Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.DOI: http://dx.doi.org/10.7554/eLife.11349.001

We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

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I hereby declare that this dissertation with the title "Structural Characterization of the Eukaryotic Translation Initiation by Electron Cryo-Microscopy" has been written independently and with no other aids or sources than quoted. This thesis (wholly or in part) has not been submitted elsewhere for any academic award or qualification.