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Use of helical assembly scaffolds as tools for alleviating preferred orientation problem in Cryo-Electron Microscopy

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dc.contributor.advisor PANANGHAT, GAYATHRI
dc.contributor.author SHAHARE, AISHWARYA
dc.date.accessioned 2023-05-22T10:58:53Z
dc.date.available 2023-05-22T10:58:53Z
dc.date.issued 2023-05
dc.identifier.citation 57 en_US
dc.identifier.uri http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/7980
dc.description.abstract The preferred orientation issue has currently emerged as a major challenge for the structural analysis of proteins in cryo-electron microscopy (cryo-EM). Smaller proteins (<100 kDa) are particularly challenging to identify and align in noisy low-dose pictures of cryo-EM. Many smaller proteins and their liganded complexes play a significant role in a biological process but their structure is unknown. Therefore, a tool that assists in structure determination of smaller proteins will be useful for structural biology applications. Megabodies have been recently used to solve the preferred orientation issue of smaller proteins, which consists of a nanobody attached to a large scaffold protein connected by a linker. It increases attached protein size and makes it easy to align and observe in 2D images. To overcome the problem of orientational preference, we have developed a megabody, consisting of a helical filament scaffold that can provide views from all orientations of the protein due to its helical symmetry. We have used ParM, a bacterial actin that polymerizes in the presence of ATP, as the choice for helical scaffold. As a proof-of-principle, we have attached a nanobody that binds to ALFA tag, a helical peptide sequence, to ParM. This enables any protein with the ALFA tag to decorate the helical megabody of nanobody-tagged ParM filaments. Towards this goal, we have explored the use of linker lengths of three different lengths between nanobody and ParM. Polymerization assay through pelleting has been used for monitoring efficiency of filament formation and co-pelleting of the ALFA-tagged small protein (SofG) was carried out for validating the tool design. Moreover, our results show that ParM helical megabody can also be used as a solubility purification tag for nanobodies. en_US
dc.language.iso en en_US
dc.subject Megabody en_US
dc.subject Nanobody en_US
dc.subject SofG protein en_US
dc.title Use of helical assembly scaffolds as tools for alleviating preferred orientation problem in Cryo-Electron Microscopy en_US
dc.type Thesis en_US
dc.description.embargo One Year en_US
dc.type.degree BS-MS en_US
dc.contributor.department Dept. of Biology en_US
dc.contributor.registration 20181093 en_US


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  • MS THESES [1705]
    Thesis submitted to IISER Pune in partial fulfilment of the requirements for the BS-MS Dual Degree Programme/MSc. Programme/MS-Exit Programme

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