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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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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Structural studies of the IFNλ4 receptor complex using cryoEM enabled by protein engineering

Abstract IFNλ4 has posed a conundrum in human immunology since its discovery in 2013, with its expression linked to complications with viral clearance. While genetic and cellular studies revealed the detrimental effects of IFNλ4 expression, extensive structural and functional characterization has been limited by the inability to express and purify the protein, complicating explanations of its paradoxical behavior. In this work, we report a method for robust production of IFNλ4. We then use yeast surface display to affinity-mature IL10Rβ and solve the 72 kilodalton structures of IFNλ4 (3.26 Å) and IFNλ3 (3.00 Å) in complex with their receptors IFNλR1 and IL10Rβ using cryogenic electron microscopy. Comparison of the structures highlights differences in receptor engagement and reveals a distinct 12-degree rotation in overall receptor geometry, providing a potential mechanistic explanation for differences in cell signaling, downstream gene induction, and antiviral activities. Further, we perform a structural analysis using molecular modeling and simulation to identify a unique region of IFNλ4 that, when replaced, enables secretion of the protein from cells. These findings provide a structural and functional understanding of the IFNλ4 protein and enable future comprehensive studies towards correcting IFNλ4 dysfunction in large populations of affected patients.

Science & Technology - Other Topics↗
DOE OSTI2022

Plant-specific features of respiratory supercomplex I + III2 from Vigna radiata

Abstract The last steps of cellular respiration—an essential metabolic process in plants—are carried out by mitochondrial oxidative phosphorylation. This process involves a chain of multi-subunit membrane protein complexes (complexes I–V) that form higher-order assemblies called supercomplexes. Although supercomplexes are the most physiologically relevant form of the oxidative phosphorylation complexes, their functions and structures remain mostly unknown. Here we present the cryogenic electron microscopy structure of the supercomplex I + III 2 from Vigna radiata (mung bean). The structure contains the full subunit complement of complex I, including a newly assigned, plant-specific subunit. It also shows differences in the mitochondrial processing peptidase domain of complex III 2 relative to a previously determined supercomplex with complex IV. The supercomplex interface, while reminiscent of that in other organisms, is plant specific, with a major interface involving complex III 2 ’s mitochondrial processing peptidase domain and no participation of complex I’s bridge domain. The complex I structure suggests that the bridge domain sets the angle between the enzyme’s two arms, limiting large-scale conformational changes. Moreover, complex I’s catalytic loops and its response in active-to-deactive assays suggest that, in V. radiata , the resting complex adopts a non-canonical state and can sample deactive- or open-like conformations even in the presence of substrate. This study widens our understanding of the possible conformations and behaviour of complex I and supercomplex I + III 2 . Further studies of complex I and its supercomplexes in diverse organisms are needed to determine the universal and clade-specific mechanisms of respiration.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Frenkel excitons in heat-stressed supramolecular nanocomposites enabled by tunable cage-like scaffolding

Delocalized Frenkel excitons—coherently shared excitations among chromophores—are responsible for the remarkable efficiency of supramolecular light-harvesting assemblies within photosynthetic organisms. The translation of nature’s design principles to applications in optoelectronic devices has been limited by the fragility of the supramolecular structures used and the delicate nature of Frenkel excitons, particularly under mildly changing solvent conditions and elevated temperatures and upon deposition onto solid substrates. Here, we overcome those functionalization barriers through composition of stable supramolecular light-harvesting nanotubes enabled by tunable (~4.3–4.9 nm), uniform (±0.3 nm) cage-like scaffolds. High-resolution cryogenic electron microscopy, combined with scanning electron microscopy, broadband femtosecond transient absorption spectroscopy and near-field scanning optical microscopy revealed that excitons within the cage-like scaffolds are robust, even under extreme heat stress, and control over nanocomposite dimensions is maintained on solid substrates. Our bio-inspired nanocomposites provide a general framework for the development of next-generation organic devices made from stable supramolecular materials.

14 SOLAR ENERGY↗
DOE OSTI2023

Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates

As genetic code expansion advances beyond l-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. The Escherichia coli ribosome tolerates non-l-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of the E. coli ribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is <4 Å from the peptidyl-tRNA carbonyl with a Bürgi–Dunitz angle of 76–115°. Monomers with free energy minima that fall outside this conformational space do not react efficiently. This insight should accelerate the in vivo and in vitro ribosomal synthesis of sequence-defined, non-peptide heterooligomers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

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↗
DOE OSTI2024

De novo design of pH-responsive self-assembling helical protein filaments

Abstract Biological evolution has led to precise and dynamic nanostructures that reconfigure in response to pH and other environmental conditions. However, designing micrometre-scale protein nanostructures that are environmentally responsive remains a challenge. Here we describe the de novo design of pH-responsive protein filaments built from subunits containing six or nine buried histidine residues that assemble into micrometre-scale, well-ordered fibres at neutral pH. The cryogenic electron microscopy structure of an optimized design is nearly identical to the computational design model for both the subunit internal geometry and the subunit packing into the fibre. Electron, fluorescent and atomic force microscopy characterization reveal a sharp and reversible transition from assembled to disassembled fibres over 0.3 pH units, and rapid fibre disassembly in less than 1 s following a drop in pH. The midpoint of the transition can be tuned by modulating buried histidine-containing hydrogen bond networks. Computational protein design thus provides a route to creating unbound nanomaterials that rapidly respond to small pH changes.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2021

Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution

Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1,2,3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure–function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Additionally, newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure–function relationships in ribozymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structures of a phycobilisome in light-harvesting and photoprotected states

Phycobilisome (PBS) structures are elaborate antennae in cyanobacteria and red algae 1,2 . These large protein complexes capture incident sunlight and transfer the energy through a network of embedded pigment molecules called bilins to the photosynthetic reaction centres. However, light harvesting must also be balanced against the risks of photodamage. A known mode of photoprotection is mediated by orange carotenoid protein (OCP), which binds to PBS when light intensities are high to mediate photoprotective, non-photochemical quenching 3-6 . Here we use cryogenic electron microscopy to solve four structures of the 6.2 MDa PBS, with and without OCP bound, from the model cyanobacterium Synechocystis sp. PCC 6803. The structures contain a previously undescribed linker protein that binds to the membrane-facing side of PBS. For the unquenched PBS, the structures also reveal three different conformational states of the antenna, two previously unknown. The conformational states result from positional switching of two of the rods and may constitute a new mode of regulation of light harvesting. Further, only one of the three PBS conformations can bind to OCP, which suggests that not every PBS is equally susceptible to non-photochemical quenching. In the OCP-PBS complex, quenching is achieved through the binding of four 34kDa OCPs organized as two dimers. The complex reveals the structure of the active form of OCP, in which an approximately 60Å displacement of its regulatory carboxy terminal domain occurs. Finally, by combining our structure with spectroscopic properties 7 , we elucidate energy transfer pathways within PBS in both the quenched and light-harvesting states. Collectively, our results provide detailed insights into the biophysical underpinnings of the control of cyanobacterial light harvesting. The data also have implications for bioengineering PBS regulation in natural and artificial light-harvesting systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

From primordial clocks to circadian oscillators

Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator1. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and a central component of the clock. Subsequent additions of KaiB and KaiA regulate the phosphorylation state of KaiC for time synchronization. The canonical KaiABC system in cyanobacteria is well understood but little is known about more ancient systems that only possess KaiBC. However, there are reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here we investigate the primordial circadian clock in Rhodobacter sphaeroides , which contains only KaiBC, to elucidate its inner workings despite missing KaiA. Using a combination of X-ray crystallography and cryogenic electron microscopy, we find a new dodecameric fold for KaiC, in which two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the carboxy-terminal extension of KaiC and serves as an ancient regulatory moiety that is later superseded by KaiA. A coiled-coil register shift between daytime and night-time conformations is connected to phosphorylation sites through a long-range allosteric network that spans over 140 Å. Our kinetic data identify the difference in the ATP-to-ADP ratio between day and night as the environmental cue that drives the clock. They also unravel mechanistic details that shed light on the evolution of self-sustained oscillators.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

De novo design of protein structure and function with RFdiffusion

Abstract There has been considerable recent progress in designing new proteins using deep-learning methods 1–9 . Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models 10,11 have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence–structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.

Science & Technology - Other Topics↗
DOE OSTI2024

O-GlcNAc forces an α-synuclein amyloid strain with notably diminished seeding and pathology

Amyloid-forming proteins such α-synuclein and tau, which are implicated in Alzheimer’s and Parkinson’s disease, can form different fibril structures or strains with distinct toxic properties, seeding activities and pathology. Understanding the determinants contributing to the formation of different amyloid features could open new avenues for developing disease-specific diagnostics and therapies. Here we report that O-GlcNAc modification of α-synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by cryogenic electron microscopy, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson’s disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc-modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that posttranslational modifications, such as O-GlcNAc modification, of α-synuclein are key determinants of α-synuclein amyloid strains and pathogenicity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structural basis for intermodular communication in assembly-line polyketide biosynthesis

Assembly-line polyketide synthases (PKSs) are modular multi-enzyme systems with considerable potential for genetic reprogramming. Understanding how they selectively transport biosynthetic intermediates along a defined sequence of active sites could be harnessed to rationally alter PKS product structures. Here, to investigate functional interactions between PKS catalytic and substrate acyl carrier protein (ACP) domains, we employed a bifunctional reagent to crosslink transient domain–domain interfaces of a prototypical assembly line, the 6-deoxyerythronolide B synthase, and resolved their structures by single-particle cryogenic electron microscopy (cryo-EM). Together with statistical per-particle image analysis of cryo-EM data, we uncovered interactions between ketosynthase (KS) and ACP domains that discriminate between intra-modular and inter-modular communication while reinforcing the relevance of conformational asymmetry during the catalytic cycle. Our findings provide a foundation for the structure-based design of hybrid PKSs comprising biosynthetic modules from different naturally occurring assembly lines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Measurement of atom resolvability in cryo-EM maps with Q-scores

Cryogenic electron microscopy (cryo-EM) maps are now at the point where resolvability of individual atoms can be achieved. However, resolvability is not necessarily uniform throughout the map. We introduce a quantitative parameter to characterize the resolvability of individual atoms in cryo-EM maps, the map Q-score. Q-scores can be calculated for atoms in proteins, nucleic acids, water, ligands and other solvent atoms, using models fitted to or derived from cryo-EM maps. Q-scores can also be averaged to represent larger features such as entire residues and nucleotides. Averaged over entire models, Q-scores correlate very well with the estimated resolution of cryo-EM maps for both protein and RNA. Here, assuming the models they are calculated from are well fitted to the map, Q-scores can be used as a measure of resolvability in cryo-EM maps at various scales, from entire macromolecules down to individual atoms. Q-score analysis of multiple cryo-EM maps of the same proteins derived from different laboratories confirms the reproducibility of structural features from side chains down to water and ion atoms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Improvement of cryo-EM maps by density modification

A density-modification procedure for improving maps from single-particle electron cryogenic microscopy (cryo-EM) is presented here. The theoretical basis of the method is identical to that of maximum-likelihood density modification, previously used to improve maps from macromolecular X-ray crystallography. Key differences from applications in crystallography are that the errors in Fourier coefficients are largely in the phases in crystallography but in both phases and amplitudes in cryo-EM, and that half-maps with independent errors are available in cryo-EM. These differences lead to a distinct approach for combination of information from starting maps with information obtained in the density-modification process. The density-modification procedure was applied to a set of 104 datasets and improved map-model correlation and increased the visibility of details in many of the maps. The procedure requires two unmasked half-maps and a sequence file or other source of information on the volume of the macromolecule that has been imaged.

47 OTHER INSTRUMENTATION↗
DOE OSTI2023

Improving resolution and resolvability of single-particle cryoEM structures using Gaussian mixture models

Cryogenic electron microscopy is widely used in structural biology, but its resolution is often limited by the dynamics of the macromolecule. Here, we developed a refinement protocol based on Gaussian mixture models that integrates particle orientation and conformation estimation, and improves the alignment for flexible domains of protein structures. We demonstrated this protocol on multiple datasets, resulting in improved resolution and resolvability, locally and globally, by visual and quantitative measures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of the human SAGA coactivator complex

The SAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. While structures of yeast SAGA (ySAGA) have been reported, there are noteworthy functional and compositional differences for this complex in metazoans. Here we present the cryogenic-electron microscopy (cryo-EM) structure of human SAGA (hSAGA) and show how the arrangement of distinct structural elements results in a globally divergent organization from that of yeast, with a different interface tethering the core module to the TRRAP subunit, resulting in a dramatically altered geometry of functional elements and with the integration of a metazoan-specific splicing module. Our hSAGA structure reveals the presence of an inositol hexakisphosphate (InsP 6 ) binding site in TRRAP and an unusual property of its pseudo-(Ψ)PIKK. Finally, we map human disease mutations, thus providing the needed framework for structure-guided drug design of this important therapeutic target for human developmental diseases and cancer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

CRISPR–Cas9 bends and twists DNA to read its sequence

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

59 BASIC BIOLOGICAL SCIENCES↗