The use of cryo-EM and three-dimensional image reconstruction is becoming increasingly common. Our vision for this technique is to provide a straightforward manner in which users can proceed from raw data to a reliable 3D reconstruction through a pipeline that both facilitates management of the processing steps and makes the results at each step more transparent. Tightly integrated with a relational SQL database, Appion is a modular and transparent pipeline that extends existing software applications and procedures. The user manages and controls the software modules via web-based forms, and all results are similarly available using web-based viewers directly linked to the underlying database, enabling even naive users to quickly deduce the quality of their results. The Appion API was designed with the principle that applications should be compatible with a broad range of specimens and that libraries and routines are modular and extensible. Presented here is a description of the design and architecture of the working Appion pipeline prototype and some results of its use.
Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries, such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER). COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and Sec13 and Sec31 (Sec13/31), which has a structural role. Whereas recent results have shown that Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles, we now show that Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24. We present a three-dimensional reconstruction of these Sec13/31 cages at 30 A resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers. Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the self-assembly of Sec13/31 to form a cage lattice driving ER cargo export.
SUMMARY
Using cryo-electron microscopy, we have solved the structure of a novel icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) co-assembled from purified cargo adaptor Sec23–24 and Sec13–31 lattice forming complexes. The coat structure shows a tetrameric assembly of the Sec23–24 adaptor layer that is positioned beneath the vertices and edges of the Sec13–31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 β-propeller domains present in Sec13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo including procollagen and chylomicrons that are sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a new mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.
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