@article {64833, title = {Cryo-EM fibril structures from systemic AA amyloidosis reveal the species complementarity of pathological amyloids.}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {2019 Mar 07}, pages = {1104}, abstract = {

Systemic AA amyloidosis is a worldwide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from the acute phase protein serum amyloid A. Here, we report the purification and electron cryo-microscopy analysis of amyloid fibrils from a mouse and a human patient with systemic AA amyloidosis. The obtained resolutions are 3.0 {\r A} and 2.7 {\r A} for the murine and human fibril, respectively. The two fibrils differ in fundamental properties, such as presence of right-hand or left-hand twisted cross-β sheets and overall fold of the fibril proteins. Yet, both proteins adopt highly similar β-arch conformations within the N-terminal ~21 residues. Our data demonstrate the importance of the fibril protein N-terminus for the stability of the analyzed amyloid fibril morphologies and suggest strategies of combating this disease by interfering with specific fibril polymorphs.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-09033-z}, author = {Liberta, Falk and Loerch, Sarah and Rennegarbe, Matthies and Schierhorn, Angelika and Westermark, Per and Westermark, Gunilla T and Hazenberg, Bouke P C and Grigorieff, Nikolaus and F{\"a}ndrich, Marcus and Schmidt, Matthias} } @article {49087, title = {cisTEM, User-friendly software for single-particle image processing.}, journal = {eLife}, volume = {7}, year = {2018}, month = {2018 Mar 07}, abstract = {

We have developed new open-source software calledTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging.TEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction. Some of these steps implement newly-developed algorithms; others were adapted from previously published algorithms. The software is optimized to enable processing of typical datasets (2000 micrographs, 200k - 300k particles) on a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that can be adapted for most computing environments.TEM is available for download from cistem.org.

}, issn = {2050-084X}, doi = {10.7554/eLife.35383}, author = {Grant, Timothy and Rohou, Alexis and Grigorieff, Nikolaus} } @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 {47506, title = {Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {113}, year = {2016}, month = {2016 May 16}, pages = {6200-5}, abstract = {

Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles.

}, issn = {1091-6490}, doi = {10.1073/pnas.1522282113}, author = {Schmidt, Andreas and Annamalai, Karthikeyan and Schmidt, Matthias and Grigorieff, Nikolaus and F{\"a}ndrich, Marcus} } @article {46998, title = {CTFFIND4: Fast and accurate defocus estimation from electron micrographs.}, journal = {Journal of Structural Biology}, volume = {192}, year = {2015}, month = {2015 Aug 13}, pages = {216-21}, abstract = {

CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope{\textquoteright}s contrast transfer function (CTF) to an image{\textquoteright}s amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.

}, issn = {1095-8657}, doi = {10.1016/j.jsb.2015.08.008}, author = {Rohou, Alexis and Grigorieff, Nikolaus} }