Abstract Three-dimensional protein crystals are ordered, porous macroscopic materials with potential applications in catalysis, biosensing, and biomedicine. However, most protein crystals are obtained by empirical screening, providing limited control over the lattice architecture, pore geometry or component composition that determine material function. Here, we present a modular strategy for the programmable design of highly porous, framework-like protein crystals using predefined protein-protein interactions. This strategy yielded over 30 distinct protein crystals, including single-component and multicomponent P 2 1 3 and I 2 1 3 lattices that grow to over 100 µm in size. Small-angle X-ray scattering and electron microscopy showed close agreement between experimental lattices and computational models. RFdiffusion-guided design generated isomorphous variants with matched lattice parameters, enabling coherent protein crystal alloys, epitaxial core–shell growth and reversible shell assembly. The designed crystals exhibit tunable mesoporous architectures, with limiting apertures of 2–18 nm, and support genetically encoded incorporation of fluorescent protein guests. These results establish a general route to programmable lattice engineering of protein crystals and position them as genetically encoded, compositionally tunable mesoporous materials.

Lipid nanoparticles (LNPs) are versatile carriers for nucleic acid (NA) therapeutics, including ASOs, siRNA, mRNA, and poly-IC. While lipid composition is known to influence LNP properties, the impact of NA length on morphology and internal structure is less understood, particularly during the stages of carrier-cargo assembly. Here, we examine NA length and lipid composition immediately after mixing using high-throughput SAXS, dynamic light scattering, and cryogenic electron microscopy. All LNPs form ordered NA/lipid compartments, with longer NAs promoting inverse hexagonal (HII) phases and larger intercompartment distances. In contrast, short NAs, especially in formulations with SM102 ionizable lipid, favor lamellar phases. SAXS peak deconvolution quantifies ordered versus disordered phases via a Robustness of Ordered Phase factor, which correlates with particle size and encapsulation efficiency. Formulations with MC3 ionizable and DOPE helper lipids exhibit the most stable HII-phase packing, highlighting the role of helper-lipid curvature in compartment stabilization. Variations in NA compartmentalization indicate differences in payload capacity, offering a framework for rational LNP design across diverse nucleic acid cargos.

Deep learning has revolutionized structural biology by prediction with near experimental accuracy static protein folds from amino acid sequence alone. However, proteins function as dynamic ensembles of protein conformation states, and current sequence-only models often fail to capture the specific conformational states and heterogeneity dictated by cellular environments or ligand binding. While recent generative models can sample broad conformational landscapes, they remain unconstrained by physical reality, often hallucinating plausible but experimentally invalid states. Here, we present AlphaSAXS, an end-to-end framework that constrains artificial intelligence (AI) inference using Small Angle X-ray Scattering (SAXS) experimental solution scattering data. By integrating real-space pair distance distributions ( P r ) directly into the AlphaFold architecture, AlphaSAXS effectively steers the structural hypothesis toward the experimentally observed structures. We demonstrate that AlphaSAXS resolves documented failure modes of sequence-only models in Apo-Holo transitions, successfully distinguishing between states with identical sequences but distinct scattering profiles. Furthermore, we introduce a hybrid inference protocol that couples deep learning with biophysical hydration modeling, enabling the reconstruction of solution state protein ensembles compatible with experimental data. This work establishes a paradigm for experimentally guided AI, bridging the gap between probabilistic sampling and biophysical measurement.

Lipid nanoparticles (LNPs) are under intensive development following their tremendous success in vaccine development during the COVID-19 pandemic. While shown to be effective drug carriers for oligonucleotide therapeutics, LNPs are challenging to optimize due to their high structural and performance variability based on factors such as lipid composition and formulation method. To achieve a comprehensive understanding of the relationship between LNP formulation, structure, and efficacy, a high-throughput workflow would unlock the ability to test the vast parameter space involved in LNP development with more efficiency. In this talk, we will discuss our efforts in LNP characterization by leveraging a high-throughput small angle X-ray scattering (HT-SAXS) platform. This system can be used to rapidly assess LNP internal structures, observe correlations with LNP activity and trends across experimental conditions such as formulation methods, and triage samples for complementary methods such as cryogenic transmission electron microscopy (cryo-TEM). The resulting data are stored in the open data repository Simple Scattering (simplescattering.com), where we envision building a collective knowledge base of LNP structure-activity relationship. Through this workflow, we seek to develop an in-depth understanding of LNP structure and activity to enable a more effective design and optimization of LNP-based therapeutics.

The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor NUB1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system with human components and revealed that NUB1 uses the intrinsic instability of FAT10 to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin-independent and p97-independent manner. Using hydrogen-deuterium exchange, structural modeling and site-directed mutagenesis, we identified the formation of an intricate complex with FAT10 that activates NUB1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic NUB1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These findings identified a previously unknown mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.

Solution scattering (SAXS) can be used to screen for small molecule inhibitors of proteins, providing several advantages. SAXS is a direct measure of structure and can be used to identify conformational or assembly changes induced by small molecules. This capability compliments the many other solution techniques that probe binding (DSF, MST, SPR…). While SAXS is higher throughput than crystallography or cryoEM, throughput and sample quantity remain challenges for wider spread applications. At the SIBYLS beamline at the Advanced Light Source, we have developed a higher throughput and small volume SAXS data collection capability. I will describe the application of this system to the protein Apoptosis Inducing Factor (AIF). The system is somewhat unique in that small molecules can trigger multimeric changes. Still further, natively, multimerization is controlled by reduction of co-factors. X-rays will reduce this co-factor in a time dependent and exposure associated manner. Small molecules were identified that provided protection or hastened X-ray induced multimerization changes, providing significantly more information about the mechanism of small molecule binding to AIF. To extend SAXS small molecule studies to other systems where protein abundance is limited we have also conducted studies using Gold labels. The protein PARP1 is a major drug target for a host of cancer related phenomenon. PARP1 recognizes nicked DNA. During recognition PARP1 bends DNA and in the presence of the co-factor NAD+, initiates signaling for further repair mechanisms. By attaching Gold to both ends of double stranded DNA with a central nick, we have boosted the SAXS signal of these event by over 1000 fold. The boost in signal, reduces sample requirements so that a 1000 member small molecule library could be screened. The technique and results will be further discussed.

ALS-ENABLE is an integrated NIH P30 resource at the Advanced Light Source synchrotron at Lawrence Berkeley National Laboratory in Berkeley, California, USA. The resource provides a single portal to the combined mature structural biology technologies of macromolecular crystallography, small-angle X-ray scattering and X-ray footprinting mass spectrometry, and includes beamlines 2.0.1, 3.3.1, 4.2.2, 5.0.1, 5.0.2, 5.0.3, 8.2.1, 8.2.2, 8.3.1 and 12.3.1. This paper describes the organizational structure and the technologies of ALS-ENABLE. A case study showcasing the main technologies of the resource applied to the characterization of the SpyCatcher–SpyTag protein system is presented.

Single-walled carbon nanotubes (SWCNTs) are near-infrared fluorescent substrates used in biological sensing, imaging, and delivery applications. These constructs are composed of SWCNTs in concert with an associated corona phase surrounding the nanotube. In this manner, noncovalent adsorption of polymers onto SWCNTs is typically implemented to disperse hydrophobic SWCNTs in solution, while also providing a functional handle such as a molecular recognition element that confers analyte specificity, giving rise to a nanosensor. For example, single-stranded DNA (ssDNA)-functionalized SWCNTs have been developed in which distinct ssDNA sequences confer specificity to different neurotransmitters for real-time brain sensing. However, this initial corona phase may be replaced by other biomolecules, predominantly proteins, once the SWCNT-based constructs are applied in the actual biological system of interest. Consequently, function of these and other nanoparticle-based biosensors is often hindered by protein corona formation, in which adsorbed proteins from the surrounding biofluid preclude interaction of the sensor with the target analyte. Establishing rational design rules to inform development and refinement of such nanosensors requires an understanding of both the initial nanoscale architectures and compositional changes upon application in complex biofluids. Herein, we describe methods and outcomes to investigate the carbon nanotube corona phase, both initially and upon interaction with proteins in the surrounding solution. To study the as-designed corona, we demonstrate a direct mode of measuring in-solution nucleic acid geometries on SWCNTs via X-ray scattering interferometry (XSI). This technique leverages the interference pattern produced by ordered gold nanoparticle tags conjugated to the DNA on the SWCNT surface. Using XSI, we quantify surface-adsorbed nucleic acid morphologies, ionic strength-dependent conformational changes, and the dopamine sensing mechanism of a DNA-SWCNT nanosensor, supported by ab initio modeling. To study the protein corona-covered SWCNT, we expand upon our previous work profiling protein adsorption on mixed-chirality SWCNTs that revealed the composition and driving forces of protein corona formation. We now characterize the protein corona across a panel of different nanosensor-relevant ssDNA sequences and single-chirality SWCNT substrates. Taken together, we aim to elucidate both the initial and end-state SWCNT surface to guide future nanobiotechnology design strategies.

Staphylococcus aureus expresses three high-affinity neutrophil serine protease (NSP) inhibitors known as the extracellular adherence protein domain (EAPs) proteins. Whereas EapH1 and EapH2 are comprised of a single EAP domain, the modular extracellular adherence protein (Eap) from S. aureus strain Mu50 consists of four EAP domains. We recently reported that EapH2 can simultaneously bind and inhibit cathepsin-G (CG) and neutrophil elastase (NE), which are the two most abundant NSPs. This unusual property of EapH2 arises from independent CG and NE-binding sites that lie on opposing faces of its EAP domain. Here we used X-ray crystallography and enzyme assays to show that all four individual domains of Eap (i.e. Eap1, Eap2, Eap3, and Eap4) exhibit an EapH2-like ability to form ternary complexes with CG and NE that inhibit both enzymes simultaneously. We found that Eap1, Eap2, and Eap3 have similar functional profiles insofar as NSP inhibition is concerned, but that Eap4 displays an unexpected ability to inhibit two NE enzymes simultaneously. Using X-ray crystallography, we determined that this second NE-binding site in Eap4 arises through the same region of its EAP domain that also comprises its CG-binding site. Interestingly, small angle X-ray scattering data showed that stable tail-to-tail dimers of the NE/Eap4/NE ternary complex exist in solution. This arrangement is compatible with NSP-binding at all available sites in a two-domain fragment of Eap. Together, our work implies that Eap is a polyvalent inhibitor of NSPs. It also raises the possibility that higher-order structures of NSP-bound Eap may have unique functional properties.

The surge in RNA therapeutics has revolutionized treatments for infectious diseases like COVID-19 and shows the potential to expand into other therapeutic areas. However, the typical requirement for ultra-cold storage of mRNA-LNP formulations poses significant logistical challenges for global distribution. Lyophilization serves as a potential strategy to extend mRNA-LNP stability while eliminating the need for ultra-cold supply chain logistics. Although recent advancements have demonstrated the promise of lyophilization, the choice of lyoprotectant is predominately focused on sucrose, and there remains a gap in comprehensive evaluation and comparison of lyoprotectants and buffers. Here, we aim to systematically investigate the impact of a diverse range of excipients including oligosaccharides, polymers, amino acids, and various buffers, on the quality and performance of lyophilized mRNA-LNPs. From the screening of 45 mRNA-LNP formulations under various lyoprotectant and buffer conditions for lyophilization, we identified previously unexplored formulation compositions, e.g., polyvinylpyrrolidone (PVP) in Tris or acetate buffers, as promising alternatives to the commonly used oligosaccharides to maintain the physicochemical stability of lyophilized mRNA-LNPs. Further, we delved into how physicochemical and structural properties influence the functionality of lyophilized mRNA-LNPs. Leveraging high-throughput small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM), we showed that there is complex interplay between mRNA-LNP structural features and cellular translation efficacy. We also assessed innate immune responses of the screened mRNA-LNPs in human peripheral blood mononuclear cells (PBMCs), and showed minimal alterations of cytokine secretion profiles induced by lyophilized formulations. Our results provide valuable insights into the structure-activity relationship of lyophilized formulations of mRNA-LNP therapeutics, paving the way for rational design of these formulations. This work creates a foundation for a comprehensive understanding of mRNA-LNP properties and in vitro performance change resulting from lyophilization.

We provide the first crystallographic structure of a green mononuclear monodomain cupredoxin. Analysis of the structure suggests that the coupled distortion model might not explain the behaviour of some cupredoxins.

Influenza A and B viruses overcome the host antiviral response to cause a contagious and often severe human respiratory disease. Here, integrative structural biology and biochemistry studies on non-structural protein 1 of influenza B virus (NS1B) reveal a previously unrecognized viral mechanism for innate immune evasion. Conserved basic groups of its C-terminal domain (NS1B-CTD) bind 5'triphosphorylated double-stranded RNA (5'-ppp-dsRNA), the primary pathogen-associated feature that activates the host retinoic acid-inducible gene I protein (RIG-I) to initiate interferon synthesis and the cellular antiviral response. Like RIG-I, NS1B-CTD preferentially binds blunt-end 5'ppp-dsRNA. NS1B-CTD also competes with RIG-I for binding 5'ppp-dsRNA, and thus suppresses activation of RIG-I's ATPase activity. Although the NS1B N-terminal domain also binds dsRNA, it utilizes a different binding mode and lacks 5'ppp-dsRNA end preferences. In cells infected with wild-type influenza B virus, RIG-I activation is inhibited. In contrast, RIG-I activation and the resulting phosphorylation of transcription factor IRF-3 are not inhibited in cells infected with a mutant virus encoding NS1B with a R208A substitution it its CTD that eliminates its 5'ppp-dsRNA binding activity. These results reveal a novel mechanism in which NS1B binds 5'ppp-dsRNA to inhibit the RIG-I antiviral response during influenza B virus infection, and open the door to new avenues for antiviral drug discovery.

Lipid nanoparticles (LNPs) are being intensively researched and developed to leverage their ability to safely and effectively deliver therapeutics. To achieve optimal therapeutic delivery, a comprehensive understanding of the relationship between formulation, structure, and efficacy is critical. However, the vast chemical space involved in the production of LNPs and the resulting structural complexity make the structure to function relationship challenging to assess and predict. New components and formulation procedures, which provide new opportunities for the use of LNPs, would be best identified and optimized using high-throughput characterization methods. Recently, a high-throughput workflow, consisting of automated mixing, small-angle X-ray scattering (SAXS), and cellular assays, demonstrated a link between formulation, internal structure, and efficacy for a library of LNPs. As SAXS data can be rapidly collected, the stage is set for the collection of thousands of SAXS profiles from a myriad of LNP formulations. In addition, correlated LNP small-angle neutron scattering (SANS) datasets, where components are systematically deuterated for additional contrast inside, provide complementary structural information. The centralization of SAXS and SANS datasets from LNPs, with appropriate, standardized metadata describing formulation parameters, into a data repository will provide valuable guidance for the formulation of LNPs with desired properties. To this end, we introduce Simple Scattering, an easy-to-use, open data repository for storing and sharing groups of correlated scattering profiles obtained from LNP screening experiments. Here, we discuss the current state of the repository, including limitations and upcoming changes, and our vision towards future usage in developing our collective knowledge base of LNPs.

Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF). By monitoring oxidized and X-ray-reduced AIF states, TR-HT-SAXS leverages structure and kinetics to generate a multidimensional screening dataset that identifies fragment chemotypes allosterically stimulating AIF dimerization. Fragment-induced dimerization rates, quantified with time-resolved SAXS similarity analysis (k VR), capture structure-activity relationships (SAR) across the top-ranked 4-aminoquinoline chemotype. Crystallized AIF-aminoquinoline complexes validate TR-SAXS-guided SAR, supporting this conformational chemotype for optimization. AIF-aminoquinoline structures and mutational analysis reveal active site F482 as an underappreciated allosteric stabilizer of AIF dimerization. This conformational discovery pipeline illustrates TR-HT-SAXS as an effective technology for targeting chemical leads to important macromolecular states. A discovery pipeline integrating time-resolved HT-SAXS and fragment screening identifies chemical leads targeting exemplary allosteric states of mitochondrial oxidoreductase apoptosis-inducing factor (AIF).

The recent clinical and commercial success of lipid nanoparticles (LNPs) for nucleic acid delivery has incentivized the development of new technologies to manufacture LNPs. As new technologies emerge, researchers must determine which technologies to assess and how to perform comparative evaluations. In this article, we use a quality-by-design approach to systematically investigate how the mixer technology used to form LNPs influences LNPstructure. Specifically, a coaxial turbulent jet mixer and a staggered herringbone microfluidic mixer were systematically compared via matched formulation and process conditions. A full-factorial design-of-experiments study with three factors and three levels was executed for each mixer to compare process robustness in the production of antisense oligonucleotide (ASO) LNPs. ASO-LNPs generated with the coaxial turbulent jet mixer were consistently smaller, had a narrower particle size distribution, and had a higher ASO encapsulation as compared to the microfluidic mixer, but had a greater variation in internal structure with less ordered cores. A subset of the study was replicated for mRNA-LNPs with comparable trends in particle size and encapsulation, but more frequent bleb features for LNPs produced by the coaxial turbulent jet mixer. The study design used here provides a road map for how researchers may compare different mixer technologies (or process changes more broadly) and how such studies can inform process robustness and manufacturing control strategies.

With three FDA-approved products, lipid nanoparticles (LNPs) are under intensive development for delivering wide-ranging nucleic acid therapeutics. A significant challenge for LNP development is insufficient understanding of structure-activity relationship (SAR). Small changes in chemical composition and process parameters can affect LNP structure, significantly impacting performance in vitro and in vivo. The choice of polyethylene glycol lipid (PEG-lipid), one of the essential lipids for LNP, has been proven to govern particle size. Here we find that PEG-lipids can further modify the core organization of antisense oligonucleotide (ASO)-loaded LNPs to govern its gene silencing activity. Furthermore, we also have found that the extent of compartmentalization, measured by the ratio of disordered vs ordered inverted hexagonal phases within an ASO-lipid core, is predictive of in vitro gene silencing. In this work, we propose that a lower ratio of disordered/ordered core phases correlates with stronger gene knockdown efficacy. To establish these findings, we developed a seamless high-throughput screening approach that integrated an automated LNP formulation system with structural analysis by small-angle X-ray scattering (SAXS) and in vitro TMEM106b mRNA knockdown assessment. We applied this approach to screen 54 ASO-LNP formulations while varying the type and concentration of PEG-lipids. Representative formulations with diverse SAXS profiles were further visualized using cryogenic electron microscopy (cryo-EM) to help structural elucidation. The proposed SAR was built by combining this structural analysis with in vitro data. Our integrated methods, analysis, and resulting findings on PEG-lipid can be applied to rapidly optimize other LNP formulations in a complex design space.