Journal of Structural Biology 173 (2011) 358–364

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Journal of Structural Biology

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The structure of a COPII tubule

Jason O’Donnell a, Kerry Maddox a, Scott Stagg a,b,⇑a Institute for Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USAb Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 July 2010Received in revised form 1 September 2010Accepted 2 September 2010Available online 7 September 2010

Keywords:Secretory pathwayCryo-electron microscopyCryo-electron tomographyCOPIICargoSubvolume averaging

1047-8477/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jsb.2010.09.002

⇑ Corresponding author. Address: Institute of MolecUniversity, 91 Chieftan Way, Tallahassee, FL 32306-47244.

E-mail addresses: sstagg@fsu.edu, sstagg@mac.com

Nearly a third of all eukaryotic proteins are transported from the ER to the Golgi apparatus through thesecretory pathway using COPII coated vesicles. Evidence suggests that this transport occurs via 500–900 Å vesicles that bud from the ER membrane. It has been shown that procollagen molecules utilizethe COPII proteins for transport, but it is unclear how the COPII coat can accommodate these �3000 Ålong molecules. We now present a cryogenic electron tomographic reconstruction of a Sec13/31 tubulethat is approximately 3300 Å long containing a hollow cylindrical interior that is 300 Å in diameter,dimensions that are consistent with those that are required to encapsulate a procollagen moleculewrapped in a membrane and accessory COPII components. This structure suggests a novel mechanismthat the COPII coat may employ to transport elongated cargo.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

A defining characteristic of eukaryotic cells is their elaborateintracellular, endomembrane system which is composed of spe-cialized compartments that are in continuous communication withone another and the environment around them. A major form ofcommunication between these compartments is conveyed by ves-icles that have one or more layers or ‘‘coats” of protein that encap-sulate membrane-enclosed cargo and mediate its transfer. Three ofthe best characterized protein complexes that are involved incoated vesicle formation and transport are clathrin, coat proteincomplex I (COPI), and coat protein complex II (COPII).

Vesicle formation from the ER for transport to the Golgi appara-tus is mediated by the COPII proteins Sar1, Sec23/Sec24 (Sec23/24),and Sec13/Sec31 (Sec13/31) (Barlowe et al., 1994). Vesicle buddingis initiated by the activation of Sar1, a member of the Ras super-family of small GTPases, by the guanine nucleotide-exchange fac-tor (GEF), Sec12. Upon exchange of GDP for GTP, Sar1 localizes tothe ER membrane where a 20–23 amino-acid amphipathic a-helixis inserted into the bilayer (Barlowe and Schekman, 1993). Thecytosolic heterodimer Sec23/24 is then recruited to the membranewhere it plays a role in cargo selection by binding exit code motifson cargo proteins (Barlowe et al., 1994; Matsuoka et al., 1998;

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ular Biophysics, Florida State380, USA. Fax: +1 (850) 644

(S. Stagg).

Mancias and Goldberg, 2008; Mossessova et al., 2003; Milleret al., 2002). Together, Sar1 GTP and Sec23/24 form ‘‘pre-buddingcomplexes” containing concave surfaces that recruit cargo andmay be involved in membrane deformation (Bickford et al., 2004;Bi et al., 2002; Bielli et al., 2005; Lee et al., 2005). The next stepin the formation of vesicles is the recruitment of the cytosolic het-erotetramer Sec13/31 by the pre-budding complex. Sec13/31 hasthe ability to self assemble into polyhedrons and this likely con-tributes to further membrane deformation (Barlowe et al., 1994;Lee et al., 2005; Stagg et al., 2006, 2008). The last step of vesicle for-mation involves a fission event where vesicles pinch-off, and this isbelieved to involve Sar1 GTP hydrolysis (Bielli et al., 2005; Leeet al., 2005).

Electron microscopy (EM) studies have gradually revealed thestructural diversity of COPII coats. Early studies by serial-thin sec-tioning of plastic embedded cells revealed buds with an averagediameter of �780 ± 60 Å located at the tips of tubular projectionsemanating from the ER (Bannykh et al., 1996). Later, cryoEM andsingle particle analysis of a macromolecular complex formed fromthe self-assembly of purified mammalian Sec13/31 led to thethree-dimensional (3D) reconstruction of a polyhedron structureapproximately 600 Å in diameter (Stagg et al., 2006). The geometryof the reconstruction was that of a cuboctahedron built by 24 edgeswhere each edge represents one Sec13/31 heterotetramer. A sec-ond reconstruction from purified self-assembled Sec23/24 andSec13/31 yielded an icosidodecahedral cage structure with twoclearly defined structural layers of protein (Stagg et al., 2008).The innermost layer was composed of Sec23/24 and the outer layerconsisted of 60 Sec13/31 edges.

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The structures of the icosidodecahedral and cuboctahedralcages suggested a mechanism by which the coat can adapt to cargoof varying shapes and sizes (Stagg et al., 2008). It was suggestedthat a single angle designated b dictates the diameter and geome-try of the coat. A b angle of 90� between adjacent Sec13/31 edges inthe cage resulted in the 600 Å diameter cuboctahedron while a bangle of 105� resulted in the 1000 Å diameter icosidodecahedron.Sec23/24 in the icosidodecahedron structure were in proximityto the Sec13/31 proteins that form the b angle and it was hypoth-esized that Sec23/24 can control the b angle in vivo in response tothe needs of the particular cargo that is being transported.

It has been controversial, however, whether COPII coats areflexible enough to accommodate cargos with diameters greaterthan 1000 Å (Mellman and Warren, 2000; Fromme and Schekman,2005). Cargo such as collagen and chylomicrons are up to 3000 and10,000 Å in diameter, respectively, yet are known to require COPIIproteins for transport (Jones et al., 2003; Shoulders et al., 2004;Stephens and Pepperkok, 2002). In the case of procollagen, numer-ous studies have demonstrated the existence of large tubular struc-tures that are involved in its trafficking between the ER and Golgi(Stephens and Pepperkok, 2002; Mironov et al., 2003; Presley et al.,1997; Scales et al., 1997). However, it is unclear whether COPII isinvolved in generating these structures and if so, how it is ableto do so. Here, we present a cryogenic electron tomographic (cryo-ET) reconstruction of a tubule formed from the self-assembly ofpurified Sec13/31. The tubule is formed from interlinked Sec13/31 cages and is �3300 Å in length with a hollow cylindrical interiorthat is 300 Å in diameter. It has a cavity that is large enough toencapsulate a procollagen molecule wrapped in a bilayer mem-brane and accessory complex proteins Sar1 and Sec23/24. We pro-pose that the COPII tubule structure provides a possible novelmechanism for the transport of elongated cargoes.

2. Results

2.1. Assembly of the Sec13/31 tubule

We previously proposed a geometry where Sec13/31 could forma filamentous structure (Stagg et al., 2008), and we reasoned thatwe could favor the assembly of the filamentous form over the cageform by increasing the Sec13/31 concentration during assembly.The distribution of structures in an assembly reaction was deter-mined by velocity analytical ultracentrifugation (AUC). This re-vealed that the reaction contained a mixture of assemblies withsedimentation coefficients of 5 S and 60 S, and higher-order assem-blies with sedimentation coefficients of 75–250 S (Fig. 1a). Weinterpreted the 60 S species to be cuboctahedral Sec13/31 cages

Fig.1. Velocity analytical ultracentrifugation (AUC) plot and images of negatively stcoefficients from a velocity analytical ultracentrifugation run of the Sec13/31 sample. Thithe distribution of sedimentation coefficients. A mixture of assemblies with 5 and 60 Snegatively stained Sec13/31 tubules. Two differently sized tubules (arrow) and an indiv

and this was borne out by negative stain EM. The negative stainedmicrographs further revealed that the higher-order species corre-sponded to elongated filamentous structures (Fig. 1b). These struc-tures showed a clear repeating pattern but were flattened due tothe negative staining. To overcome the flattening, the Sec13/31sample was vitrified on a holey carbon grid and visualized by cryo-EM. Inspection of the cryoEM micrographs revealed that the prep-aration contained cages corresponding to self-assembled Sec13/31cuboctahedrons that were �600 Å in diameter and similar to whatwas previously seen (Fig. 2a black arrows). In addition to individualcages, multiple filamentous structures were observed with lengthsranging from 1000 to 7500 Å and a constant diameter of �600 Å(Fig. 2a white arrow). The filament structures showed a clearrepeating pattern, though their Fourier transforms did not showobservable reflections (data not shown). Thus, a tomographic ap-proach was undertaken to determine the molecular structures ofthe Sec13/31 filaments.

2.2. Tomography of the Sec13/31 filaments

We performed cryo-electron tomography to determine thestructure of the Sec13/31 filament. Several tilt series were ac-quired, and one had a filament lying along the tilt axis (see Supple-mentary Fig. 1a). This tilt series was aligned and reconstructed inthree-dimensions using the PROTOMO software package (Winklerand Taylor, 2006) (Fig. 2). The reconstructed filament had featuresthat were similar to the cuboctahedral Sec13/31 cage with charac-teristic ‘square’ and ‘diamond’-shaped views (Fig. 2b and c). Basedon these similarities, it appeared that the filament was composedof Sec13/31 cages that connect to form a tubule. However, giventhe low-resolution of the tomographic volume and the effect ofthe missing wedge, the topology of the individual Sec13/31 hetero-tetramers was unclear.

In order to overcome these obstacles, we performed subvolumeaveraging on segments of the Sec13/31 tubule (Winkler, 2007).Additionally, two free-floating Sec13/31 cages that were visiblein the tilt series (Fig. 2a black arrows) and not connected to anyother cages were aligned to one another and octahedrally averagedto generate a map of the Sec13/31 cage (hereafter referred to as thesubvolume averaged cage (SVcage) at the same scale as the subvo-lume averaged tubule (SVtubule). The resolution for both mapswas estimated to be �85–90 Å based on the location of the firstzero in the Fourier transformation of the 0� tilt image and thiswas verified by Fourier neighbor correlation using the programRMEASURE (Sousa and Grigorieff, 2007). Although the SVcagewas of lower resolution than the previously determined Sec13/31cage reconstruction (Stagg et al., 2006), density corresponding to

ained Sec13/31 tubules. (a) Van Holde–Weischet distributions of sedimentations plot shows that the sample contains a mixture of assemblies, which correspond toexist, as well as higher-order assemblies between 75 and 250 S. (b) An image of

idual cuboctahedron cage (arrowhead) are visible. Scale bar = 500 Å.

Fig.2. 2D and 3D representation of the Sec13/31 tubule. (a) An image from the tilt series taken at the 0� angle. Scale bar = 500 Å. Black arrows point to individual Sec13/31cages and the white arrow points to the Sec13/31 tubule. (b) The tomogram generated by alignment and back projection of all the images in the tilt series. Arrowheadscorrespond to individual Sec13/31 cages, which are also pointed out with black arrows in panel a. The dashed box is magnified in panel c. (c) A magnified view the dashed boxin panel b. The square and star point to areas in the filament that correspond to vertices and edges, respectively.

Fig.3. Three-dimensional reconstruction of the SVcage, SVtubule, and their differ-ence map. (a) The SVcage (blue) generated by extracting the two filament-freecages (arrowheads in Fig. 2), and octahedrally symmetrized. Scale bar = 500 Å. (b)The SVtubule map (red) corresponding to subvolume averaged cages in the filamentand D4-symmetrized. This map is different from that which is shown in panel a inthat the triangular faces are filled with density and connects with C4 symmetryrelated triangular faces to form a ‘‘crown” of density above and below the map. (c)The difference map (green) highlighting differences between the SVcage and theSVtubule map. The difference map (shown here at 1.9 r) is composed of globules ofdensity that are connected with one another by two strands of density. Thesestrands are similar to the edge components in both length and a bend in the middle.The location of the globules are in the same location as the globules or tips of thecrown that are located in the SV tubule map. (d) The difference map shown in panelc superimposed onto the SVcage in panel a. (e) Two copies of the SVcage (cyan andmagenta) were fitted into the difference map and superimposed onto the SVcagethat was used for the difference mapping. Each cage in the filament is linked withone another through the triangular faces. (f) The map in panel e but rotated 90�along the axis of arrow that points from panel e to f.

360 J. O’Donnell et al. / Journal of Structural Biology 173 (2011) 358–364

edges and vertices could be identified unambiguously, and thecharacteristic curve in the center of the edge was visible (Fig. 3a).The subvolume averaging was performed on the tubule by cuttingout segment volumes corresponding to apparent asymmetric unitsin the tubule. The individual subvolumes were globally averaged toobtain an initial model. This map then served as a reference foralignment of the individual subvolumes. The aligned subvolumeswere averaged to produce a new reference, and this procedurewas iterated until the subvolume average ceased to change instructure.

2.3. The structure of the Sec13/31 tubule

The SVcage and SVtubule maps shared many similarities; theywere both �600 Å in diameter with vertices and open square facesin similar locations (Fig. 3a and b). The SVtubule, however, had ex-tra density which spanned across the entire area of the triangularfaces (Fig. 3b). This extra density extended towards symmetry re-lated triangular faces and formed a ‘‘crown” of density, where atthe tips of the crown are globules of density commensurate withthe size and topology of vertices found within the SVcage. To bettervisualize any disparities between the two maps, a difference mapwas created by subtracting the SVcage map from the SVtubulemap. The strongest positive difference peaks in the resultingmap, were located within the triangular faces of superimposedSVcages (Fig. 3c and d). Elongated lobes of difference density fromsymmetry related triangular faces extended up and connected withone another, and this corresponded to the crown densities in theSVtubule. These elongated difference peaks resembled the Sec13/31 edges of the SVcage map in size, shape, and topology (Fig. 3a).This was verified by fitting two copies of the SVcage map intothe difference map (Fig. 3e). This clearly showed that the elongatedlobes of difference density and the globular masses at their pointsof connection corresponded to edge elements and vertices ofSec13/31 cages. Remarkably, these analyses revealed that theSec13/31 tubule is formed from topologically interlocked cages(Fig. 3e). Neighboring cages in the tubule are separated by a320 Å translation and 45� rotation about the long axis of the fila-ment. In this way, four vertices from one cage are located in fourtriangular faces of a neighboring cage. The cages that comprisethe tubule are all aligned along their 4-fold symmetry axes so thatthe tubule contains a cylindrical cavity 300 Å in diameter that runsthrough the entire length of the tubule (Fig. 3f). This was confirmedby fitting 10 cages into the original tomographic filament map(Fig. 4).

For additional verification that the 10-cage filament was com-posed of interlocked cages we repeated the subvolume averagingprocedure using 3 full cages. We call the resulting subvolume aver-aged map the ‘‘3-Cage-SVTubule”. This new subvolume was D4-symmetrized and compared with a synthetic interlocking filamentcontaining three copies of the Sec13/31 cuboctahedron (Stagget al., 2006). This synthetic filament was generated by taking thepreviously published cage structure and repeating it with a 320 Åtranslation and 45� rotation between each cage. Comparisons werethen made between the synthetic filament, the original tomogram,and the 3-Cage-SVTubule by looking at their isosurface maps andrespective 2D projection images (Supplementary Fig. 2). In theprojection image and isosurface map of the synthetic filament

Fig.4. An Sec13/31 tubule and fitted SVcages. (a) The raw tomogram cropped toshow only the tubule. (b) Ten copies of the SVcage were fitted into the rawtomogram. Each map is colored differently for ease of visualization. On average,each cage is related to a neighbor by a 320 Å translation and a 45� rotation aboutthe long axis of the filament. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

J. O’Donnell et al. / Journal of Structural Biology 173 (2011) 358–364 361

(Supplementary Fig. 2a and a’), the density of each cage is clearlyvisible as being interlocked with one anther and the cages maybe found in one of two orientations. One orientation pertains tothe central cage while the other orientation can be visualized inthe flanking cages. Approximately, 1=3 of each flanking cage volumeoverlaps with the central cage. Similar orientations and degree ofoverlap may be visualized in the projection images and maps ofthe 3-Cage-SVTubule and tomogram. In all three isosurface mapsand projection images, the degree of overlap between each cageand the topology of vertices and square faces are similar. This dem-onstrates identical interlocking mechanisms between the syntheticfilament and experimental data, that four vertices from one cageare located in four triangular faces of a neighboring cage.

3. Discussion

The COPII protein complex, Sec13/31, is remarkably versatile atforming assemblies with a variety of shapes and sizes. Two previ-ous cryoEM reconstructions of purified Sec13/31, forming an�600 Å cuboctahedron, and purified Sec23/24 and Sec13/31, form-ing an �1000 Å icosadodecahedron, have laid down the first set oforganizing principles in vesicle formation. Comparing the commoncomponent between these two cages, the Sec13/31 edge, clearlyshowed that the angle formed between edges is variable and givesthe COPII coat flexibility to accommodate cargo of varying size byutilizing different geometries. Indeed, to date, three differentlysized cages have been directly visualized by electron microscopyand range in size from 600 Å to 1000 Å (Stagg et al., 2008). Whilethese structures are well adapted for generating spherical cargotransport vesicles in vivo, they are too small to accommodate elon-gated cargo such as procollagen (Stephens and Pepperkok, 2002).

We have now demonstrated for the first time that Sec13/31 canform a tubular structure. Electron tomographic reconstruction andsubvolume averaging have shown that the tubule is composed ofinterlocking cuboctahedral cages. The three-dimensional recon-

struction shows a 3300 Å long filament with helical symmetry con-structed by 10 cages, each of which are related to their neighborsby a 45� rotation and 320 Å translation along the long filamentaxis. In the raw tomogram, density corresponding to edges andvertices could be clearly visualized and subvolume averaging anddifference mapping showed that the tubule was composed of cub-octahedral cages that were linked with one another through theirtriangular faces. Furthermore, the entire length of the interior isan empty cylindrical cavity that could sheath elongated cargo suchas procollagen for transportation.

Several pieces of data have suggested that COPII proteins are in-volved in transporting large and irregular cargos such as procolla-gen and chylomicron particles from the ER. For instance, mutationsin Sar1b can result in chylomicron retention disease (Jones et al.,2003; Shoulders et al., 2004) where chylomicron particles fail toexit from the ER. This suggests a direct role for COPII in transport-ing these >4000 Å particles. Furthermore, Martinez-Menárguezet al. showed that immunogold labeled COPII coated membranesare often tubular in appearance (Martínez-Menárguez et al.,1999). Additionally, Bonfanti et al. have shown tubule-like forma-tions emerging from the ER which stain heavily for procollagen(Bonfanti et al., 1998).

The exact role of COPII proteins in transporting procollagen hasbeen a controversial topic in the vesicle transport field. Mironovet al. suggested that procollagen was transported by COPII inde-pendent en-bloc transport. In their study, large and irregularlyshaped procollagen-containing saccules that were not associatedwith COPII proteins formed in sites that were different from ER exitsites (ERES) and protruded from the ER membrane (Mironov et al.,2003). It was suggested that the 600–800 Å COPII coats were toosmall to accommodate such elongated cargo. Other and morerecent data, however, strongly suggest that COPII proteins areinvolved in procollagen transport. Immunofluorescence micros-copy allowed the visualization of procollagen exiting from COPII-labeled ERES, and inhibition of COPII function with dominantnegative mutants resulted in the buildup of procollagen withinthe ER (Stephens and Pepperkok, 2002). Furthermore, the diseasecranio-lenticulo-sutural dysplasia which is characterized byimproper collagen trafficking was demonstrated to result from amutation in Sec23A (Boyadjiev et al., 2006). Finally, Saito et al.recently identified that the protein TANGO1 is a transmembranereceptor that interacts with both collagen VII and Sec23/24 (Saitoet al., 2009). Their results suggested that TANGO1 is responsiblefor loading collagen VII into COPII coated vesicles. Our resultsnow provide a potential mechanism by which 600 Å COPII cagescan concatenate into an interlocking tubular structure that couldserve as the procollagen transporter in vivo.

3.1. Model for the assembly of the Sec13/31 tubule

While it is possible that the COPII proteins in the cell may formelongated coats using a different mechanism and geometry, ours isthe first data showing that the COPII proteins can assemble into atubular structure with the potential for providing a carrier for elon-gated cargo. In our hands, tubule formation in vitro is favored overindividual cage assembly by increasing the Sec13/31 concentra-tion. There are a number of mechanisms by which the tubular car-riers may be assembled in the cell. For example, it may be that aspecific combination of Sec23 or Sec24 isoforms favor their assem-bly, or there may be an as yet undiscovered accessory protein thatparticipates in their assembly. Regardless, for the structure pre-sented here to form in the cell, the Sec13/31 edges must overcomemultiple steric constraints while concatenating into the interlock-ing structure of the Sec13/31 tubule. We hypothesize that tubuleformation in the cell may be favored by differential assemblykinetics of tubular carriers. It is known that Sec23/24 interacts

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with Sar1b on the ER to form ERES and ‘‘pre-budding complexes”(Aridor et al., 1998). TANGO1 could localize procollagen to an ERESby simultaneously binding collagen inside the ER lumen andSec23/24 on the outside. TANGO1 has a proline-rich domain thatinteracts with Sec23/24 in a similar way to Sec13/31, and thismay serve to stall Sec13/31 assembly. The reduced velocity of cageassembly may allow Sec13/31 the extra time necessary to over-come the steric constraints from the topologically interlockedcages (Fig. 5).

How does Sec23/24 interact with Sec13/31 in the COPII tubule?When Sec13/31 heterotetramers interact to form the tubule, someof the Sec23/24 binding sites are blocked. Sec23/24 binds Sec13/31at the vertices of the cage (Stagg et al., 2008), and two-thirds of thevertices in the tubule overlap with an edge from a neighboringcage (Fig. 3e). However, the remaining one-third of the verticesare free for Sec23/24 binding and lie far enough away from wherethe tubular membrane would be that it would allow Sec23/24 tobind Sec13/31 and have its membrane facing surface be in proxim-ity to the membrane. Thus, the Sec13/31 tubule is well suited forbinding Sec23/24 and a tubular membrane despite blocking some

Fig.5. A hypothetical scenario for the sequential steps of Sec13/31 tubule formationand procollagen transport. (a) Free COPII proteins Sar1 (light blue rectangle), Sec23/24 (yellow), and Sec13/31 (green) next to an ER membrane (dark blue). Within themembrane is the transmembrane protein, TANGO1 (cyan), and inside the lumen ofthe ER is an elongated cargo, procollagen (red). (b) Pre-budding complex at the ERESfacilitated by TANGO1. TANGO1 localizes procollagen to the membrane by bindingto Sec23/24 in the cytosol with a proline-rich domain (PRD) and to procollagen inthe ER lumen with an SH3 domain. (c) Self-assembly of an initial cuboctahedronoccurs around the pre-budding complex. (d) As the collagen exits the ER, themembrane wraps around it forming a tubular shape. The initial cuboctahedron isnow fully assembled. Juxtaposed and between the initial cuboctahedron and the ERmembrane (non-tubular portion) are free Sec13/31 heterotetramers that start toassemble into a second cuboctahedron. (e) A second fully assembled cuboctahedronhas formed around the membrane and is linked with the initial cuboctahedron.Between the second cuboctahedron and the ER membrane (non-tubular portion)are free Sec13/31 heterotetramers which commence the assembly of another linkedcuboctahedron. This process continues until the procollagen molecule is fullyencapsulated.

of the Sec23/24 binding sites on individual Sec13/31 heterotetra-mers. Interestingly, in the endocytic pathway, clathrin has alsobeen shown to polymerize into tubules in the presence of theGGA adaptor molecule (Zhang et al., 2007). Analogously, it is pos-sible in the COPII system that Sec23/Sec24, in combination withTANGO1 and procollagen, promote the formation of a Sec13/31tubular carrier in vivo, though this hypothesis would be quite dif-ficult to test experimentally.

Finally, we note that Sec13/31 may have a potential use innanotechnology. It has now been shown that Sec13/31 can selfassemble into a variety of different macromolecular complexes. Itcan form the 600 Å diameter cuboctahedron, the �875 Å diameterD5 cage, and the 1000 Å diameter icosidodecahedron. Our currentstudy shows that Sec13/31 can self assemble into a chain of inter-locking cuboctahedron cages that form tubules up to 0.75 lm inlength (see Supplementary Fig. 1b). Furthermore, we have previ-ously proposed that Sec13/31 may be able to form planar sheetstructures (Stagg et al., 2008). Careful site-directed mutagenesisof the Sec13/31 residues that form the vertices of the cage struc-tures may give the possibility of directing the self-assembly inmore complex structures that could be useful as nanotechnologicaldevices similar to what is currently being designed using DNA(Aldaye et al., 2008).

4. Methods

4.1. Recombinant protein production and purification

Mammalian Sec13/31 heterotetramers were produced andpurified using a recombinant protein expression system asdescribed elsewhere (Gurkan and Balch, 2005). Briefly, an expres-sion construct encoding human SEC13R and SEC31 Li genes waskindly provided by the laboratory of William Balch. Recombinantco-expression was carried out in Sf9 insect cells. The initial purifi-cation of Sec13/31 was by immobilized metal affinity chromatog-raphy (IMAC) using an N-terminal hexa-histidine tag present onSEC13R. This was followed by anion exchange and size exclusionchromatography, and dialysis of the Sec13/31-rich pool in low saltbuffer (20 mM Tris–Cl, pH 7.5, 300 mM NaCl, 1 mM MgOAc, 10 mMdithiothreitol (DTT)) against assembly buffer (20 mM Tris–Cl, pH7.5, 700 mM KOAc, 1 mM MgOAc, 10 mM DTT) before storage at�80�.

4.2. Analytical ultracentrifugation

Analytical ultracentrifugation experiments were performed in aBeckman XL-I centrifuge (Beckman Coulter, Inc., Fullerton, CA)using absorbance optics by measuring intensity scans at 280 nm.Sample at a concentration of 0.4 O.D (at 280 nm) was preparedfor centrifugation by dialysis into assembly buffer (see above) ex-cept for a substitution of DTT with 10 mM TCEP to prevent exces-sive buffer background absorbance. The experiments wereperformed at 20 �C in two-channel epon centerpieces with anAN60 Ti rotor at 20,000 rpm with no delays between scans. Datawere analyzed using the UltraScan II version 9.9 software suite(Demeler and van Holde, 2004). Data were first analyzed withthe two-dimensional spectrum analysis with simultaneous timeinvariant noise subtraction according to Schuck and Demeler(Schuck and Demeler, 1999). After noise subtraction, the datawas analyzed with the enhanced van Holde-Weischet analysis.Further analysis of noise-corrected data was performed with en-hanced van Holde-Weischet and fitted by genetic algorithmMonteCarlo analysis. The partial specific volume at 20 �C ofSec13/31 sample was 0.7309 cm3/g and estimated from the pep-tide sequence as described by Durchschlag (Durchschlag, 1986).

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All computations were performed on the TIGRE cluster at theUniversity of Texas Health Science Center at San Antonio and theTexas Advanced Computing Center at the University of Texasin Austin.

4.3. Electron microscopy

Vitrified, thin-layered samples were prepared on Quantifoil R2/1 grids (Quantifoil Micro Tools) that had been plasma cleaned for10 s using a Gatan Solarus plasma cleaner (Gatan, Pleasanton,CA). Three microliters of sample was applied to the grids, blottedand plunged into liquid ethane using an FEI Vitrobot in conditionsof 100% humidity and 4 �C. Grids were transferred to a Gatan 626cryoholder (Gatan, Pleasanton, CA) and imaged under lowdose conditions (.7–1.6 e�/Å2) with a Philips (FEI, Eindhoven,Netherlands) CM-300FEG at 300 keV. Single axis tilt series imageswere collected onto a 4096 � 4096 Tietz Tem-Cam F415/MP slowscan CCD (2-fold binning) at a magnification of 24,000� usingthe Leginon automated electron microscopy package (Carragheret al., 2000). At this magnification, the pixel size at the specimenlevel is 9.2 Å. Images were collected at an angular increment of2� and spanned an angular range of ±64�. Each image was CTF cor-rected (phase flipping) using CTFIT of EMAN based on the meandefocus value (Ludtke et al., 1999).

4.4. Image analysis

Images in the tilt series were aligned by cross correlation in thePROTOMO software package. Tomograms were reconstructedusing weighted back projection. To enhance the signal to noise ra-tio, individual volumes were extracted from the tomogram andsubjected to an iterative cycle of alignment and averaging. Here,the volumes were selected from a projection image of the tomo-gram by manually locating the center of each cage within the fila-ment. Only those cages which appeared linked between two cageswere used for averaging. The size of the volume, 80 � 80 � 80, waslarge enough to encompass an entire cage and a portion of eachneighbor. This process was also repeated for 3 full cages. For theinitial round of refinement a global average was used as a refer-ence. To align the cages to this reference, a half-cone width of55� with a step size of 2� was used for an orientation search. Thealigned cages were then averaged to create a 3D map, which wasthen used for a new round of refinement. This process was iterateduntil the averaged 3D map ceased improving in quality. Since thetilt series was confined to an angular range of ±60�, the raw tomo-gram contained a wedge of missing information. If uncorrected, theaveraging would be done with areas that do and do not containdata. To avoid this, the averaging was performed in Fourier spaceand excluded Fourier coefficients that corresponded to the missingwedge. For the final map, D4 symmetry was applied to increase thesignal to noise ratio. A control map (SVcage) was created from twoindividual cages lying outside the filament in the tomogram. Thesewere aligned to one another and octahedrally averaged. A differ-ence map was created using the PROC3D program of EMAN pack-age by subtracting the SVCage from the subvolume averaged map(SVtubule) that had been symmetrized according to D4 symmetry.Prior to subtraction, each map was low pass filtered to 85 Å andnormalized so the mean density level of each voxel was zero andthe standard deviation was 1. It should be noted that althoughthe filament contained interlinked cuboctahedrons, the filamentsubvolume contained D4 symmetry and thus was the appropriatesymmetry to apply. Isosurface map representations were createdwith the molecular graphics program, CHIMERA (Pettersen et al.,2004).

The resolution of the reconstruction was estimated by two dif-ferent methods to be �85–90 Å. In one method the resolution

(85 Å) was approximated by locating the first zero of the 0� tiltedimage with CTFIT program of the EMAN package. To verify thismeasurement, a different method of calculating resolution wasperformed with the program RMEASURE on the SVCage whichmeasures the correlation between neighboring Fourier pixels in asingle reconstruction to distinguish signal from noise (Sousa andGrigorieff, 2007). This measurement estimated the resolution tobe 90 Å.

4.5. Accession Codes

Cryo-ET data corresponding to the original tomogram and sub-volume averaged maps has been deposited in the EMDB withaccession codes EMD-1784, -1785, and -1786.

Acknowledgments

The studies were supported by a grant from the American HeartAssociation (#0835300N). We thank Dr. Joan Hare and MatteusFajer for assistance with the insect cell expression and Dr. ClaudiusMundoma for assistance with the AUC.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jsb.2010.09.002.

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