2 Function of the COP II Mediate cargo export from ER to Golgi complex
3 Componets of COPII Sar1 GTPase Sec 23/24 Sec 13/31 Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles. Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24.
4 Structure of the “cage” 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.
5 Total introduction 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.
6 Methods 1 、 Recombinant production and purification 2 、 Dynamic light scattering （ DLS ） 3 、 cryo-electron microscopy (cryo-EM) 4 、 Single particle reconstruction. 5 、 gel electrophoresis (PAGE) 6 、 gel filtration chromatography (GFC)
7 cryo-EM analysis Purified Sec13/31 forms a relatively homogeneous population of assemblies as judged by GFC, analytical ultracentrifugation, DLS, GFC-MALS and electron microscopy analyses of the negatively stained samples We characterized the same samples using cryo-electron microscopy (cryo-EM) and single particle analysis.
8 Images of the specimen preserved in vitreous ice showed a population of cage- like particles, most of which were symmetric and with an average diameter of,600 A. These dimensions are in good agreement with the size of COPII cages/vesicles observed in vitro4,12 and in vivo cryo-EM analysis
9 a total of 9,777 individual cage particles were selected from a set of 516 defocus pairs of micrographs. Particles were first subjected to a reference-free alignment algorithm as implemented in the EMAN package to generate averages with an improved signal-to-noise ratio. single particle analysis
10 single particle analysis Of the 104 resulting class averages, ten that showed the best signal-to-noise ratio and symmetry were used as reference images in a multi-reference alignment procedure. The resulting class averages exhibited two-fold, three-fold and four-fold symmetry and geometry consistent with that of a cuboctahedron
11 Cuboctahedrons roughly spherical polyhedrons E=24 V=12 F=14 8 triangles 6 squares exhibit 4 3 2 or octahedral symmetry four edges intersect at each vertex （ clathrin geometries are defined by vertices formed from only three edges ）
12 Cuboctahedrons the four-fold rotational axes of symmetry run down the middle of the square faces, the three-fold rotational axes run through the middle of the triangular faces the two-fold rotational axes run through the vertices
13 Reconstruct cage structure Use a simple cuboctahedron constructed with continuous density for the edges as an initial model The cage structure was refined to a resolution of 30 Å
14 Reconstruct cage structure There is excellent agreement between projections of the final model and the individual raw particle images as well as the class averages
15 Reconstruct cage structure molecular mass5.4-9.6 MDa diameter along its longest diagonal 600 Å the length of an edge300 Å the width of an edge40 Å
16 The asymmetric unit (ASU) the smallest unit that can be repeated to generate the full structure
17 The asymmetric unit (ASU) Two roughly spherical lobes of density at either end （ 1 、 2 、 5 、 6 ） connected by a continuous curving stretch of density with a diameter of 40 Å （ 3 、 4 ）
18 The asymmetric unit (ASU) The lobes at either end are not identical, they appear to be related to each other by a 180°rotation around the centre of the density connecting the two ends. ASU is a dimer The centre of symmetry of the ASU is not in the centre of the edge
19 The asymmetric unit (ASU) We propose that the 24 off-centre, dimeric ASUs comprising the Sec13/31 cage correspond to 24 Sec13/31 heterotetramers.
20 Positions of Sec13/31 The positions occupied by Sec13 and Sec31 in the cage remain to be determined. From a structural perspective, Sec13 contains WD40 repeat motifs that are implicated in protein-protein interactions. Biochemical and computational analyses indicate that the Sec13 structure may comprise a single domain, b-propeller fold with six blades
21 Positions of Sec13/31 heterotetramer model two Sec13 proteins would form part of the continuous density in the centre of the ASU (3 and 4 ) but cannot be resolved as distinct entities at the present resolution. such a model would suggest that Sec13 dimerization is critical for cross-bridging the two halves of the edge.
22 An alternative model Sec13 forms the vertices of the cuboctahedron Sec13/31 heterotetramer is arranged as Sec13/Sec31-Sec31/ Sec13 and corresponds to the ASU that constitutes the edges of the cuboctahedron
23 Regions 1 and 6 contain Sec13. The Sec1 3 subunits would interact with each other at the vertices of the cage in two unique ways 1 、 edge-vertex contacts 2 、 vertex-vertex contacts An alternative model
24 larger globular domains (2 and 5 ） would correspond to the predicted b -propeller fold comprising the seven WD40 motif repeats/blades of the Sec31 N-terminal domain smaller globular domains (1 and 6 ） would correspond to the predicted b- propeller fold comprising the six WD40 motif repeats/blades of the entire Sec13 subunit An alternative model
25 Considering that Sec13 interacts with the N-terminal WD40 repeat domain of Sec31 ， this new model would place the N-terminal domain of Sec31 near the vertex of the cuboctahedral cage. It follows that Sec31 dimerization at the centre of the ASU would be critical for cross-bridging the two halves of the cuboctahedral edge. An alternative model
26 An alternative model Sec23 is expected to bind near the Sec31-Sec31 dimer interface, whereas Sec24 should bind towards the ends of the ASU
27 Clathrin can also self-assemble in vitro to form empty cages lacking the adaptor components and cargo, all of which comprise the clathrin coat. These are strikingly different from the Sec13/31 cage
28 Contrast of clathrin and sec13/31 ClathrinSec13/31 intersect to form the vertices threefour cage formed from four overlapping clathrin heavy chains a Sec1 3/31 heterotetramer diameter100 A40 A interdigitatedNot extensivelyextensively
29 overview 1 、 The function of Sec13/31 analogous to that of clathrin, which self-assembles to form a cage independent of its adaptor proteins. This is in contrast with a recent study that suggested that the Sec23/24 adaptor is required for the self-assembly of a minimal COPII cage.
30 overview 2 、 A model for COPII coat formation where Sec23/24, like the clathrin adaptor proteins, coordinates cargo selection with the self-assembly of the Sec13/31 cage to promote budding from the ER.
31 overview 3 、 The discovery of the self-assembling properties of Sec13/31 to generate a cage structure provides a new focus for elucidating the biological mechanisms of cargo selection, concentration and budding for transport of nearly one- third of all proteins encoded by the eukaryotic genome.
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