STRUCTURE DETERMINATION OF THE SAR/SEC23/PEPTIDE

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 这是一系列旨在获得酵母COPII囊泡外壳结构信息的建议中的第三个。COPII是一个420 kDa的五聚体复合物，负责从内质网（ER）出芽运输囊泡，并将特定的货物内容物递送到高尔基复合体。在第一个提案中，我们重点关注了COPII涂层的内壳，即Sec 23/24-Sar 1复合物。我们能够通过MAD和分子置换从X-25和CHESS收集的数据中解决这个210 kDa的结构（手稿已出版）。第二个建议集中在COPII涂层的货物和ER-高尔基体陷阱融合蛋白的选择性。我们能够确定Sec 24与货物和SNARE序列结合的三种复合物的结构（提交的手稿）。 在目前的提案中，我们侧重于COPII的外壳，包括Sec 13/31复合体。Sec 13/31在功能上（但不是结构上）与网格蛋白同源-它使ER膜上的COPII内壳聚合并引起膜变形。因为，像网格蛋白，Sec 13/31是一个长的，灵活的结构，能够寡聚化（如EM图像所示），它是不可能的，完整的复合物可以结晶。因此，我们将蛋白质分解为我们认为功能上有趣的两个片段，这两个片段构成了我们在此光束线应用中提出的两个项目。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This is the third in a series of proposals that aims at obtaining structural information on the yeast COPII vesicular coat. COPII is a 420 kDa pentameric complex responsible for budding transport vesicles from the endoplasmic reticulum (ER) and delivering the specific cargo contents to the Golgi complex. In the first proposal we focused on the inner shell of the COPII coat, the Sec23/24-Sar1 complex. We were able to solve this 210 kDa structure by MAD and molecular replacement from data collected at X-25 and at CHESS (manuscript published). The second proposal focused on the selectivity of the COPII coat for cargo and for ER-to-Golgi SNARE fusogenic proteins. We were able to determine the structures of three complexes of Sec24 bound to cargo and SNARE sequences (manuscript submitted). In the current proposal we focus on the outer shell of COPII, comprising the Sec13/31 complex. Sec13/31 is functionally (but not structurally) homologous to clathrin-it polymerizes the COPII inner shell on the ER membrane and causes membrane deformation. Since, like clathrin, Sec13/31 is a long, flexible structure capable of oligomerizing (as seen in EM images) it is unlikely that the intact complex can be crystallized. Therefore, we have dissected the protein into what we believe are the two functionally-interesting fragments, and these constitute the two projects that we propose in this beamline application.