scholarly journals SARS-CoV-2 can recruit a haem metabolite to evade antibody immunity

2021 ◽  
pp. eabg7607
Author(s):  
Annachiara Rosa ◽  
Valerie E. Pye ◽  
Carl Graham ◽  
Luke Muir ◽  
Jeffrey Seow ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Viral Antigen ◽  
X Ray ◽  
X Ray Crystallography ◽  
Flexible Loop ◽  
Cryo Electron Microscopy ◽  
Allosteric Mechanism ◽  
Dominant Epitope

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through the recruitment of a metabolite.

scholarly journals SARS-CoV-2 recruits a haem metabolite to evade antibody immunity

2021 ◽  
Author(s):  
Annachiara Rosa ◽  
Valerie E. Pye ◽  
Carl Graham ◽  
Luke Muir ◽  
Jeffrey Seow ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Humoral Immunity ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Viral Antigen ◽  
Antibody Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Flexible Loop ◽  
Cryo Electron Microscopy

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that the virus co-opts the haem metabolite for the evasion of humoral immunity via allosteric shielding of a sensitive epitope and demonstrate the remarkable structural plasticity of the NTD.

scholarly journals Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape

Science ◽  
2021 ◽  
Vol 371 (6530) ◽  
pp. eabe6230 ◽  
Author(s):  
Paul-Albert Koenig ◽  
Hrishikesh Das ◽  
Hejun Liu ◽  
Beate M. Kümmerer ◽  
Florian N. Gohr ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Passive Immunization ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Escape Mutants

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo–electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Using cryo-electron microscopy maps for X-ray structure determination of homologues

2020 ◽  
Vol 76 (1) ◽  
pp. 63-72
Author(s):  
Lingxiao Zeng ◽  
Wei Ding ◽  
Quan Hao
Keyword(s):  
Electron Microscopy ◽  
Hybrid Method ◽  
Model Building ◽  
Test Cases ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sequence Identity ◽  
Whole Process ◽  
Cryo Electron Microscopy

The combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography reflects an important trend in structural biology. In a previously published study, a hybrid method for the determination of X-ray structures using initial phases provided by the corresponding parts of cryo-EM maps was presented. However, if the target structure of X-ray crystallography is not identical but homologous to the corresponding molecular model of the cryo-EM map, then the decrease in the accuracy of the starting phases makes the whole process more difficult. Here, a modified hybrid method is presented to handle such cases. The whole process includes three steps: cryo-EM map replacement, phase extension by NCS averaging and dual-space iterative model building. When the resolution gap between the cryo-EM and X-ray crystallographic data is large and the sequence identity is low, an intermediate stage of model building is necessary. Six test cases have been studied with sequence identity between the corresponding molecules in the cryo-EM and X-ray structures ranging from 34 to 52% and with sequence similarity ranging from 86 to 91%. This hybrid method consistently produced models with reasonable R work and R free values which agree well with the previously determined X-ray structures for all test cases, thus indicating the general applicability of the method for X-ray structure determination of homologues using cryo-EM maps as a starting point.

scholarly journals The Combination of X-Ray Crystallography and Cryo-Electron Microscopy Provides Insight into the Overall Architecture of the Dodecameric Rvb1/Rvb2 Complex

PLoS ONE ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. e0146457 ◽  
Author(s):  
Noella Silva-Martin ◽  
María I. Daudén ◽  
Sebastian Glatt ◽  
Niklas A. Hoffmann ◽  
Panagiotis Kastritis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Insight Into

scholarly journals HIV-1 Env structure and vaccine design

2014 ◽  
Vol 70 (a1) ◽  
pp. C117-C117
Author(s):  
Peter Kwong
Keyword(s):  
Electron Microscopy ◽  
Neutralizing Antibodies ◽  
Bound States ◽  
Vaccine Design ◽  
Atomic Level ◽  
Alpha Helices ◽  
X Ray ◽  
X Ray Crystallography ◽  
Gp120 Subunit ◽  
Hiv 1

Roughly one third of the HIV-1 genome is devoted to the HIV-1 envelope (Env) glycoprotein spike, which comprises three gp120 and three gp41 subunits. Structural characterization of the HIV-1 Env by electron microscopy, NMR, and X-ray crystallography reveals considerable conformational alterations, not only between trimeric ground state, CD4 receptor-bound conformation, and postfusion conformation of the spike, but also between monomeric and trimeric configurations of the subunits as well as between free- and antibody–bound states. One important structure, however, that of the prefusion HIV-1 spike, has resisted atomic level determination. This structure has been on the 10 list of most wanted structure for more than 20 years, because it is the target of the majority of broad HIV-1-neutralizing antibodies – and therefore of importance to vaccine design. In late 2013, the structure of a prefusion HIV-1 spike, based on a BG505 SOSIP.R6.664 construct, was reported by both X-ray crystallography (4.7 Å) and electron microscopy (5.8 Å). While these structures described the trimeric configuration of most of the HIV-1 gp120 subunit, the description of the gp41 subunit was limited to two helical regions comprising only about half the gp41 ectodomain, and the sequence register for the alpha helices was not reported. Recently, we were able to obtain x-ray diffraction data to 3.5 Å resolution on a prefusion crystal structure of the entire HIV-1 spike. The structure utilizes the same BG505 SOSIP.R6.664 construct as previously published, but crystallized in space group P6(3) with the antigen-binding fragments (Fab) of two antibodies, PGT122 and 35O22. The new structure provides atomic-level details for the complete prefusion structure of gp120 and the majority of the trimeric ectodomain of gp41 (up to residue 664). Also visualized are details of the gp120-gp41 interface and of antibodies such as 35O22. In addition to the complete HIV-1 Env ectodomain structure, implications for HIV-1 vaccine design will be described.

scholarly journals From X-ray crystallography to cryo-electron microscopy: computing infrastructure in structural biology

2018 ◽  
Vol 74 (a1) ◽  
pp. a224-a224
Author(s):  
Jason Key ◽  
Peter A. Meyer ◽  
Carol Herre ◽  
Michael Timony ◽  
Dimitry Filonov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Computing Infrastructure

scholarly journals A basic introduction to single particles cryo-electron microscopy

2021 ◽  
Vol 9 (1) ◽  
pp. 5-20
Author(s):  
Vittoria Raimondi ◽  
◽  
Alessandro Grinzato ◽  
◽  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Protein Complexes ◽  
Computational Power ◽  
Single Particles ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
First Choice ◽  
New Generation

<abstract> <p>In the last years, cryogenic-electron microscopy (cryo-EM) underwent the most impressive improvement compared to other techniques used in structural biology, such as X-ray crystallography and NMR. Electron microscopy was invented nearly one century ago but, up to the beginning of the last decades, the 3D maps produced through this technique were poorly detailed, justifying the term “blobbology” to appeal to cryo-EM. Recently, thanks to a new generation of microscopes and detectors, more efficient algorithms, and easier access to computational power, single particles cryo-EM can routinely produce 3D structures at resolutions comparable to those obtained with X-ray crystallography. However, unlike X-ray crystallography, which needs crystallized proteins, cryo-EM exploits purified samples in solution, allowing the study of proteins and protein complexes that are hard or even impossible to crystallize. For these reasons, single-particle cryo-EM is often the first choice of structural biologists today. Nevertheless, before starting a cryo-EM experiment, many drawbacks and limitations must be considered. Moreover, in practice, the process between the purified sample and the final structure could be trickier than initially expected. Based on these observations, this review aims to offer an overview of the principal technical aspects and setups to be considered while planning and performing a cryo-EM experiment.</p> </abstract>

scholarly journals GPU-accelerated analysis and visualization of large structures solved by molecular dynamics flexible fitting

2014 ◽  
Vol 169 ◽  
pp. 265-283 ◽  
Author(s):  
John E. Stone ◽  
Ryan McGreevy ◽  
Barry Isralewitz ◽  
Klaus Schulten
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Hybrid Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Interactive Computation ◽  
Biomolecular Complexes ◽  
Quality Of Fit

Hybrid structure fitting methods combine data from cryo-electron microscopy and X-ray crystallography with molecular dynamics simulations for the determination of all-atom structures of large biomolecular complexes. Evaluating the quality-of-fit obtained from hybrid fitting is computationally demanding, particularly in the context of a multiplicity of structural conformations that must be evaluated. Existing tools for quality-of-fit analysis and visualization have previously targeted small structures and are too slow to be used interactively for large biomolecular complexes of particular interest today such as viruses or for long molecular dynamics trajectories as they arise in protein folding. We present new data-parallel and GPU-accelerated algorithms for rapid interactive computation of quality-of-fit metrics linking all-atom structures and molecular dynamics trajectories to experimentally-determined density maps obtained from cryo-electron microscopy or X-ray crystallography. We evaluate the performance and accuracy of the new quality-of-fit analysis algorithmsvis-à-visexisting tools, examine algorithm performance on GPU-accelerated desktop workstations and supercomputers, and describe new visualization techniques for results of hybrid structure fitting methods.

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