Dmitry Lyumkis

Crystal Structure of a Soluble Cleaved HIV-1 Envelope Trimer

Knowing the Enemy Infection of host cells by HIV-1 is mediated by an envelope glycoprotein (Env) trimeric spike on the surface of the virus. Proteins comprising the Env trimer must be cleaved for infectivity, and thus viral fusion involves three Env conformations. The flexibility of the Env trimer has made it a challenge to determine a high-resolution structure, although such a structure is key both for understanding trimer function and for guiding vaccine design. Lyumkis et al. (p. 1484) and Julien et al. (p. 1477) studied soluble cleaved trimers stabilized by specific mutations but that have kept a near-native antigenicity profile. Lyumkis et al. present a high-resolution structure of the trimer in complex with a broadly neutralizing antibody, and Julien et al. present a crystal structure of the trimer in complex with another broadly neutralizing antibody. Key structural features dictate how the HIV envelope protein functions and interacts with the human immune system. HIV-1 entry into CD4+ target cells is mediated by cleaved envelope glycoprotein (Env) trimers that have been challenging to characterize structurally. Here, we describe the crystal structure at 4.7 angstroms of a soluble, cleaved Env trimer that is stabilized and antigenically near-native (termed the BG505 SOSIP.664 gp140 trimer) in complex with a potent broadly neutralizing antibody, PGT122. The structure shows a prefusion state of gp41, the interaction between the component gp120 and gp41 subunits, and how a close association between the gp120 V1/V2/V3 loops stabilizes the trimer apex around the threefold axis. The complete epitope of PGT122 on the trimer involves gp120 V1, V3, and several surrounding glycans. This trimer structure advances our understanding of how Env functions and is presented to the immune system, and provides a blueprint for structure-based vaccine design.

Cryo-EM Structure of a Fully Glycosylated Soluble Cleaved HIV-1 Envelope Trimer

Knowing the Enemy Infection of host cells by HIV-1 is mediated by an envelope glycoprotein (Env) trimeric spike on the surface of the virus. Proteins comprising the Env trimer must be cleaved for infectivity, and thus viral fusion involves three Env conformations. The flexibility of the Env trimer has made it a challenge to determine a high-resolution structure, although such a structure is key both for understanding trimer function and for guiding vaccine design. Lyumkis et al. (p. 1484) and Julien et al. (p. 1477) studied soluble cleaved trimers stabilized by specific mutations but that have kept a near-native antigenicity profile. Lyumkis et al. present a high-resolution structure of the trimer in complex with a broadly neutralizing antibody, and Julien et al. present a crystal structure of the trimer in complex with another broadly neutralizing antibody. Key structural features dictate how the HIV envelope protein functions and interacts with the human immune system. The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo–electron microscopy reconstruction and structural model of a cleaved, soluble Env trimer (termed BG505 SOSIP.664 gp140) in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 angstrom resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1, and HR2 domains, as well as shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.

Movies of ice-embedded particles enhance resolution in electron cryo-microscopy.

Low-dose images obtained by electron cryo-microscopy (cryo-EM) are often affected by blurring caused by sample motion during electron beam exposure, degrading signal especially at high resolution. We show here that we can align frames of movies, recorded with a direct electron detector during beam exposure of rotavirus double-layered particles, thereby greatly reducing image blurring caused by beam-induced motion and sample stage instabilities. This procedure increases the efficiency of cryo-EM imaging and enhances the resolution obtained in three-dimensional reconstructions of the particle. Using movies in this way is generally applicable to all cryo-EM samples and should also improve the performance of midrange electron microscopes that may have limited mechanical stability and beam coherence.

Likelihood-based classification of cryo-EM images using FREALIGN

We describe an implementation of maximum likelihood classification for single particle electron cryo-microscopy that is based on the FREALIGN software. Particle alignment parameters are determined by maximizing a joint likelihood that can include hierarchical priors, while classification is performed by expectation maximization of a marginal likelihood. We test the FREALIGN implementation using a simulated dataset containing computer-generated projection images of three different 70S ribosome structures, as well as a publicly available dataset of 70S ribosomes. The results show that the mixed strategy of the new FREALIGN algorithm yields performance on par with other maximum likelihood implementations, while remaining computationally efficient.

Initial evaluation of a Direct Detection Device detector for single particle cryo-electron microscopy

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

Significance All organisms have systems in place to ensure that aberrant nascent polypeptide chains are promptly dealt with before being released from ribosomes and posing harm to the cell. The ribosome-associated quality control complex (RQC), composed of the Ltn1 E3 ubiquitin ligase catalytic subunit and cofactors, has become a paradigm for understanding quality control in eukaryotes. However, exactly how RQC functions has remained unknown. Here, we determine the structure of the 60S subunit-bound RQC complex. The data provide critical insights into how RQC is able to selectively target aberrant nascent chains, while ignoring nascent chains in normally translating ribosomes. Furthermore, the structure shows the architecture of a ribosome-bound E3 ligase poised to mark nascent chains for degradation. All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

A Toolbox for ab initio 3-D reconstructions in single-particle electron microscopy

Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a toolbox of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.

Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike

For many enveloped viruses, binding to a receptor(s) on a host cell acts as the first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. Although Env can tolerate a high degree of mutation in five variable regions (V1–V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env, and are targets of broadly neutralizing antibodies. Thus, they are attractive immunogens for vaccine development. Here we present high-resolution cryo-electron microscopy structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of 3.7u2009Å and 3.6u2009Å, respectively. We compare these to cryo-electron microscopy reconstructions of B41 SOSIP Env trimers with no ligand or in complex with either CD4 or the CD4-binding-site antibody PGV04 at 5.6u2009Å, 5.2u2009Å and 7.4u2009Å resolution, respectively. Consequently, we present the most complete description yet, to our knowledge, of the CD4–17b-induced intermediate and provide the molecular basis of the receptor-binding-induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.

Model Building and Refinement of a Natively Glycosylated HIV-1 Env Protein by High-Resolution Cryoelectron Microscopy.

Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle cryoelectron microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 Å resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.

Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome.

High-resolution insights into the intasome An essential step in the life cycle of lentiviruses such as HIV-1 is when viral DNA integrates into the host genome, establishing a permanent infection of the host cell. The viral integrase enzyme catalyzes this process and is a major drug target. During viral integration, integrase binds the ends of viral DNA, forming a higher-order structure called the intasome. Passos et al. and Ballandras-Colas et al. used cryo—electron microscopy to solve the structures of the intasomes from HIV-1 and maedi-visna virus (ovine lentivirus), respectively. These structures reveal how integrase self-associates to form a functional intasome and help resolve previous conflicting models of intasome assembly. Science, this issue p. 89, p. 93 Cryo–electron microscopy reveals how lentiviral DNA and the viral integrase assemble to promote retroviral integration into host cell DNA. Like all retroviruses, HIV-1 irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase (IN) and the ends of linear vDNA, mediates integration. Productive integration into host chromatin results in the formation of the strand transfer complex (STC) containing catalytically joined vDNA and tDNA. HIV-1 intasomes have been refractory to high-resolution structural studies. We used a soluble IN fusion protein to facilitate structural studies, through which we present a high-resolution cryo–electron microscopy (cryo-EM) structure of the core tetrameric HIV-1 STC and a higher-order form that adopts carboxyl-terminal domain rearrangements. The distinct STC structures highlight how HIV-1 can use the common retroviral intasome core architecture to accommodate different IN domain modules for assembly.