High‐Resolution Imaging of Human Viruses in Liquid Droplets

Jonaid, G. M.; Dearnaley, W. J.; Casasanta, Michael A.; Kaylor, Liam; Berry, Samantha; Dukes, Madeline J.; Spilman, Michael; Gray, Jennifer L.; Kelly, Deborah F.

doi:10.1002/adma.202103221

Cited by 23 publications

(37 citation statements)

References 35 publications

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“…The AAV purification protocol was recently described (Rutledge et al, 1998; Jonaid et al, 2021). Briefly, HEK293 cells were transfected using at a 2:1:1 ratio of AAV helper (260 μ g), AAV cis (130 μ g), and AAV trans (130 μ g) plasmids and incubated at 37°C with 5% atmospheric CO 2 for 24 h. Viruses were purified using affinity chromatography methods that employed AVB sepharose HP (GE Healthcare) and AAV preparations were evaluated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The structure of a new AAV-based COVID vaccine candidate was recently determined at ∼3.22 Å resolution using liquid-EM (Jonaid et al, 2021). To accomplish this feat, we used the Poseidon Select system and SiN microchips containing integrated microwells.…”

Section: Case Studies: Aav and The Sars-cov-2 N Proteinmentioning

confidence: 99%

“…It is highly desirable to pair high-resolution structural information with real-time dynamics, as many events occur in the order of milliseconds or faster. Such rapid movements may include changes in pliable protein loops that serve as immune epitopes on human viruses (Jonaid et al, 2021). Improved knowledge of flexible protein structures can inspire the design of new antibody therapies or anti-viral agents to combat emerging variants of influenza, ebola, or SARS-CoV-2 (Kiss et al, 2014; Pallesen et al, 2016; Casasanta et al, 2021; Jonaid et al, 2021; Watanabe et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

“…Such rapid movements may include changes in pliable protein loops that serve as immune epitopes on human viruses (Jonaid et al, 2021). Improved knowledge of flexible protein structures can inspire the design of new antibody therapies or anti-viral agents to combat emerging variants of influenza, ebola, or SARS-CoV-2 (Kiss et al, 2014; Pallesen et al, 2016; Casasanta et al, 2021; Jonaid et al, 2021; Watanabe et al, 2021). Importantly, viewing materials in a fluid, stochastic setting provides unique insight of their performance in the body.…”

Section: Introductionmentioning

confidence: 99%

“…Here, we describe how automated tools can advance high-resolution imaging in liquid. We examine two recent case studies, ranging from large virus assemblies, adeno-associated virus subtype 3 (AAV) to small individual proteins, the SARS-CoV-2 nucleocapsid (N) (Casasanta et al, 2021; Jonaid et al, 2021). These macromolecules were specifically chosen, as they have very different dimensions and symmetries.…”

Section: Introductionmentioning

confidence: 99%

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Automated Tools to Advance High-Resolution Imaging in Liquid

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Liquid-electron microscopy (EM), the room-temperature correlate to cryo-EM, is a rapidly growing field providing high-resolution insights of macromolecules in solution. Here, we describe how liquid-EM experiments can incorporate automated tools to propel the field to new heights. We demonstrate fresh workflows for specimen preparation, data collection, and computing processes to assess biological structures in liquid. Adeno-associated virus (AAV) and the SARS-CoV-2 nucleocapsid (N) were used as model systems to highlight the technical advances. These complexes were selected based on their major differences in size and natural symmetry. AAV is a highly symmetric, icosahedral assembly with a particle diameter of ~25 nm. At the other end of the spectrum, N protein is an asymmetric monomer or dimer with dimensions of approximately 5–7 nm, depending upon its oligomerization state. Equally important, both AAV and N protein are popular subjects in biomedical research due to their high value in vaccine development and therapeutic efforts against COVID-19. Overall, we demonstrate how automated practices in liquid-EM can be used to decode molecules of interest for human health and disease.

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Section: Methodsmentioning

confidence: 99%

Section: Case Studies: Aav and The Sars-cov-2 N Proteinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Automated Tools to Advance High-Resolution Imaging in Liquid

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Conformation Dynamics of Single Polymer Strands in Solution

Bae

Bang

et al. 2022

Advanced Materials

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structures. For example, intrinsically disordered proteins, which account for one-third of the proteins in the human proteome, do not adopt a standardized 3D structure, and their spontaneous interconversion between unfolded states is crucial in dynamic biological processes. [4] In addition, various synthetic polymers, which are highly regulated by complex molecular interactions and the resulting conformational changes, form a variety of highorder structures via the self-structuring of individual molecules. [5][6][7][8] Thus, it is important to understand the intrinsic structural diversity and dynamic behaviors of individual macromolecules at the single-chain level.Over the last few decades, the conformation and dynamics of a single chain have been widely studied both theoretically and experimentally. Theoretical and computational modeling of single-chain dynamics in ideal solutions are well established in the field of polymer physics. [9][10][11][12] Moreover, enhanced sampling simulation techniques can efficiently locate candidates for energetically stable structures and calculate the free energy differences between their distinct states. [13][14][15] Nonetheless, it is still challenging to understand realistic single-chain behaviors using computational methods, as it is not trivial to simulate ion-solute interactions, crowding, or confinement in polymer solutions. [16][17][18] Furthermore, it is not rare to find a Conformational changes in macromolecules significantly affect their functions and assembly into high-level structures. Despite advances in theoretical and experimental studies, investigations into the intrinsic conformational variations and dynamic motions of single macromolecules remain challenging. Here, liquid-phase transmission electron microscopy enables the real-time tracking of single-chain polymers. Imaging linear polymers, synthetically dendronized with conjugated aromatic groups, in organic solvent confined within graphene liquid cells, directly exhibits chain-resolved conformational dynamics of individual semiflexible polymers. These experimental and theoretical analyses reveal that the dynamic conformational transitions of the single-chain polymer originate from the degree of intrachain interactions. In situ observations also show that such dynamics of the single-chain polymer are significantly affected by environmental factors, including surfaces and interfaces.

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Radiolysis‐Driven Evolution of Gold Nanostructures – Model Verification by Scale Bridging In Situ Liquid‐Phase Transmission Electron Microscopy and X‐Ray Diffraction

et al. 2022

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Utilizing ionizing radiation for in situ studies in liquid media enables unique insights into nanostructure formation dynamics. As radiolysis interferes with observations, kinetic simulations are employed to understand and exploit beam‐liquid interactions. By introducing an intuitive tool to simulate arbitrary kinetic models for radiation chemistry, it is demonstrated that these models provide a holistic understanding of reaction mechanisms. This is shown for irradiated HAuCl4 solutions allowing for quantitative prediction and tailoring of redox processes in liquid‐phase transmission electron microscopy (LP‐TEM). Moreover, it is demonstrated that kinetic modeling of radiation chemistry is applicable to investigations utilizing X‐rays such as X‐ray diffraction (XRD). This emphasizes that beam‐sample interactions must be considered during XRD in liquid media and shows that reaction kinetics do not provide a threshold dose rate for gold nucleation relevant to LP‐TEM and XRD. Furthermore, it is unveiled that oxidative etching of gold nanoparticles depends on both, precursor concentration, and dose rate. This dependency is exploited to probe the electron beam‐induced shift in Gibbs free energy landscape by analyzing critical radii of gold nanoparticles.

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High‐Resolution Imaging of Human Viruses in Liquid Droplets

Cited by 23 publications

References 35 publications

Automated Tools to Advance High-Resolution Imaging in Liquid

Automated Tools to Advance High-Resolution Imaging in Liquid

Conformation Dynamics of Single Polymer Strands in Solution

Radiolysis‐Driven Evolution of Gold Nanostructures – Model Verification by Scale Bridging In Situ Liquid‐Phase Transmission Electron Microscopy and X‐Ray Diffraction

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