Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography

Abstract

We used cryo-electron tomography in conjunction with single-particle averaging techniques to study the structures of frozen-hydrated envelope glycoprotein (Env) complexes on intact Moloney murine leukemia retrovirus particles. Cryo-electron tomography allows 3D imaging of viruses in toto at a resolution sufficient to locate individual macromolecules, and local averaging of abundant complexes substantially improves the resolution. The averaging of repetitive features in electron tomograms is hampered by a low signal-to-noise ratio and anisotropic resolution, which results from the ''missingwedge'' effect. We developed an iterative 3D averaging algorithm that compensates for this effect and used it to determine the trimeric structure of Env to a resolution of 2.7 nm, at which individual domains can be resolved. Strikingly, the 3D reconstruction is shaped like a tripod in which the trimer penetrates the membrane at three distinct locations Ϸ4.5 nm apart from one another. The Env reconstruction allows tentative docking of the x-ray crystal structure of the receptorbinding domain. This study thus provides 3D structural information regarding the prefusion conformation of an intact unstained retrovirus surface protein.cryo-electron microscopy ͉ single-particle analysis ͉ Moloney murine leukemia virus T he surfaces of retroviruses are studded with a trimeric, type I transmembrane glycoprotein complex called ''Env.'' During infection, Env mediates binding of virus to its receptor in the cell plasma membrane and subsequent fusion of the viral and cellular membranes. Env undergoes large-scale conformational changes during these events (1, 2). Although the high-resolution structures of Env fragments from various retroviruses have been determined (3-9), the structure of intact Env on the virion surface is known only on a gross morphological level as a trimeric projection (10-12). The quaternary structure of Env on fresh virions is the starting point for the structural rearrangements that enable entry of the virus into cells and is therefore key to our understanding of the mechanism of retrovirus infection.Certain features of intact retroviral Env hamper its structural analysis by x-ray crystallography. For example, glycosylation, which can exceed 50% of the mass of Env, is an impediment to crystallization. Single-pass transmembrane proteins are notoriously difficult to crystallize; therefore attempts to replace the transmembrane region have been made by fusing the Env ectodomain to carrier proteins (13,14). Generation of a soluble Env ectodomain lacking transmembrane segments has typically involved removal of a proteolytic processing site (13), which prevents subunit dissociation but also prevents maturation of the complex into its final functional form. The large quantities of soluble Env necessary for crystallization attempts are difficult to prepare, and the material is often unstable and heterogeneous. Electron microscopy (EM) avoids many of these difficulties and provides useful structural information albeit at lower re...

Classification and 3D averaging with missing wedge correction in biological electron tomography

“…Although in some cases the search may be restricted by exploiting the geometry of the problem, e.g., by only searching for in-plane rotations (Winkler and Taylor, 1999;Förster et al, 2005), this is still a very computationally demanding procedure. When the axis of rotation between the volumes can be determined, the use of cylindrical coordinates can accelerate the rotational search (at least in one of the three Euler angles) by locating the peak of the cross-correlation function on a cylindrically transformed domain (Walz et al, 1997;Förster et al, 2005). This procedure however, does not account for the missing wedge and as a consequence the result of the alignment will be biased.…”

“…More recently, Förster et al (2008) have also studied this case but only in the context of classification (not for alignment), extending the constrained cross-correlation initially proposed in Frangakis et al (2002) to account for the missing wedge of both volumes. The approaches taken in Frangakis et al (2002), Förster et al (2005), Schmid et al (2006) and Förster et al (2008), all rely almost exclusively on exhaustive search of the rotational space to determine the best matching orientation between the two volumes, precluding the analysis of very large data sets. Although in some cases the search may be restricted by exploiting the geometry of the problem, e.g., by only searching for in-plane rotations (Winkler and Taylor, 1999;Förster et al, 2005), this is still a very computationally demanding procedure.…”

“…The approaches taken in Frangakis et al (2002), Förster et al (2005), Schmid et al (2006) and Förster et al (2008), all rely almost exclusively on exhaustive search of the rotational space to determine the best matching orientation between the two volumes, precluding the analysis of very large data sets. Although in some cases the search may be restricted by exploiting the geometry of the problem, e.g., by only searching for in-plane rotations (Winkler and Taylor, 1999;Förster et al, 2005), this is still a very computationally demanding procedure. When the axis of rotation between the volumes can be determined, the use of cylindrical coordinates can accelerate the rotational search (at least in one of the three Euler angles) by locating the peak of the cross-correlation function on a cylindrically transformed domain (Walz et al, 1997;Förster et al, 2005).…”

“…A reference-based approach to refinement using a single external reference was used in Walz et al (1997) and Förster et al (2005). Here an initial reference is selected and all raw volumes are rotationally and translationally aligned to this reference.…”

Influenza virus pleiomorphy characterized by cryoelectron tomography

“…The virion structure is of interest not only in the context of virus assembly but also in light of the possibility that pleiomorphic variations may correlate with infectivity and/or pathogenicity. As a step toward addressing these questions, we have used cryoelectron tomography, a technique capable of rendering the 3D structures of individual macromolecular particles in their native states (19)(20)(21)(22)(23)(24)(25), to visualize influenza virions of the type A eggadapted X-31 strain (26).…”