Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM
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59 BASIC BIOLOGICAL SCIENCES↗
SEARCH · Engineering Papers
Results for “cryo-electron microscopy”
Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.
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Combining Spatial and Temporal Resolution in Cryo-TEM of Device Materials: Preprint
Cryogenic transmission electron microscopy (cryo-TEM) has been instrumental to reducing the effects of electron-beam sample interactions and enabling higher spatial resolution studies of beam sensitive materials. However, the process of thinning and freezing samples for cryo-TEM characterization is often complex and frequently involves removing materials from their environment or stimuli of interest for extended times. This can allow for structural relaxation, diffusion, and other dynamic processes to occur prior to characterization; for energy materials like battery electrodes, this makes it difficult to precisely correlate the imaged structure with native structures that evolve in-situ during device processing, operation, or aging. Here, we present methods to prepare cryo-TEM samples from energy materials during these critical processes in the relevant device lifetimes. This offers insights into improving the temporal resolution of cryo-TEM, while still exploiting its ability to achieve high spatial resolutions in characterizing beam sensitive materials.
BASIC BIOLOGICAL SCIENCES,MATERIALS SCIENCE↗
Influence of Electrolyte Additives on Interfacial Stability of Manganese-Rich Lithium-Ion Battery Cathodes
Affordable, long-lasting energy storage has become critical to support increased electricity demand in recent years. Cobalt-free, lithium- and manganese-rich lithium nickel manganese oxide (LMR-NM) cathodes stand to reduce cost and supply-chain concerns associated with traditional cobalt-containing cathodes for lithium-ion batteries by leveraging more earth-abundant materials; however, they have shown issues with long-term cycling stability. Here, we investigate lithium difluoro(oxalate)borate (LiDFOB), tris(trimethylsilyl) phosphite (TMSPi), and vinylene carbonate (VC) electrolyte additives for their ability to improve cycling performance of LMR-NM (0.3 Li 2 MnO 3 + 0.7 LiMn 0.5 Ni 0.5 0 2 ) cells. Cryogenic scanning transmission electron microscopy (cryo-STEM) with electron energy loss spectroscopy enables the construction of a structure–function relationship between cathode electrolyte interphase (CEI) characteristics and the electrochemical performance of cells aged with these additives. We find the combination of 2 wt % TMSPi + 1 wt % LiDFOB performs better than any single additive, achieving a 28% improvement in specific capacity over the baseline electrolyte after long-term cycling. We attribute this to LiDFOB mitigating Mn ion dissolution, with cryo-STEM showing Mn stabilized up to the CEI surface, coupled with improved CEI structure and chemistry enabled by TMSPi, evidenced by a moderately thick (∼7–15 nm) CEI that appears to protect against further electrolyte reactions with the particle. These results, achieved through site-specific nanoscale characterization, directly reveal mechanisms through which electrolyte engineering can improve the performance of earth-abundant cathodes, enabling informed development of more affordable and reliable batteries to meet future energy storage needs.
25 ENERGY STORAGE↗
Mechanism-Informed Breakdown: Understanding Degradation by Controlling Voltage-Hold Patterns in Proton Exchange Membrane Water Electrolyzers
Low catalyst loadings pose challenges to performance stability in proton exchange membrane (PEM) water electrolysis over extended operation. To study the impact of degradation mechanisms and voltage loss rates, different stress tests are applied to membrane electrode assemblies. Potential cycling conditions were observed to induce higher degrees of iridium (Ir) oxide crystallization, ionomer degradation, and catalyst layer (CL) thinning, which likely contributed to higher kinetic loss rates. On the other hand, while Ir migrating into the PEM (Ir band) generally impairs performance, the interconnected and more uniform Ir band formed under a constant 2 V hold may allow for Ir at the catalyst/membrane interface to remain electronically connected and kinetically accessible, as well as indicate greater Ir site access during the applied stressor. The 2 V hold also demonstrates improved kinetic durability through a lower Tafel slope, faster polarization kinetics, and reduced charge transfer resistance. In contrast, potential cycling caused the migration of disconnected Ir agglomerates into the membrane bulk and created a steady increase in charge transfer resistance, a more dramatic decrease in capacitance (46.7% loss), and significant damage to the surrounding ionomer, indicating a decline in both the quality and quantity of active sites in the anode CL. This work underscores the distinct degradation pathways associated with load holds versus cycling, highlighting the role of catalyst-ionomer interactions in kinetic performance and long-term stability. These insights can inform operational strategies for PEM electrolyzers powered by intermittent energy sources, aiming to minimize efficiency losses over extended operation.
36 MATERIALS SCIENCE↗
Continuity of Mitochondrial Budding: Insights from BS-C-1 Cells by In Situ Cryo-electron Tomography
Mitochondrial division is a fundamental biological process essensial for cellular functionality and vitality. The prevailing hypothesis that dynamin related protein 1 (Drp1) provides principal control in mitochondrial division, in which it also involves the endoplasmic reticulum (ER) and the cytoskeleton, does not account for all the observations. Therefore. the hypothesis may be incomplete. Our previous study in HeLa cells led to a new hypothesis of mitochondrial division by budding. To follow-up our previous study, we employed in situ cryo-electron tomography to visualize mitochondrial budding in the intact healthy monkey kidney cells (BS-C-1 cells). Our findings reaffirm single and multiple mitochondrial budding, consistent with our observations in HeLa cells. Notably, the budding regions vary significantly in diameter and length, which may represent different stages of budding. More interestingly, neither rings nor ring-like structures, nor the wrapping of ER tubes was observed in the budding regions, suggesting mitochondrial budding is independent from Drp1 and ER. Meanwhile, we uncovered direct interactions between mitochondria and large vesicles that are distinct from small mitochondrial-derived vesicles and extracellular mitovesicles. In conclusion, we propose that these interacting vesicles may have mitochondrial origins.
(Cryo-EM)↗
The His-tag as a decoy modulating preferred orientation in cryoEM
The His-tag is a widely used affinity tag that facilitates purification by means of affinity chromatography of recombinant proteins for functional and structural studies. We show here that His-tag presence affects how coproheme decarboxylase interacts with the air-water interface during grid preparation for cryoEM. Depending on His-tag presence or absence, we observe significant changes in patterns of preferred orientation. Our analysis of particle orientations suggests that His-tag presence can mask the hydrophobic and hydrophilic patches on a protein’s surface that mediate the interactions with the air-water interface, while the hydrophobic linker between a His-tag and the coding sequence of the protein may enhance other interactions with the air-water interface. Our observations suggest that tagging, including rational design of the linkers between an affinity tag and a protein of interest, offer a promising approach to modulating interactions with the air-water interface.
59 BASIC BIOLOGICAL SCIENCES↗
Alphavirus Particles Can Assemble with an Alternate Triangulation Number
Alphaviruses are spherical, enveloped RNA viruses primarily transmitted by mosquitoes, and cause significant arthritogenic and neurotropic disease in humans and livestock. Previous reports have shown that—in contrast to prototypical icosahedral viruses—alphaviruses incorporate frequent defects, and these may serve important functions in the viral life cycle. We confirm the genus-wide pleomorphism in live viral particles and extend our understanding of alphavirus assembly through the discovery of an alternate architecture of Eastern equine encephalitis virus (EEEV) particles. The alternate T = 3 icosahedral architecture differs in triangulation number from the classic T = 4 icosahedral organization that typifies alphaviruses, but the alternate architecture maintains the quasi-equivalence relationship of asymmetric units. The fusion spike glycoproteins are more loosely apposed in the T = 3 form with corresponding changes in the underlying capsid protein lattice. This alternate architecture could potentially be exploited in engineering alphavirus-based particles for delivery of alphaviral or other RNA.
59 BASIC BIOLOGICAL SCIENCES↗
CryoSheet Jet™: A Novel Deposition and Rapid Cooling Method for Cryogenic Sample Preparation (Final Report)
Explore the source record for details and available documents.
59 BASIC BIOLOGICAL SCIENCES↗
Characterizing Dynamic Structure in Battery Electrodes by Time-Resolved Cryo-TEM
In recent years, cryogenic transmission electron microscopy (cryo-TEM) has enabled high-resolution characterization of sensitive battery materials by minimizing electron beam-induced artifacts and damage. Success of this technique relies on the preparation of thin, rapidly frozen samples, generally by disassembling batteries under inert atmosphere, transferring materials of interest to a TEM grid, and finally plunge freezing into a cryogen. In material degradation studies, this extensive time between electrochemical cycling and cryo-TEM characterization leaves room for structural relaxation, diffusion, and other dynamic processes that make it difficult to precisely correlate the imaged structure with the native structure that evolves during battery cycling or aging. Here, we present a method to integrate battery cycling with fast preparation of electrode samples for cryo-TEM. This enables higher fidelity between the structures characterized and the electrochemical state of interest, which we use to study deformation in silicon nanoparticle anodes for lithium-ion batteries.
CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSICS↗
Understanding In-Situ Structure and Chemistry at Battery Interfaces Through Time-Resolved Cryo-EM
Transmission and scanning transmission electron microscopy are vital tools for characterizing interfacial structure and chemistry at high spatial resolutions but are limited by the fact that many materials are quickly damaged by electron beam irradiation. This is a challenge particularly for materials relevant to energy applications, such as lithium-containing battery materials, organic-inorganic hybrids, and liquid electrolytes. Cryogenic electron microscopy (cryo-EM) offers a partial solution to this issue by stabilizing beam-sensitive solid-solid or solid-liquid interfaces for characterization, but typical cryo-EM sample preparations are ex situ and thus cannot capture interfaces in their electrified state of interest to applications. Here, we present a method to prepare cryo-EM samples of electrified battery interfaces in situ to preserve the time-resolved structures and chemistry that arise under applied current or bias.
batteries↗
Ion-Depleted Microenvironments During Lithium Deposition Revealed by Operando Freezing Cryogenic Electron Microscopy
Local structures and chemistry at active electrochemical interfaces are critical to determining safety, lifetime, and energy density in lithium metal batteries and other devices, but they are challenging to characterize at the nanoscale. We address this issue by developing operando freezing cryogenic electron microscopy (cryo-EM) to preserve battery interfaces in an active state for subsequent high-resolution characterization. We find that ion-depleted microenvironments form locally in the electrolyte adjacent to the lithium deposition interface in lithium metal batteries and are linked to heterogenous growth morphologies. These depleted environments arise locally even under conditions for which ion depletion is not predicted at steady state; this provides a mechanistic explanation for why dangerous lithium morphologies can still propagate in such systems and lead to thermal runaway. Operando freezing cryo-EM thus provides a method to directly visualize nanoscale heterogeneities that arise locally at electrochemical interfaces and play key roles in device failure.
CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSICS,E↗
Microfluidic synthesis of multilayered lipid–polymer hybrid nanoparticles for the formulation of low solubility drugs
Hybrid phospholipid/block copolymer membranes where polymers and lipids are molecularly mixed or phase-separated into polymer-rich and lipid-rich domains are promising drug delivery materials. Harnessing the chemical diversity of polymers and the biocompatability of lipids is a compelling approach to design the next generation of drug carriers. Here, we report on the development of a microfluidics-based strategy analogous to produce lipid nanoparticles (LNPs) for the nanomanufacturing of multilayered hybrid nanoparticles (HNPs). In conclusion, using X-ray scattering, Cryo-electron, and polarized microscopy we show that phosphatidylcholine (PC) and PBD-b-PEO (poly(butadiene-block-ethylene oxide)) hybrid membranes can be nanomanufactured by microfluidics into HNPs with dense and multilayered cores which are ideal carriers of low-solubility drugs of the Biopharmaceutical Classification System (BCS) II and IV such as antimalarial DSM265 and Paclitaxel, respectively.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Low Temperature Electron Microscopy with High Field Superconducting Lenses
Cryo-electron microscope with high field superconducting lenses
Fernandez-Moran, H.↗
Structure, folding and flexibility of co-transcriptional RNA origami
RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Furthermore, exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices.
77 NANOSCIENCE AND NANOTECHNOLOGY↗
Aqueous Self‐Assembly of Cylindrical and Tapered Bottlebrush Block Copolymers
The self‐assembly of amphiphilic bottlebrush block copolymers (BCPs), featuring backbones densely grafted with two types of side chains, is less well understood compared to linear BCPs. In particular, the solution self‐assembly of tapered bottlebrush BCPs—cone‐shaped BCPs with hydrophilic or hydrophobic tips—remains unexplored. This study investigates eight tapered and four cylindrical bottlebrush BCPs with varied ratios of hydrophobic polystyrene (PS) and hydrophilic poly(acrylic acid) (PAA) side chains, synthesized via sequential addition of macromonomers using ring‐opening metathesis polymerization (SAM‐ROMP). Self‐assembled nanostructures formed in water were analyzed using cryogenic transmission electron microscopy, small‐angle neutron scattering, and dynamic light scattering. Most BCPs generated multiple nanostructures with surface protrusions, including spherical micelles, cylindrical micelles, and vesicles, alongside transitional forms like ellipsoids and semi‐vesicles. Coarse‐grained molecular dynamics simulations supported the experimental findings, which revealed two distinct self‐assembly pathways. The first involved micelle fusion, producing elliptical and cylindrical aggregates, sometimes forming Y‐junctions. The second pathway featured micelle maturation into semivesicles, which developed into vesicles or large compound vesicles. This work provides the first experimental evidence of vesicle formation via semivesicles in bottlebrush BCPs and demonstrates the significant influence of cone directionality on self‐assembly behavior in these cone‐shaped polymeric amphiphiles.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Evolution of standardization and dissemination of cryo-EM structures and data jointly by the community, PDB, and EMDB
Cryogenic electron microscopy (cryo-EM) methods began to be used in the mid-1970s to study thin and periodic arrays of proteins. Following a half-century of development in cryo-specimen preparation, instrumentation, data collection, data processing and modeling software, cryo-EM has become a routine method for solving structures from large biological assemblies to small biomolecules at near to true atomic resolution. This review explores the critical roles played by the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) in partnership with the community to develop the necessary infrastructure to archive cryo-EM maps and associated models. Public access to cryo-EM structure data has in turn facilitated better understanding of structure-function relationships and advancement of image processing and modeling tool development. The partnership between the global cryo-EM community and PDB and EMDB leadership has synergistically shaped the standards for metadata, one-stop deposition of maps and models, and validation metrics to assess the quality of cryo-EM structures. The advent of cryo-electron tomography (cryo-ET) for in situ molecular cell structures at a broad resolution range and their correlations with other imaging data introduces new data archival challenges in terms of data size and complexity in the years to come.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery
Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV’s complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein’s post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.
59 BASIC BIOLOGICAL SCIENCES↗
Correlative montage parallel array cryo-tomography for in situ structural cell biology
Abstract Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.
59 BASIC BIOLOGICAL SCIENCES↗