John Briggs: A closer look at HIV and coated vesicles

Clathrin forms the polyhedral cage of coated vesicles, which mediate the transfer of selected membrane components within eukaryotic cells. Clathrin cages and coated vesicles have been extensively studied by electron microscopy of negatively stained preparations and shadowed specimens. From these studies the gross morphology of the outer part of the polyhedral coat has been established and some features of the packing of clathrin trimers into the coat have also been described. However these previous studies have not revealed any internal details about the position of the terminal domain of the clathrin heavy chain, the location of the 100kd-50kd accessory coat proteins or the interactions of the coat with the enclosed membrane.

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

10.1101/2020.01.28.922591 ◽

2020 ◽

Cited By ~ 1

Author(s):

Oleksiy Kovtun ◽

Veronica Kane Dickson ◽

Bernard T. Kelly ◽

David. J. Owen ◽

John A. G. Briggs

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Protein Composition ◽

Binding Mode ◽

Coated Vesicles ◽

Conformational Rearrangement ◽

Multiple Interfaces ◽

Cryo Electron Microscopy ◽

Subtomogram Averaging ◽

Key Steps

AbstractClathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

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

Science Advances ◽

10.1126/sciadv.aba8381 ◽

2020 ◽

Vol 6 (30) ◽

pp. eaba8381 ◽

Cited By ~ 3

Author(s):

Oleksiy Kovtun ◽

Veronica Kane Dickson ◽

Bernard T. Kelly ◽

David J. Owen ◽

John A. G. Briggs

Keyword(s):

Electron Microscopy ◽

Adaptor Protein ◽

Protein Composition ◽

Binding Mode ◽

Coated Vesicles ◽

Conformational Rearrangement ◽

Multiple Interfaces ◽

Cryo Electron Microscopy ◽

Subtomogram Averaging ◽

Key Steps

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo–electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate)–containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

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Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

Journal of Virology ◽

10.1128/jvi.01453-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9653-9664 ◽

Cited By ~ 22

Author(s):

Gabriela N. Condezo ◽

Roberto Marabini ◽

Silvia Ayora ◽

José M. Carazo ◽

Raúl Alba ◽

...

Keyword(s):

Electron Microscopy ◽

Human Adenovirus ◽

Virus Genome ◽

Proteolytic Processing ◽

Coat Proteins ◽

Human Pathogens ◽

X Ray ◽

Cryo Electron Microscopy ◽

Capsid Shell ◽

Light Particles

ABSTRACTAdenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating.IMPORTANCEAdenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.

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The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy

eLife ◽

10.7554/elife.45345 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Rebecca L Newcomer ◽

Jason R Schrad ◽

Eddie B Gilcrease ◽

Sherwood R Casjens ◽

Michael Feig ◽

...

Keyword(s):

Electron Microscopy ◽

Covalent Binding ◽

Three Dimensional ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Coat Proteins ◽

Binding Interface ◽

Cryo Electron Microscopy ◽

Ob Fold ◽

Icosahedral Capsid

The major coat proteins of dsDNA tailed phages (order Caudovirales) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary ‘decoration’ (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

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The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy

10.1101/420992 ◽

2018 ◽

Cited By ~ 1

Author(s):

Rebecca L. Newcomer ◽

Jason R. Schrad ◽

Eddie B. Gilcrease ◽

Sherwood R. Casjens ◽

Michael Feig ◽

...

Keyword(s):

Electron Microscopy ◽

Covalent Binding ◽

Three Dimensional ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Coat Proteins ◽

Binding Interface ◽

Cryo Electron Microscopy ◽

Ob Fold ◽

Icosahedral Capsid

AbstractThe major coat proteins of dsDNA tailed phages and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary “decoration” (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that precisely distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. Lastly, the NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

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New challenges to image processing posed by cryo-Electron microscopy of single particles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086416 ◽

1991 ◽

Vol 49 ◽

pp. 422-423

Author(s):

Joachim Frank

Keyword(s):

Electron Microscopy ◽

Phase Contrast ◽

Particle Orientation ◽

Single Particles ◽

Contrast Transfer Function ◽

Virtual Absence ◽

Cryo Electron Microscopy ◽

Spatial Frequencies ◽

New Challenges ◽

Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

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Enhanced information from biological materials through technological improvements in cryo-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124343 ◽

1992 ◽

Vol 50 (1) ◽

pp. 788-789

Author(s):

Marc J.C. de Jong ◽

Wim M. Busing ◽

Max T. Otten

Keyword(s):

Electron Microscopy ◽

Electron Beam ◽

Biological Materials ◽

Electron Crystallography ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

The Many ◽

Technological Improvements ◽

Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

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Study of eukaryotic flagellar components by cryo-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142141 ◽

1986 ◽

Vol 44 ◽

pp. 98-101

Author(s):

John M. Murray ◽

Rob Ward

Keyword(s):

Electron Microscopy ◽

Primary Motion ◽

Cryo Electron Microscopy ◽

Cross Links ◽

Cilia And Flagella

The eukaryotic flagellum is constructed from 11 parallel tubular elements arranged as 9 peripheral fibers (doublet microtubules) and 2 central fibers (singlet microtubules). The primary motion generating component has been found to be arranged as axially periodic “arms” bridging the adjacent doublets. The dynein, comprising the arms, has been isolated and characterized from several different cilia and flagella. Various radial and azimuthal cross-links stabilize the axially aligned microtubules, and probably play some role in controlling the form of the flagella beat cycle.

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CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179993 ◽

1990 ◽

Vol 48 (1) ◽

pp. 248-249

Author(s):

John Trinickt ◽

Howard White

Keyword(s):

Electron Microscopy ◽

Atp Hydrolysis ◽

Myosin Head ◽

Bound State ◽

Cross Linking ◽

Weakly Bound ◽

Cryo Electron Microscopy ◽

Myosin Subfragment 1 ◽

Attached State ◽

High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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