1

Structure of the Sec13/31 COPII coat cage

2

Function of the COPII

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 preservedin vitreous ice showeda population of cage-like particles, most of which were symmetricand 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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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

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Cuboctahedrons

roughly spherical polyhedronsE=24 V=12 F=14 8 triangles 6 squaresexhibit 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 mass 5.4-9.6 MDa

diameter along its longest diagonal

600 Å

the length of an edge

300 Å

the width of an edge 40 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

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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

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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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Contrast of clathrin and sec13/31

Clathrin Sec13/31

intersect to form the vertices

three four

cage formed from

four overlapping clathrin heavy chains

a Sec1 3/31 heterotetramer

diameter 100 A 40 A

interdigitated

Not extensively extensively

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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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.