@article {48302, title = {Conformational states of a soluble, uncleaved HIV-1 envelope trimer.}, journal = {Journal of Virology}, volume = {91}, year = {2017}, month = {2017 Mar 01}, pages = {e00175-17}, abstract = {

HIV-1 envelope spike [Env; trimeric (gp160)3, cleaved to (gp120/gp41)3] induces membrane fusion, leading to viral entry. It is also the viral component targeted by neutralizing antibodies. Vaccine development requires production, in quantities suitable for clinical studies, of a recombinant form that resembles functional Env. HIV-1 gp140 trimers - the uncleaved ectodomains of (gp160)3 - from a few selected viral isolates adopt a compact conformation with many antigenic properties of native Env spikes. One is currently being evaluated in a clinical trial. We report here low-resolution (20{\r A}) cryoEM (electron cryomicroscopy) structures of this gp140 trimer, which adopts two principal conformations, one closed and the other slightly open. The former is indistinguishable at this resolution from those adopted by a stabilized, cleaved trimer (SOSIP) or by a membrane-bound Env trimer with truncated cytoplasmic tail (EnvΔCT). The latter conformation is closer to a partially open Env trimer than to the fully open conformation induced by CD4. These results show that a stable, uncleaved HIV-1 gp140 trimer has a compact structure close to that of native Env.IMPORTANCE Development of any HIV vaccine with a protein component (either prime or boost) requires production of a recombinant form to mimic the trimeric, functional HIV-1 envelope spike, in quantities suitable for clinical studies. Our understanding of the envelope structure has depended in part on a cleaved, soluble trimer, known as SOSIP.664, stabilized by several modifications including an engineered disulfide. This construct, difficult to produce in large quantities, has yet to induce better antibody responses than other envelope-based immunogens, even in animal models. The uncleaved ectodomain of the envelope protein, called gp140, has also been made as a soluble form to mimic the native Env present on the virion surface. Most HIV-1 gp140 preparations are not stable, however, and of inhomogeneous conformation. The results presented here show that gp140 preparations from suitable isolates can adopt a compact, native-like structure, supporting its use as a vaccine candidate.

}, issn = {1098-5514}, doi = {10.1128/JVI.00175-17}, author = {Liu, Yuhang and Pan, Junhua and Cai, Yongfei and Grigorieff, Nikolaus and Harrison, Stephen C and Chen, Bing} } @article {48489, title = {CryoEM structure of an influenza virus receptor-binding site antibody-antigen interface.}, journal = {Journal of Molecular Biology}, volume = {429}, year = {2017}, month = {2017 May 12}, pages = {1829-39}, abstract = {

Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes. Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain Fv (scFv) complexes, by studying a complex we failed to crystallize in pursuing an extended project of the human immune response to influenza vaccines. The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen:antibody interface, although small variation in the twist of the rod-like HA trimer limited the overall resolution to about 4.5{\r A}. Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2017.05.011}, author = {Liu, Yuhang and Pan, Junhua and Jenni, Simon and Raymond, Donald D and Caradonna, Tim and Do, Khoi T and Schmidt, Aaron G and Harrison, Stephen C and Grigorieff, Nikolaus} } @article {6320, title = {Beam-induced motion of vitrified specimen on holey carbon film.}, journal = {Journal of Structural Biology}, volume = {177}, year = {2012}, month = {2012 Mar}, pages = {630-7}, abstract = {

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.

}, keywords = {Computer-Assisted, Cryoelectron Microscopy, Image Processing}, issn = {1095-8657}, doi = {10.1016/j.jsb.2012.02.003}, author = {Brilot, Axel F and Chen, James Z and Cheng, Anchi and Pan, Junhua and Harrison, Stephen C and Potter, Clinton S and Carragher, Bridget and Henderson, Richard and Grigorieff, Nikolaus} } @article {6319, title = {Movies of ice-embedded particles enhance resolution in electron cryo-microscopy.}, journal = {Structure}, volume = {20}, year = {2012}, month = {2012 Nov 7}, pages = {1823-8}, abstract = {

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.

}, keywords = {Cryoelectron Microscopy, Ice, Rotavirus}, issn = {1878-4186}, doi = {10.1016/j.str.2012.08.026}, author = {Campbell, Melody G and Cheng, Anchi and Brilot, Axel F and Moeller, Arne and Lyumkis, Dmitry and Veesler, David and Pan, Junhua and Harrison, Stephen C and Potter, Clinton S and Carragher, Bridget and Grigorieff, Nikolaus} }