Probing Intracellular Vesicle Trafficking and Membrane Remodelling by Cryo-EM

Abstract

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins’ structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.

Graphical Abstract

Graphical Abstract: Schematic representation of vesicle trafficking and coat protein complexes. Teal box) Overview of the major players in the endocytic and secretory pathways along with inset magnified boxes. The colors of the COPI, COPII, and clathrin coats match with the colors of their respective boxes. Blue box) COPII components and their organization. Red box) COPI components and their organization. Yellow box) Clathrin components and their organization. Figure was sketched in Biorender (https://biorender.com/). Abbreviations used in this figure: endoplasmic reticulum: ER; trk-fused gene: TFG; coat protein: COP; ER exit sites: ERES; ER-Golgi intermediate compartment: ERGIC; lysosome: Lys, multivesicular bodies: MVBs; endosomes: Endo; plasma membrane: PM.

Membrane and protein trafficking in eukaryotes is classified into two major pathways: i) the secretory pathway through which the newly synthesized proteins are transported from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane (PM), and ii) the endocytic pathway where the internalized molecules are delivered to early endosomes for sorting¹. Three canonical coat protein complexes have been identified that transport cargo from the ER to the Golgi apparatus, known as anterograde transport (COPII), between Golgi stacks and from the Golgi to the ER, known as retrograde transport (COPI), and from the plasma membrane to endosomes, known as endocytosis (clathrin). The coat protein structures are highly pleiomorphic and the structures of many of their components have been determined by X-ray crystallography. However, cryo-EM has played a critical role determining the structures of the complexes as complete coats and determining how they interact with and remodel membrane. Here we review the structures of the membrane trafficking complexes and take a look at future technologies that will enable the structures to be determined in their native state in situ.

Anterograde Transport by COPII

Coat protein complex II (COPII-coated vesicle) facilitates the export from the ER exit sites (ERESs) to the ERGIC or the cis-Golgi compartments². The protein coat for COPII vesicles consists of a set of five cytosolic proteins: Sar1-GTP, dimeric Sec23/Sec24, and tetrameric Sec13/Sec31³. COPII coat formation is initiated when the guanine nucleotide exchange factor Sec12 protein (Sec12-GEF) catalyzes the exchange of GDP to GTP on Sar1 at the ER membrane. Exchange of GDP to GTP on Sar1 induces a conformational change of the Sar1 N-terminal α-helix that embeds in the ER membrane³. ER-embedded Sar1-GTPase mediates the recruitment Sec23/Sec24 to form the inner layer of the COPII coat. The Sec23/24-Sar1 prebudding complex recruits Sec13/31 that oligomerizes into a geometrical lattice clustering the Sec23/24-Sar1 complexes into a bud that is eventually cleaved to form a coated vesicle.

X-ray crystal structures have been determined for each of the COPII components^4–6. Sar1 has a typical structure of the Ras-family GTPases with Switch I and II domains and an N-terminal amphipathic α-helix that becomes membrane accessible in the GTP state^4,5 (Fig. 1A,B). Sec23 and Sec24 are structurally homologous, and they interconnect through the β -sheet structure between their trunk domains⁵ (Fig. 1C,D). Sec13 is a single domain β-propeller fold, with six WD40 repeat motifs, while Sec31 contains an N-terminal WD40 domain, followed by an extended α-solenoid fold⁶ and a large disordered region in the C-terminal (Fig. 1E–G).

Fig. 1. COPII Coat Structure.

A) Structure of Sar1-GTP from PDB: 1M2O⁵. (Switch I: purple, Switch II: pink, Linker: orange). B) Structure of Sar1-GDP from PDB:1F6B⁴ (N-terminal α-helix: red). C) Superposition of Sec23: blue, and Sec24: green and their domains, indicating the homology between two structures. Dashed lines show the domains (Trunk: red, Zn-finger: khaki, β-barrel: pink, Helical: orange, and Gelsolin-like: dark blue). The Sec23-TFG binding domain is shown in light blue/yellow. D) Structure of Sec23/24 (blue/green) in complex with Sar1 (gold). Sec23 and Sec24 connect through the beta-sheet structure of their trunk domain and form a bowtie-shaped dimer5. E) WD40 domains of Sec13 and Sec31 (the seventh blade of Sec31 is donated to the 6-bladed β-propeller of Sec13). Sec13 is colored pink, and Sec31 is purple. F) The α-solenoid motifs of two Sec31s that dimerize by folding back on each other and form a continuous bundle. G) Overall structure of the Sec13/31 heterotetramer (Sec 13 is shown in pink and salmon. Sec31 is shown in purple and yellow). H) Single-particle reconstruction of self-assembled Sec13/31 heterotetramer⁷ (cuboctahedron at 12 Å-resolution⁸) that showed the cage structure and that Sec23/24 is not necessarily part of cage assembly⁷. I) Atomic model of the cage vertex fitted in cryo-EM map. Four different contact surfaces (cI-cIV) were predicted between for Sec31 WD40 domains and the four Sec13 WD40 domains⁶. The Sec13 subunits have minimal contacts to give the cage flexibility⁸. J) The icosidodecahedron structure of the COPII coat and cage solved by single-particle cryo-EM⁹. K) Slice through the cryo-tomogram of COPII-coated tubule indicating densities of both the inner and outer coat lattices (scale bar is 50 nm). L) Schematic representation of the Sec13/31 subunit forming a lozenge pattern around the inner coat¹⁰. M) Top and side view of the 12 Å sub-tomogram average of COPII vertex¹⁰ with atomic models (PDB:2PM6⁶ and 2PM9⁶) fitted by the rigid body (Sec31: firebrick and orange, and Sec13: grey). The contacts regions are slightly different from the structure fitted in the SPA map (panel I). N) Zoomed region of the 4.9 Å cryo-ET of the membrane-assembled COPII inner coat¹¹ locally filtered (transparent) along with the fitted atomic models (PDB: 1M2O⁵ and 1M2V⁵) O) 3D map of the N-terminal domain of human TFG (EMD:6079¹⁶), reconstructed by random conical tilt. The reconstruction shows that TFG octamers assemble into a cup-shaped structure in vitro (scale bar is 25 Ź⁶. Panels A to J and O are generated in ChimeraX²⁶. Panels K to M are adopted with permission from reference¹⁰ (requested). Panels N to is adopted with permission from reference¹¹ (requested).

While crystallography revealed the structures of the individual COPII subunits, cryo-electron microscopy (cryo-EM) elucidated how the proteins come together to form a coat and interact with membrane. COPII forms various morphologies from spherical cages to tubular structures to enclose cargoes of different shapes and sizes. Single-particle cryo-EM showed that Sec13/31 heterotetramers oligomerize on their own to form symmetric cage-like structures⁷. In the absence of other factors, the Sec13/31 cage formed in the geometry of a cuboctahedron⁷ with an average diameter of ~600 Å with octahedral symmetry consisting of 24 edges and 12 vertices (Fig. 1H)⁷,⁸. In the presence of Sec23/24, the COPII structure forms a 1000 Å icosidodecahedron with 60 edges and 32 faces composed of an inner Sec23/24 layer and an outer Sec13/31 layer (Fig. 1J)⁹. For both the cuboctahedral and icosidodecahedral structures, the interactions between four Sec13/31 heterotetramers mediate the assembly of the COPII cages at the vertices (Fig. 1I,H). The inner and outer layers of COPII interact via several interfaces, and single-particle analysis showed that binding of Sec23/24 beneath the Sec13/31 vertices can influence the diameter of the cage by changing the angles between adjacent edges⁹. Cryo-electron tomography (cryo-ET) extended these results and revealed that in the presence of the membrane, Sar1-Sec23/24 assembly alone can drive membrane remodelling into extended tubular structures^10,11 that can be decorated with a geometric Sec13/31 coat (Fig. 1K–N). In these COPII tubules, the two layers form separate, distinct lattices, and the outer coat builds a rhomboidal lattice where Sec31 N-terminal β-propeller interacts to form a lozenge pattern¹⁰ (Fig. 1L). It remains to be determined what triggers the change from the tubular-like structures to spherical vesicle-like structures observed in cells.

Scission of COPII vesicle was initially suggested to be driven by the conformational change of the Sar1 N-terminal α-helix¹². Further studies suggested that GTPase Sar1 constricts vesicle necks on the ER membrane by disrupting local lipid packing to promote vesicle scission¹³. The exact role that Sar1 plays in scission remains controversial. A model studied by cryo-EM suggested that Sar1 oligomerizes into a lattice of dimers on membranes with low curvature to constrict the neck of nascent buds and induce scission¹⁴. However, the atomistic details of the Sar1 lattice and its role in vesicle scission have yet to be determined.

COPII-coated vesicles are uncoated to fuse with the target membrane. One protein that is involved in both COPII trafficking and uncoating is the trk-fused gene (TFG). TFG binds the Sec23 through its C-terminus and competes with Sec31 binding, and it ultimately helps displace the outer coat of the vesicle and helps mediate binding of the inner coat to the ERGIC¹⁵. The structure of the assembly unit of TFG was revealed by 3D electron microscopy showing that the N-terminal of TFG oligomerizes into a cup-shaped octamer (Fig. 1O) via interactions of its N-terminus PB1 and coiled-coil domains¹⁶. Whereas the C-terminus proline/glutamine (PQ)-rich regions contribute to form a homo-oligomeric protein complex which assembles into a meshwork between ER and ERGIC regions¹⁵.

Retrograde Transport by COPI

Coat protein complex I (COPI-coated vesicles) mediate the retrieval of proteins from the cis-Golgi to the ER. Analogous to the Sar1 mechanism in recruiting COPII, Arf1-GTP functions to recruit COPI to the Golgi membrane. However, unlike COPII, Arf1-GTP recruits the COPI proteins to the membrane as an en-bloc heteroheptameric complex, known as coatomer¹⁷. COPI coatomer is composed of two interconnected subcomplexes, i) a trimer that is composed of ε (Sec28), α (Ret1), and β’ (Sec27) subunits and ii) a tetramer that is composed of β (Sec26), γ (Sec21), δ (Ret2) and ζ (Ret3)¹⁸.

Crystal structures of each subunit have been determined, and cryo-ET enabled the reconstruction of the COPI subunits assembled together as a coat on the membrane^17–19. In contrast to COPII and clathrin, COPI does not form a regular geometric coat on membranes. Instead, the COPI coatomer forms a leaf-like structure (Fig. 2A–D). The leaves come together in four unique linkages (Fig. 2G, H) that decorate the surface of the vesicle, making a continuous coat but with no underlying geometrical pattern. The leaves, along with three duplicates of Arf1, assemble into a three-fold triad to form the repeating unit of the COPI. Two subcomplexes of coatomer configure intertwined arches in each triad to shape the COPI vesicles in various morphologies¹⁷. A crystal structure of βδ-COP and ArfGAP2, which regulates Arf1 GTPase activity, in association with coatomer were solved which provided further insights into the overall topology of the COPI leaf (Fig. 2D–F). In that study, the leaf structure was resolved by sub-tomogram averaging (STA) to higher resolution (9 Å) and the atomic models were generated that describe the four linkages that form the basis for interactions mediating the formation of COPI coated vesicles¹⁸ (Fig. 2G,H).

Fig. 2. COPI Coat Structure.

A) Cryo-ET reconstruction of the COPI leaf at 9.2 Å-resolution at the side (left) and top view (right). Densities are colored by distance from the membrane: from orange (closest to the membrane) to dark blue (furthest to the membrane) (EMDB: 3720¹⁸). B) Cryo-ET reconstruction of the COPI leaf with an extra density corresponding to the ArfGAP2: dashed box (EMDB: 3721¹⁸). C) A structural model of the COPI coat after flexible-fitting and homology modeling of the structures: α-COP: purple, β-COP: light coral, γ-COP: yellow and lime, ζ-COP: grey, β’-COP: cyan, δ-COP: dark blue, Arf1: pink (PDB: 5NZR¹⁸). α-/ε-COP C-terminus and the δ-COP MHD are not visible since they constitute inner-triad linkage. D) Structural model of the COPI coat leaf in the presence of Arf-GAP2: gold (PDB: 5NZS¹⁸). E) 2.57 Å crystal structure of βδ-COP (PDB: 5MU7¹⁸) was used to solve COPI leaf’s complete atomic model. F) Zoomed view of Arf-GAP2 structure. The domain is positioned near the Arf1-nucleotide-binding site. G) Cryo-ET structures of COPI linkages. Linkage I to IV are shown from left to right at 17, 15, 30, and 17.3 Å resolution, respectively (Linkage I: EMD 3722¹⁸, Linkage II: EMD 3723¹⁸, Linkage III: EMD 2988¹⁷, Linkage IV: EMD 3724¹⁸). Maps are colored by distance from the membrane: from orange (closest to the membrane) to dark blue (furthest to the membrane). Linkage III was the least abundant. H) Atomic models of the linkages I-VI (PDBs: 5NZT¹⁸, 5NZU¹⁸, 5A1X¹⁷, 5NZV¹⁸). Structures are colored according to panel-B’s theme, and ε-COP is shown in orange. The subunits that mediate the primary contacts are αε-COP (purple-orange), and δ-COP MHD (dark blue)^17,18.Panels are generated in ChimeraX.

Clathrin-mediated trafficking

Clathrin-coated vesicles (CCVs) are involved in various trafficking routes, including endocytosis and Golgi-endosome trafficking. Analogous to COPII vesicles, CCVs form a geometrical lattice composed of two distinctive layers. The outermost layer is a triskelion with three copies of clathrin heavy chains (CHCs), each bearing a clathrin light chain (CLC). The clathrin triskelion is the repeating unit of CCVs, and copies of triskelia come together to form polygonal cages with pentagonal and hexagonal faces with various geometries and sizes (Fig. 3A). The C-terminal domain of clathrin heavy chains is centered on the vertices of the cage followed by long α-solenoid legs that project inwards and intertwine with legs from adjacent vertices. The N-terminal domain of the clathrin heavy chain is a β-propeller that possesses binding sites for adaptor proteins (AP1–4) as well as other mediating proteins. The inner layer of CCVs is formed from adaptor proteins, which interact with membrane and cargo proteins²⁰. Each membranous compartment has its own cognate AP for CCV formation.

Fig. 3. Architecture of Clathrin.

A) Cryo-EM structure of clathrin coats in 6 geometries. Top row from left to right, tetrahedral mini coat at 8.5 Å resolution (EMDB: 21608²⁴), D3 football at 15.1 Å (EMDB: 21612²⁴), C2 basket, which is an unprecedented geometry at 15.6 Å (EMDB: 21610²⁴), and bottom row from left to right: D2 tennis ball at 13.75 Å (EMDB: 0118²³), D6 barrel at 12.18 Å (EMDB:0116²³), and big apple at 23.68 Å resolution (EMDB: 0120²³)^23,24. B) Sub-particle refinement of tetrahedral coat asymmetric vertex, which improved the resolution to 6.3 Å, is shown at the top and side views. In the side view, one CHC and CLC were removed to show the trimerization helices. Structure is colored based on the different domains²⁴. C) Densities corresponding to the β2 appendage (shown by red arrow) appear in the hexagonal faces of the clathrin mini-coat (EMDB: 21609²⁴), whereas the pentagonal faces lack the β2 appendage²⁴. D) EM map (EMD: 10754²¹) of clathrin legs in agreement with panel C observation, indicating that the β2 appendage is at higher occupancy in hexagonal faces²¹,along with magnified view of the β2 appendage and fitted atomic model (PDB: 6YAI²¹). E) Left: The atomic model of the full vertex colored by chain (PDB: 6WCJ²⁴); middle: the unique interactions of the two CHC distal legs (green and cyan) with the trimerization domain of a third CHC (magenta) highlighted in the same atomic model; top right: a side view of the same model, and bottom right: the three interacting CHCs²⁴. Transparent density removed for better visualization.F) EM density for the tripod interaction (left) and the atomic model (right), which shows the position of the QLML residues of the QLMLT motif that is recognized by auxilin for clathrin uncoating extending down from the trimerization helix. G) Cryo-EM micrograph of helical dynamin tubes (left) and the 3D map of the membrane assembled dynamin in the GTP-bound state at 3.75 Å (right), EMDB: 7957²⁵. H) Model of tetramer dynamin with surrounding density and domains (GTPase domain: green: BSE: pink, Stalk: blue, PHD: gold). The assembly interfaces are labeled 1–3, PDB: 6DLU²⁵. Panels A to D are generated in ChimeraX. Panels E and F are adopted with permission from reference²⁴ (requested). Panel G and H are adopted with permission from reference²⁵ (requested).

AP2 is the most abundant clathrin adaptor and mediates the endocytosis from the plasma membrane. AP2 like the rest of the APs is a heterotetrameric complex consisting of four subunits; α, β2, μ2, and σ2²¹. It acts as a binding scaffold for various proteins, including endophilin, amphiphysin BAR proteins, and Eps15, facilitating CCVs invagination²². To generate the CCVs, first, the α-adaptin subunit of AP2 binds the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂ ) on the parent membrane. Next, the β2 subunit of AP2 recruits clathrin to induce the assembly of the polyhedral coat. Meanwhile, the μ2 subunit interacts with the Yxxφ sequences of cargo membrane proteins and assembles them in the clathrin-coated regions²².

Structures of the individual CCV components have been determined through a host of crystallographic studies. Recent cryo-EM studies have combined the different components to create a coherent picture for the CCV’s structure. Near-atomic resolution reconstruction of the clathrin cage vertices solved by single-particle cryo-EM, revealed the interactions between the CHC and CLC and provided insight into the mechanisms of CCVs (Fig 3B/E and F)^23,24 including revealing the position of the QLMLT motif that is recognized by auxilin for clathrin uncoating (Fig. 3E,F). Two different studies illustrated the interaction of AP2 and clathrin together on the membrane. Both studies agree that AP2 β2 has a preferential binding to hexagonal faces (Fig. 3C,D)^21,24, while the curvature of the clathrin is more determined by the pentagons²⁴. One difference, however, is that the two studies show the binding of the β2 appendage domain to slightly different regions of clathrin which could indicate differences in the maturation of the CCVs in the two studies (Fig. 3C, D). The conformational change of AP2 when it binds the membrane protein PtdIns(4,5)P₂ to unlock the cargo binding site was also observed²¹. Lastly, the structures of natively assembled CCVs in various geometries, including an unprecedented cage geometry with C2 symmetry (C2 basket) was revealed by cryo-EM SPA (Fig. 3A)²⁴.

The final stage of CCV formation is scission where the neck of the CCV bud must be constricted such that the membrane comprising the bud neck fuses, freeing the nascent vesicle. The major protein involved in neck constriction is the GTPase dynamin. Dynamin binds PtdIns(4,5)P₂ and forms a helical collar around the bud neck. Subsequently, GTP hydrolysis of the dynamin narrows the neck and induces coated vesicles scission of the membrane. Kong et al.²⁵ have reconstructed the 3.75 Å resolution structure of the membrane-associated dynamin polymer by cryo-EM. The structure displays the helical symmetry of dynamin 1 bound to a nonhydrolyzable GTP analog and the locations of its oligomeric interfaces. This high-resolution reconstruction provides a detailed molecular model of the dynamin tetramer (Fig. 3G,H), and shows how dynamin binds to the membrane via the pleckstrin homology domain (PHD) and how conformational changes at the GTPase and bundle signalling element (BSE) domains drive constriction in response to GTP hydrolysis²⁵.

Membrane Trafficking Visualized in situ

Cryo-EM has revealed the higher-order structures of the COPII, COPI, and clathrin vesicle trafficking complexes in vitro. However, the knowledge of the structures in their native states inside cells is missing. Recently focused-ion-beam (FIB) milling has been applied to cryogenically prepared whole cells and has enabled cryo-EM structure determination of macromolecular complexes in their native contexts. FIB milling functions by irradiating samples with a focused ion beam, usually gallium (Ga⁺), in a multistep process. This stepwise irradiation ablates material in the path of the ion beam and enables the preparation of a 200–400 nm thin layer of material (lamella), that can be imaged by TEM²⁷. The technique is still in its infancy and is prone to various limitations²⁸. In recent years, studies have demonstrated the suitability of this technique to determine the in situ structure of proteins at near-atomic resolution. However, the number of studies that use cryo-FIB milling to determine the structure of vesicle coats and membrane remodelling components in situ is limited to date. Among the present in situ studies of vesicle trafficking components, cryo-FIB-milling was used to observe the structure of the COPI coat in the process of a cargo transport between the Golgi stacks¹⁹. The native morphology of the ER, Golgi apparatus, and the three characteristic protein coats were also shown (Fig. 4A–E). Through this study, it was noted that the cargo binds beneath β’ COP (Fig. 4H). Furthermore, the morphology, and cargo components of COPI vesicles vary through their transport between cis and trans-Golgi.

Fig. 4. Membrane trafficking in situ.

A) Architecture of the Chlamydomonas reinhardtii Golgi apparatus and transport vesicles by cryo-ET: a slice through a tomogram showing a representative Golgi stack¹⁹. B) Corresponding 3D segmentation, displaying the in situ morphology of organelles. ER is colored yellow. Four cis cisternae colored green, cis vesicles in light green, four medial cisternae in magenta, and medial vesicles colored light pink. The trans cisterna is blue, trans vesicles is cyan, and the trans-Golgi-network (TGN) is purple. Other membranes, the nuclear envelope, nuclear pore complexes, and ribosomes are colored in grey. C–E) Slices through tomograms: side views of C: clathrin, D: COPII, and E: COPI coated transport vesicles. Yellow arrowheads indicate the cytoplasmic boundary of the coat, and blue arrowheads point to vesicle or bud membrane. Scale bars are 200 nm in A-B and 50 nm in C-E¹⁹. F–I) Structure of the native COPI coat structure: F) Isosurface view of the COPI triad density map within the cell (EMDB: 3968¹⁹): cytoplasmic side view, along with the COPI model (PDB: 5A1U¹⁷). Dashed rectangle indicates the position of cross-sectional view in panel K¹⁹. G) Vesicle lumen side, showing three cargo density protrusions. H) Cross-sectional view, indicating the position of the cargo density: shown by black arrowhead relative to the N-terminal β–propellers of α–COP position: shown by purple arrowhead and β’-COP: shown by cyan arrowhead. I) Examples of coat arrangement on vesicles and buds in situ. In each row, tomographic slice are shown on the left; tomographic slice overlaid with triangles at the positions of each triad: middle (colored green to red corresponding to the correlations: high to low); rotated view of the triad indicating the budding: right¹⁹. The vesicle in the last row is an uncomplete coat and most likely indicating the uncoating. Scale bar is 50 nm. J–M)¹⁹ Organization of the retromer–Vps5 coats on membrane tubules. J) A slice through tomographic reconstructions of a C. reinhardtii cell in with retromer-coated membranes shown by arrowheads. K) Zoomed view of two retromer-coated membranes that form arches. L) 35 Å maps of the retromer structures determined by STA in situ (within the cell) and in vitro. M) Ribbon model of the retromer–Vps5 complex superimposed on EM density map, from an intermediate STA alignment indicating the two-fold dimeric interface formed by Vps26, and how the coat can propagate around the membrane tubule (Vps35: yellow, Vps29: red, Vps26: green, and Vps5: blue)²⁹. N) A retromer–Vps5-coated tubule: a model of the Vps5–Vps26 layers (on the left), and the complete coat (in the middle and on the right, respectively showing the side view and along the tube axis view). Structures were solved by STA. Magnified segments of retromers are shown corresponding to the dashed boxes. Vps26 dimers are placed in six relative positions on the underlying Vps5 array, as shown by pink arrows. Panels A to E, and I are adopted with permission from reference¹⁹ (requested). Panels F to H are generated in ChimeraX. Panels J to N are adopted with permission from reference²⁹ (requested).

In another study, cryo-ET and subtomogram averaging (STA) were used to provide the molecular details of the reconstituted retromer complex in association with the membrane²⁹. Retromer complex contributes with BAR domains to generate cargo-selective tubulovesicular carriers from endosomal membranes. By using cryo-FIB milling of Chlamydomonas reinhardtii cells the authors identified and compared the structure of sorting nexin protein Vps5 bound to retromer complex in situ with its in vitro model (Fig. 4J–M). Both models suggest that the retromer forms an arch-shaped arrangement that extends away from a membrane-associated Vps5 array. Vps35 builds the legs of the arch, and Vps29 forms the tip of the arch and interconnects with regulatory factors. Vps26 connects the neighboring arches together and contacts with Vps5 on the membrane (Fig 4M, N). This study provides an insight on how retromer promotes tubule formation by directing the distribution of sorting nexin (Vsp5)²⁹.

Conclusion

The structures of COPII, COPI, and clathrin-coated vesicles have been studied for over a decade, yet there is still much to learn about their structures, mechanisms, and dynamics. New discoveries in recent years have been facilitated through a combination of multiple technologies. X-ray crystallography combined with cryo-EM and mass spectrometry have been particularly powerful in elucidating the complicated and flexible structures of the coat complexes^8,18,24. As the field progresses, even more advanced technologies will be required to determine the high-resolution details of how the proteins assemble in vivo and interact with membrane and cargo. Electron tomography and cellular cryo-EM by cryo-FIB milling are expected to yield many new insights about these processes. To date, cryo-FIB milling has enabled the visualization of CPCs and associated proteins in the native environment of the cells. Despite this successful application, the number of studies using this technique to solve the structure of protein trafficking components is limited due to the complexity of the method, leaving us with minimal knowledge of the structural details of membrane trafficking in vivo. Several research groups are working on establishing workflows to improve the throughput and application of cryo-FIB in structural biology^28,30. As the FIB milling technology matures, there is enormous potential for the technique to provide novel insights into the mechanisms of vesicle trafficking.