scholarly journals Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

2020 ◽  
Author(s):  
Zunlong Ke ◽  
Joaquin Oton ◽  
Kun Qu ◽  
Mirko Cortese ◽  
Vojtech Zila ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Neutralizing Antibodies ◽  
Structural Rearrangement ◽  
Conformational Flexibility ◽  
Target Cells ◽  
Receptor Binding Site ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Virion Surface

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

scholarly journals In situ structure determination using single particle cryo-electron microscopy images

2020 ◽  
Author(s):  
Jing Cheng ◽  
Bufan Li ◽  
Long Si ◽  
Xinzheng Zhang
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Structure Refinement ◽  
Target Function ◽  
Target Protein ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Tilt Series

AbstractCryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

scholarly journals Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

2021 ◽  
Author(s):  
Nicole Dimos ◽  
Carl P.O. Helmer ◽  
Andrea M. Chanique ◽  
Markus C. Wahl ◽  
Robert Kourist ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Structure Analysis ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Fast Development ◽  
Pharmaceutical Industries ◽  
Exquisite Specificity ◽  
Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

scholarly journals Networks of HIV-1 envelope glycans maintain antibody epitopes in the face of glycan additions and deletions

2020 ◽  
Author(s):  
Gemma E. Seabright ◽  
Christopher A. Cottrell ◽  
Marit J. van Gils ◽  
Alessio D’addabbo ◽  
David J. Harvey ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Neutralizing Antibodies ◽  
Broadly Neutralizing Antibodies ◽  
Site Specific ◽  
Glycan Analysis ◽  
Cryo Electron Microscopy ◽  
Immunogen Design ◽  
The Face ◽  
Antibody Epitopes ◽  
Hiv 1

SUMMARYNumerous broadly neutralizing antibodies (bnAbs) have been identified that target the glycans of the HIV-1 envelope spike. Neutralization breadth is notable given that glycan processing can be substantially influenced by the presence or absence of neighboring glycans. Here, using a stabilized recombinant envelope trimer, we investigate the degree to which mutations in the glycan network surrounding an epitope impact the fine glycan processing of antibody targets. Using cryo-electron microscopy and site-specific glycan analysis, we reveal the hierarchy of importance of glycans in the formation of the 2G12 bnAb epitope, and show that the epitope is only subtly impacted by variations in the glycan network. In contrast, we show that the PG9 and PG16 glycan-based epitopes at the trimer apex are dependent on the presence of the highly conserved surrounding glycans. Glycan networks underpin the conservation of bnAb epitopes and are an important parameter in immunogen design.

scholarly journals Structures of SARS-CoV-2 B.1.351 neutralizing antibodies provide insights into cocktail design against concerning variants

2021 ◽  
Author(s):  
Shuo Du ◽  
Pulan Liu ◽  
Zhiying Zhang ◽  
Tianhe Xiao ◽  
Ayijiang Yasimayi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Antibody Responses ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Humoral Antibody ◽  
Cryo Electron Microscopy ◽  
Antibody Cocktail

The spread of the SARS-CoV-2 variants could seriously dampen the global effort to tackle the COVID-19 pandemic. Recently, we investigated the humoral antibody responses of SARS-CoV-2 convalescent patients and vaccinees towards circulating variants, and identified a panel of monoclonal antibodies (mAbs) that could efficiently neutralize the B.1.351 (Beta) variant. Here we investigate how these mAbs target the B.1.351 spike protein using cryo-electron microscopy. In particular, we show that two superpotent mAbs, BD-812 and BD-836, have non-overlapping epitopes on the receptor-binding domain (RBD) of spike. Both block the interaction between RBD and the ACE2 receptor; and importantly, both remain fully efficacious towards the B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. The BD-812/BD-836 pair could thus serve as an ideal antibody cocktail against the SARS-CoV-2 VOCs.

scholarly journals In situ structure determination using single particle cryo-electron microscopy images

2020 ◽  
Author(s):  
Jing Cheng ◽  
Bufan Li ◽  
Long Si ◽  
Xinzheng Zhang
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Structure Refinement ◽  
Target Function ◽  
Target Protein ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Tilt Series

Abstract Cryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

scholarly journals Cryo-electron microscopy structure of a nucleosome-bound SWI/SNF chromatin remodeling complex

2019 ◽  
Author(s):  
Yan Han ◽  
Alexis A Reyes ◽  
Sara Malik ◽  
Yuan He
Keyword(s):  
Electron Microscopy ◽  
Chromatin Remodeling ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Gene Activation ◽  
The Body ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Chromatin Remodeling Complex ◽  
High Resolution Structure

AbstractThe multi-subunit chromatin remodeling complex SWI/SNF1–3 is highly conserved from yeast to humans and plays critical roles in various cellular processes including transcription and DNA damage repair4, 5. It uses the energy from ATP hydrolysis to remodel chromatin structure by sliding and evicting the histone octamer6–10, creating DNA regions that become accessible to other essential protein complexes. However, our mechanistic understanding of the chromatin remodeling activity is largely hindered by the lack of a high-resolution structure of any complex from this family. Here we report the first structure of SWI/SNF from the yeast S. cerevisiae bound to a nucleosome at near atomic resolution determined by cryo-electron microscopy (cryo-EM). In the structure, the Arp module is sandwiched between the ATPase and the Body module of the complex, with the Snf2 HSA domain connecting all modules. The HSA domain also extends into the Body and anchors at the opposite side of the complex. The Body contains an assembly scaffold composed of conserved subunits Snf12 (SMARCD/BAF60), Snf5 (SMARCB1/BAF47/ INI1) and an asymmetric dimer of Swi3 (SMARCC/BAF155/170). Another conserved subunit Swi1 (ARID1/BAF250) folds into an Armadillo (ARM) repeat domain that resides in the core of the SWI/SNF Body, acting as a molecular hub. In addition to the interaction between Snf2 and the nucleosome, we also observed interactions between the conserved Snf5 subunit and the histones at the acidic patch, which could serve as an anchor point during active DNA translocation. Our structure allows us to map and rationalize a subset of cancer-related mutations in the human SWI/SNF complex and propose a model of how SWI/SNF recognizes and remodels the +1 nucleosome to generate nucleosome-depleted regions during gene activation11–13.

scholarly journals High-Light versus Low-Light: Effects on Paired Photosystem II Supercomplex Structural Rearrangement in Pea Plants

2020 ◽  
Vol 21 (22) ◽  
pp. 8643
Author(s):  
Alessandro Grinzato ◽  
Pascal Albanese ◽  
Roberto Marotta ◽  
Paolo Swuec ◽  
Guido Saracco ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Photosystem Ii ◽  
Structural Rearrangement ◽  
Structural Features ◽  
Thylakoid Membranes ◽  
Variable Number ◽  
High Light ◽  
Low Light ◽  
Cryo Electron Microscopy ◽  
Light Effects

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.

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