no-novel-juncs –GTF. Only uniquely mapped reads were used for further bioinformatic analysis. For identifying the P-site position within the footprints, a meta-gene analysis using 5′ end of the footprints around the start codon was performed. Based on this analysis, the first peak appeared 12 nt upstream of the start codon. A-, P- and E-site codons for all footprints were assigned by shifting 16, 13 and 10 nt, respectively. The number of footprints per amino acid in each site was calculated by summing up the shifted footprints over the corresponding codons. To normalize the occurrence, the codons in the second position downstream of the A-site were summed up and the occurrence for each amino acid was calculated. The values for the amino acids in A-, P- and E-site where then divided by the occurrence for each corresponding amino acid in this second position accordingly. For all the footprints mapping to ORFs, the normalized occurrence of A-, P- and E-site amino acids were then plotted in Supplementary Figure S1. [...] The ribosomes from the TEV-eluate were adjusted to a concentration of 4 A260/ml (0.8 μM 80S ribosomes), applied to 2 nm pre-coated Quantifoil R3/3 holey carbon supported grids and vitrified using a Vitrobot Mark IV (FEI Company). Data collection was performed on a Titan Krios TEM (FEI Company) equipped with a Falcon II direct electron detector, operated at 300 keV using the EPU software (FEI). The magnification settings resulted in a pixel size of 1.084 Å/pixel. The dataset was provided with the semi-automatic software EPU (FEI Company) with a dose of 2.4 e−/Å−2 per frame for 13 frames in total. The frames were aligned using the Motion Correction software (). Data were collected at a defocus range between −0.8 and −3.4 μm. Only micrographs that showed clearly visible Thon rings below 5.5 Å on the level of the rotationally averaged power spectra profiles were used for further analysis. Automatic particle detection was performed by the program SIGNATURE (). Initial in silico sorting of the dataset consisting of 246.555 particles in total was performed using the SPIDER software package (). Classes were obtained by competitive projection matching in SPIDER (,). The vast majority (>95%) of the particles we found were programmed with tRNAs. This dataset could be subdivided into two main classes, both containing A- and P-tRNAs and either with or without the Ski complex (76 816 and 88 640 particles, respectively). The large number of ribosomal particles without density for the Ski complex suggests that the Ski complex was not stably bound to these particles. To our surprise, both classes contained ribosome-bound eIF-5A. Nevertheless, only the class without Ski complex displayed a highly resolved density for eIF-5A that allowed model building. The density for eIF-5A in the Ski complex bound ribosome class was partially disordered and the density for the factor was fragmented. For high-resolution refinement, the dataset containing the eIF-5A particles was further cleaned by removing particles with low cross-correlation. The cleaned dataset (62 532 particles) was then processed further using RELION (). To do this, the particle boxes were extracted using the coordinates obtained by SIGNATURE and normalized in RELION. The contrast transfer function (CTF) estimation was repeated using CTFFIND3 () and the dataset was subjected to auto-refinement in RELION using a ribosomal reference low-pass filtered to 70 Å. After auto-refinement, the dataset was subjected to movie processing and the particle-polishing feature in RELION. Here, only the first eight frames were used for the calculations, resulting in an accumulated dose of 24 e−/Å−2. Subsequent auto-refinement of ‘shiny’ particles resulted in a final reconstruction of 3.9 Å resolution according to a gold standard fourier shell correlation (FSC) cutoff of 0.143. This map was sharpened using automatic b-factor estimation in RELION and used for interpretation and model building. Local resolution was calculated using ResMap () and maps were visualized in UCSF Chimera (). RELION data were processed on the Leibnitz-Rechenzentrum (LRZ) Munich. [...] For modeling the large ribosomal subunit (LSU), the crystal structure of the yeast ribosome (PDB ID: 4V88) () was taken as a template. Peptidyl A- and deacylated P-tRNA were modeled based on the crystal structure of the Thermus thermophilus 70S ribosome in the post-catalysis state of peptide bond formation (containing dipeptidyl-tRNA in the A site and deacylated tRNA in the P site, PDB ID: 1VY5) () and for eIF-5A a homology model was generated using HHPred (). All structures were roughly fitted into the map using UCSF Chimera. Flexible fitting and, where necessary, de novo model building was done in Coot () followed by real space refinement in PHENIX (). For the rRNA and the tRNAs, geometry restrictions were calculated using the ‘PDB to 3D restraints’ database prior to PHENIX refinement.The eIF-5A homology model was obtained after a multiple alignment using HHPred. This model was subjected to geometry minimization using PHENIX and remodeled in Coot. The well-resolved hypusine-containing β3-β4 loop (residues 47–54) was modeled de novo and for the N-terminal extension (NTE; res 1–16) a poly-Ala model was generated. For uL16, the loop containing residues 103–111 (not present in the yeast ribosome X-ray structure ()) was modeled de novo. The L1 stalk in the eIF-5A position was remodeled and a poly-alanine model for uL1 was generated using uL1 from the human 80S ribosome as a template () (PDB ID: 5AJ0). In a final step, all models were combined and subjected to real-space refinement using the PHENIX software. […]

Pipeline specifications

Software tools SPIDER, RELION, CTFFIND, ResMap, UCSF Chimera, HHPred, Coot, PHENIX
Applications cryo-EM, Protein structure analysis
Organisms Saccharomyces cerevisiae