CRYO-EM URI-COMPLEX · A combined Cryo Electron Microscopy and mass Spectrometry approach for the structural and functional characterization of the URI-complex
6РП — Действия „Мария Кюри“
- Период
- 2007-07-01 → 2009-06-30
- Финансиране от ЕС
- 173 264 €
- Участници
- 1
- Схема
- EIF
Линиите свързват координатора с партньорите. За проекти отпреди 2014 г. CORDIS не винаги дава точни координати. Тези точки са на ниво град или държава.
Накратко на български
Структурата и функциите на протеинови комплекси, като URI комплекса при бактерията Leptospira interrogans, се анализират чрез комбиниране на електронна микроскопия и масспектрометрия. Методът помага да се свърже анализът на изолирани протеини с тяхното реално наблюдение вътре в клетката.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Final Activity Report Summary - CRYO-EM URI-COMPLEX (A Combined Cryo Electron Microscopy and Mass Spectrometry Approach for the Structural and Functional Characterization of the URI-complex)
We proposed an integrated approach for the structure determination of protein complexes that combines the strength of two technologies. While mass spectrometry can identify and quantify proteins and protein interactions with high sensitivity in the bulk of lysate of many cells, cryo electron microscopy can in principle detect individual protein complexes. We chose of the human pathogen Leptospira interrogans as a model system and determined the cytoplasmic protein concentrations of several protein complexes using selected reaction monitoring mass spectrometry. This provided information aided the identification of individual protein complexes within cryo electron tomograms of Leptospira interrogans cells by template matching. Furthermore, we identified the interfaces within several protein complexes, including the URI complex, using isotope coded cross-linking reagents with sub-sequent identification of cross-linked peptides by tandem mass spectrometry. The outlined technology promises to establish the critical link from structural analysis of isolated protein complexes to their observation in the cell.
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
Here we propose a novel hybrid strategy for the structure determination of macromolecular complexes using a state of the art mass spectrometry (MS) and cryo-electron microscopy (EM) approach. This strategy allows for the accurate fitting of crystal structures into EM-maps, but also provides structural templates for the identification of macromolecular complexes within cryo-electron tomograms (CET) by pattern recognition. It promises to establish the critical link from structural analysis of isolated protein complexes to their observation in the cell. As a promising initial target for our investigations we chose the recently identified URI complex (unconventional prefoldin RBP5 interactor), a regulator in cell growth control that acts downstream of the target of rapamycin (TOR).The structure of the URI complex is unknown, although the crystal structures of the archaeal homologues of several of its components have been solved. Therefore, it is likely to form a striking jellyfish-shaped scaffold structure consisting of prefoldins (PDFs) with having RuvB-like helicases attached to it. To generate accurate fits of crystal structures into EM-maps, additional spatial information is advantageous. A novel cross-linking strategy using a bivalent isotope tagged chemical compound, in combination with MS can provide such information. It has been established in the host institution and allows for identification of the interface of interacting proteins at primary structure level.We plan to combine this technology with single particle cryo-EM and the fitting of crystal structures to resolve the structure of the URI complex to pseudo atomic level. In a second step, the structure of the URI complex will be used as a template to monitor interactions with the next neighbours in the signalling pathway by pattern recognition within CET. This approach will provide a structural insight into a branch of the TOR signalling pathway that has been linked to diseases like the metabolic syndrome.
Оригинален текст от CORDIS (на английски).
Участници
- EIDGENOSSISCHE TECHNISCHE HOCHSCHULE ZURICH · ZURICHКоординаторНиво градШвейцария
Връзки
Данни: CORDIS, © Европейски съюз