scholarly journals Structure of AAV-DJ, a Retargeted Gene Therapy Vector: Cryo-Electron Microscopy at 4.5 Å Resolution

Structure ◽  
2012 ◽  
Vol 20 (8) ◽  
pp. 1310-1320 ◽  
Author(s):  
Thomas F. Lerch ◽  
Jason K. O'Donnell ◽  
Nancy L. Meyer ◽  
Qing Xie ◽  
Kenneth A. Taylor ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gene Therapy ◽  
Gene Therapy Vector ◽  
Cryo Electron Microscopy

scholarly journals Adeno-associated Virus Receptor-binding: Flexible Domains and Alternative Conformations through cryo-Electron Tomography of AAV2 and AAV5 complexes

2022 ◽  
Author(s):  
Guiqing Hu ◽  
Mark A Silveria ◽  
Michael S Chapman ◽  
Scott M Stagg
Keyword(s):  
Electron Microscopy ◽  
Gene Therapy ◽  
Viral Entry ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Fold Axis ◽  
Adeno Associated Virus ◽  
Gene Therapy Vector ◽  
Molecular Virology ◽  
Cryo Electron Tomography

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE: With 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.

scholarly journals Cryo-EM Structure of Adeno-associated virus-4 at 2.2 Å resolution

2022 ◽  
Author(s):  
Grant M Zane ◽  
Mark A Silveria ◽  
Nancy L Meyer ◽  
Tommi A White ◽  
Michael S Chapman
Keyword(s):  
Electron Microscopy ◽  
Gene Therapy ◽  
Variable Region ◽  
Cell Receptor ◽  
Polycystic Kidney ◽  
Adeno Associated Virus ◽  
Variable Regions ◽  
Virus Receptor ◽  
Cryo Electron Microscopy ◽  
Viral Surface

Adeno-associated virus (AAV) is the vector of choice for several approved gene therapy treatments and is the basis for many ongoing clinical trials. Various strains of AAV exist (referred to as serotypes), each with their own transfection characteristics. Here, we present a high-resolution cryo-electron microscopy structure (2.2 Å) for AAV serotype 4 (AAV4). The receptor responsible for transduction of the AAV4 clade of AAV viruses (including AAV11, 12 and rh32.33) is unknown. Other AAVs interact with the same cell receptor, Adeno-associated virus receptor (AAVR), in one of two different ways. AAV5-like viruses interact exclusively with the polycystic kidney disease-like [PKD]-1 domain of AAVR while most other AAVs interact primarily with the PKD2 domain. A comparison of the present AAV4 structure with prior corresponding structures of AAV5, AAV2 and AAV1 in complex with AAVR, provides a foundation for understanding why the AAV4-like clade is unable to interact with either PKD1 or PKD2. The conformation of the AAV4 capsid in variable regions I, III, IV and V on the viral surface appears to be sufficiently different from AAV2 to ablate binding with PKD2. Differences between AAV4 and AAV5 in variable region VII appear sufficient to exclude binding with PKD1.

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

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.

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

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.

Study of eukaryotic flagellar components by cryo-electron microscopy

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.

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

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.

Lipid Polymorphism and Virus-Membrane Fusion as Observed in Vitrified Thin Films by Cryo-Electron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 492-493
Author(s):  
P.M. Frederik ◽  
K.N.J. Burger ◽  
M.C.A. Stuart ◽  
A.J. Verkleij
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Membrane Fusion ◽  
Film Formation ◽  
Molar Ratio ◽  
Influenza B ◽  
Complex Structures ◽  
Type Ii ◽  
Suspended Material ◽  
Cryo Electron Microscopy

Cellular membranes are often composed of phospholipid mixtures in which one or more components have a tendency to adopt a type II non-bilayer lipid structure such as the inverted hexagonal (H||) phase. The formation of a type II non-bilayer intermediate, the inverted lipid micel is proposed as the initial step in membrane fusion (Verkleij 1984, Siegel, 1986). In the various forms of cellular transport mediated by carrier vesicles (e.g. exocytosis, endocytosis) the regulation of membrane fusion, and hence of inverted lipid micel formation, is of vital importance.We studied the phase behaviour of simple and complex lipid mixtures by cryo-electron microscopy to gain more insight in the ultrastructure of different lipid phases (e.g. Pβ’, Lα, H||) and in the complex membrane structures arising after Lα < - > H|| phase changes (e.g. isotropic, cubic). To prepare hydrated thin films a 700 mesh hexagonal grid (without supporting film) was dipped into and withdrawn from a liposome suspension. The excess fluid was blotted against filter paper and the thin films that form between the bars of the specimen grid were immediately (within 1 second) vitrified by plunging of the carrier grids into ethane cooled to its melting point by liquid nitrogen (Dubochet et al., 1982). Surface active molecules such as phospholipids play an important role in the formation and thinning of these aqueous thin films (Frederik et al., 1989). The formation of two interfacial layers at the air-water interfaces requires transport of surface molecules from the suspension as well as the orientation of these molecules at the interfaces. During the spontaneous thinning of the film the interfaces approach each other, initially driven by capillary forces later by Van der Waals attraction. The process of thinning results in the sorting by size of the suspended material and is also accompanied by a loss of water from the thinner parts of the film. This loss of water may result in the concentration and eventually in partial dehydration of suspended material even if thin films are vitrified within 1 sec after their formation. Film formation and vitrification were initiated at temperatures between 20-60°C by placing die equipment in an incubator provided widi port holes for the necessary manipulations. Unilamellar vesicles were made from dipalmitoyl phosphatidyl choline (DPPC) by an extrusion method and showed a smooth (Lα) or a rippled (PB’.) structure depending on the temperature of the suspensions and the temperature of film formation (50°C resp. 39°C) prior to vitrification. The thermotropic phases of hydrated phospholipids are thus faithfully preserved in vitrified thin films (fig. a,b). Complex structures arose when mixtures of dioleoylphosphatidylethanol-amine (DOPE), dioleoylphosphatidylcholine (DOPC) and cholesterol (molar ratio 3/1/2) are heated and used for thin film formation. The tendency of DOPE to adopt the H|| phase is responsible for the formation of complex structures in this lipid mixture. Isotropic and cubic areas (fig. c,d) having a bilayer structure are found in coexistence with H|| cylinders (fig. e). The formation of interlamellar attachments (ILA’s) as observed in isotropic and cubic structures is also thought to be of importance in biological fusion events. Therefore the study of the fusion activity of influenza B virus with liposomes (DOPE/DOPC/cholesterol/ganglioside in a molar ratio 1/1/2/0.2) was initiated. At neutral pH only adsorption of virus to liposomes was observed whereas 2 minutes after a drop in pH (7.4 - > 5.4) fusion between virus and liposome membranes was demonstrated (fig. f). The micrographs illustrate the exciting potential of cryo-electron microscopy to study lipid-lipid and lipid-protein interactions in hydrated specimens.

Sign in / Sign up

Export Citation Format

Share Document