scholarly journals Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display

2019 ◽  
Vol 5 (9) ◽  
pp. eaaw2853 ◽  
Author(s):  
Charles Vragniau ◽  
Joshua C. Bufton ◽  
Frédéric Garzoni ◽  
Emilie Stermann ◽  
Fruzsina Rabi ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Electron Microscopy ◽  
High Performance ◽  
Vaccine Candidate ◽  
Cost Effective ◽  
Atomic Resolution ◽  
Self Assembling ◽  
Cryo Electron Microscopy ◽  
Architectural Features ◽  
Multimeric Protein

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo–electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.

scholarly journals Low cost, high performance processing of single particle cryo-electron microscopy data in the cloud

2015 ◽  
Author(s):  
Michael A. Cianfrocco ◽  
Andres E. Leschziner
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
High Performance ◽  
Low Cost ◽  
Cost Effective ◽  
Cryo Electron Microscopy ◽  
Software Packages ◽  
Electron Microscopes ◽  
Microscopy Data ◽  
New Generation

The advent of a new generation of electron microscopes and direct electron detectors has realized the potential of single particle cryo-electron microscopy (cryo-EM) as a technique to generate high-resolution structures. However, calculating these structures requires high performance computing clusters, a resource that may be limiting to many likely cryo-EM users. To address this limitation and facilitate the spread of cryo-EM, we developed a publicly available ‘off-the-shelf’ computing environment on Amazon’s elastic cloud computing infrastructure. This environment provides users with single particle cryo-EM software packages and the ability to create computing clusters that can range in size from 16 to 480+ CPUs. Importantly, these computing clusters are also cost-effective, as we illustrate here by determining a near-atomic resolution structure of the 80S yeast ribosome for $28.89 USD in ~10 hours.

scholarly journals Determination of intermediate state structures in the opening pathway of SARS-CoV-2 spike using cryo-electron microscopy

2021 ◽  
Author(s):  
Z. Faidon Brotzakis ◽  
Thomas Lohr ◽  
Michele Vendruscolo
Keyword(s):  
Infectious Disease ◽  
Electron Microscopy ◽  
Severe Acute Respiratory Syndrome ◽  
Intermediate State ◽  
Therapeutic Interventions ◽  
Cryo Electron Microscopy ◽  
Welfare Systems

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a highly infectious disease that is severely affecting our society and welfare systems. In order to develop therapeutic interventions against this...

scholarly journals Molecular dynamics-based refinement and validation for sub-5 Å cryo-electron microscopy maps

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Abhishek Singharoy ◽  
Ivan Teo ◽  
Ryan McGreevy ◽  
John E Stone ◽  
Jianhua Zhao ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Molecular Dynamics ◽  
Cost Effective ◽  
Search Model ◽  
Mean Square ◽  
Cryo Electron Microscopy ◽  
Experimental Resolution ◽  
Amazon Web Services ◽  
Determination Methods ◽  
Local Root

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5 Å cryo-electron microscopy (EM) maps with either single structures or ensembles of such structures. The methods, denoted cascade MDFF and resolution exchange MDFF, sequentially re-refine a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution. Application of sequential re-refinement enables MDFF to achieve a radius of convergence of ~25 Å demonstrated with the accurate modeling of β-galactosidase and TRPV1 proteins at 3.2 Å and 3.4 Å resolution, respectively. The MDFF refinements uniquely offer map-model validation and B-factor determination criteria based on the inherent dynamics of the macromolecules studied, captured by means of local root mean square fluctuations. The MDFF tools described are available to researchers through an easy-to-use and cost-effective cloud computing resource on Amazon Web Services.

scholarly journals A resolution record for cryoEM

2021 ◽  
Vol 10 ◽  
Author(s):  
Jonathan Ashmore ◽  
Bridget Carragher ◽  
Peter B Rosenthal ◽  
William Weis
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Structure Determination ◽  
Test Specimen ◽  
Structural Information ◽  
Atomic Resolution ◽  
Fast Growing ◽  
Cryo Electron Microscopy ◽  
New Instrument ◽  
Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

scholarly journals Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
George Ueda ◽  
Aleksandar Antanasijevic ◽  
Jorge A Fallas ◽  
William Sheffler ◽  
Jeffrey Copps ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
De Novo ◽  
Viral Glycoprotein ◽  
Influenza Hemagglutinin ◽  
Self Assembling ◽  
Cryo Electron Microscopy ◽  
Viral Glycoproteins ◽  
Protein Nanoparticles ◽  
New Generation ◽  
Tailored Design

Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.

Forge Wednesday Roundup - June 10, 2020

2021 ◽  
Author(s):  
H.L. Cu Si
Keyword(s):  
Electron Microscopy ◽  
Facial Recognition ◽  
Academic Research ◽  
Atomic Resolution ◽  
Rapa Nui ◽  
Social Distancing ◽  
Cryo Electron Microscopy

In today’s Roundup: COVID is still with us; pair of papers quantifies effects of social distancing; IBM bails on facial recognition, warns of harms; questioning “collapse” of Rapa Nui; attention turns to antibodies for COVID-19 therapies; WHO walks back confusing statement on asymptomatic COVID transmission; cryo-electron microscopy reveals proteins at atomic resolution; unstoppable newsbot runs amok, propagates bias; exploring the lure of misinformation; academic research goes on strike against racism; much more...

scholarly journals Atomic Resolution Structures of Human Bufaviruses Determined by Cryo-Electron Microscopy

Viruses ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 22 ◽  
Author(s):  
Maria Ilyas ◽  
Mario Mietzsch ◽  
Shweta Kailasan ◽  
Elina Väisänen ◽  
Mengxiao Luo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atomic Resolution ◽  
Cryo Electron Microscopy

scholarly journals Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

2015 ◽  
Vol 112 (43) ◽  
pp. 13237-13242 ◽  
Author(s):  
Lorenzo Sborgi ◽  
Francesco Ravotti ◽  
Venkata P. Dandey ◽  
Mathias S. Dick ◽  
Adam Mazur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Specific Binding ◽  
3D Structure ◽  
High Mobility ◽  
Atomic Resolution ◽  
Receptor Protein ◽  
Filament Formation ◽  
Cryo Electron Microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

scholarly journals Cryo-EM Reveals the Structural Basis of Microtubule Depolymerization by Kinesin-13s

2017 ◽  
Author(s):  
Matthieu P. M. H. Benoit ◽  
Ana B. Asenjo ◽  
Hernando Sosa
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Distinct Group ◽  
Atomic Resolution ◽  
Structural Basis ◽  
Structural Elements ◽  
Microtubule Depolymerization ◽  
Cryo Electron Microscopy ◽  
Kinesin Superfamily ◽  
Motile Activity

SummaryKinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promotes microtubule depolymerization and lacks motile activity. The molecular mechanism by which the kinesins depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue we obtained near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A constructs bound to curved or straight tubulin in different nucleotide states. The structures show how nucleotide induced conformational changes near the catalytic site are coupled with kinesin-13-specific structural elements to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.

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