Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

Oleksiy Kovtun, Veronica Kane Dickson, Bernard T. Kelly, David. J. Owen, John A. G. Briggs

Preprint posted on 28 January 2020

Article now published in Science Advances at


The Structures of Natively Assembled Clathrin Coated Vesicles

Mohammadreza Paraan, Joshua Mendez, Savanna Sharum, Danielle Kurtin, Huan He, Scott M. Stagg

Preprint posted on 29 January 2020

Article now published in Science Advances at

Coat coterie: Two preprints from Kovtun et al. and Paraan et al. show us high resolution glimpses of clathrin and friends, assembled on membranes.

Selected by Stephen Royle

Categories: cell biology


Endocytosis is the process cells use to internalize macromolecules by forming transport vesicles from the plasma membrane. This process is crucial to many aspects of cell biology because it controls many core functions including cell excitability, cell signalling, and more. The main transport vesicle formed during endocytosis is the clathrin-coated vesicle (CCV). The basic anatomy of a CCV is a cargo-laden vesicle inside a coat. The coat is composed of an outer layer of clathrin, arranged in a beautiful geodesic array of hexagons and pentagons, and an inner layer of adaptors and other proteins that allow clathrin to indirectly contact the cargo and the vesicle.

The quest to define the mechanism of CCV formation using structural methods has a long history. Back in 1969, Kanaseki and Kadota used electron microscopy to describe each CCV as a “vesicle in a basket”1 and since this time, researchers have followed two paths. In the first, atomic resolution structures of CCV proteins have been obtained predominately using X-ray crystallography. These structures, of individual domains, whole proteins and even some multi-protein complexes have provided valuable snapshots of how the endocytic machinery may go about its job. In the second, much larger assemblies, such as the clathrin cage itself, have been viewed at progressively higher resolution using electron microscopy. What has been missing, until now, was a convergence of these two paths.

What does the assembled clathrin coat on a vesicle look like? Two new preprints use cryo-electron microscopy (cryoEM) to understand CCV assembly. They each use complementary samples and approaches to provide new insight into this fascinating question.


Experimental model of a clathrin-coated bud formed on a membrane that contains a cargo-like molecule. Taken from Kovtun et al. 2020 and made available under a CC-BY-NC-ND 4.0 International license.


Key findings

Kovtun et al. used a synthetic strategy to assemble clathrin coats on membranes using purified components2. They took a membrane which contained PtdIns(4,5)P2 and a cargo-like molecule, added a form of AP2 (the main plasma membrane adaptor) and, of course, clathrin. This protein concoction caused clathrin-coated buds to form on the membrane and these invaginations could then be studied by cryoEM, using tomography and subtomogram averaging. Over the last two decades, David Owen’s group have used crystallography to study the AP2 complex in various conformations. Now, together with John Briggs’ tomographic analyses, a view of AP2 posed at the membrane in complex with cargo and relative to clathrin is revealed. We can see for the first time how AP2 interacts with the membrane via multiple PtdIns(4,5)P2 interactions, and adopts an open conformation which is compatible with cargo binding. This is all happening under the outer layer of clathrin. While the clathrin cage has a regular structure, the underlying AP2 complexes are not organised in a regular lattice-like fashion.

Paraan et al. started with clathrin-coated vesicles isolated from bovine brains3. Using cryoEM and single particle analysis we see high-resolution views of the clathrin coat. The power of this approach is in the use of a native sample. Scott Stagg’s group found a range of clathrin cage types, which echoed recent work on clathrin cages assembled in vitro by Corinne Smith’s lab4. In fact, they observed a new type of clathrin geometry, dubbed the C2 cage, which appears to break the head-to-tail exclusion model for cage assembly; a reminder that biology is a messy business where rule-breaking is the norm. Because this is a native sample, AP2 and a number of other proteins are present in the CCVs. This means that there is some uncertainty in assigning densities to certain molecules. Nevertheless, density which matches the size and shape of AP2 could be resolved in the inner layer of the coat.

AP2 has two appendage domains on the alpha and beta2 subunits which attach to the core heterotetramer via long, flexible hinges. We know that beta2 engages clathrin, but what these studies now reveal is how this happens in an assembled coat. Both studies find the beta2 appendage to be present in the hexagonal faces rather than the pentagonal ones. Since an enclosed cage must contain twelve pentagons, the creation of pentagons is key to driving vesicle curvature. The preference of beta2 appendage for hexagonal versus pentagonal faces may explain why AP2 drives vesicle curvature via clathrin, even though it has no curvature-generating properties by itself.

There is a wealth of further detail in these studies that will satisfy the clathrin aficionados (like me!). For example, the beta2 appendage position relative to the clathrin legs is different in each study. Again, highlighting that any “rules” for clathrin engagement by AP2 may be easily broken.


What I liked about these preprints

Both preprints represent a significant step forward in our understanding of CCV formation. The work underlying this advance is very technically challenging. Clathrin assemblies are large, flexible and heterogeneous. All three properties make structural approaches – which rely on averaging – much more difficult. The complementarity of the two studies is nice: Paraan et al. used native material and single particle methods, while Kovtun et al. went for a reductionist approach and tomographic imaging.

CryoEM has revolutionised structural biology. We are now at an exciting point where these high-resolution views of complex samples are not only possible, but also have the power to reveal so much, thanks to years of previous work in crystallography, cell biology and biochemistry. These preprints are perfect examples of the point that we’ve reached.



I would like to thank Sarah Smith and Katie Wood from Corinne Smith’s lab for valuable discussions around these preprints.



  1. Kanaseki & Kadota (1969) The “vesicle in a basket”. A morphological study of the coated vesicle isolated from the nerve endings of the guinea pig brain, with special reference to the mechanism of membrane movements. J Cell Biol, 42, 202-20.
  2. Kovtun et al. (2020) Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles. bioRxiv, 2020.01.28.922591.
  3. Paraan et al. (2020) The Structures of Natively Assembled Clathrin Coated Vesicles. bioRxiv, 2020.01.28.923128.
  4. Morris et al. (2019) Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self-assembly. Nat Struct Mol Biol, 26, 890-898.

Tags: ap2, clathrin, cryoem, endocytosis

Posted on: 24 February 2020


(No Ratings Yet)

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023


List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.


List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.


List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23


List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.


List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.


List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.


List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage