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1 Structure of the Sec13/31 COPII coat cage

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2 Function of the COP II Mediate cargo export from ER to Golgi complex

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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.

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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.

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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.

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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)

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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.

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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

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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

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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

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11 Cuboctahedrons roughly spherical polyhedrons E=24 V=12 F=14 8 triangles 6 squares exhibit or octahedral symmetry four edges intersect at each vertex （ clathrin geometries are defined by vertices formed from only three edges ）

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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

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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

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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

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15 Reconstruct cage structure molecular mass MDa diameter along its longest diagonal 600 Å the length of an edge300 Å the width of an edge40 Å

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16 The asymmetric unit (ASU) the smallest unit that can be repeated to generate the full structure

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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 ）

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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

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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.

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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.

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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.

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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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