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SISGR: The regulation of carbon fixation in plant and green algae: Rubisco activase and the origin of heat inactivation of CO 2 assimilation
Rubisco activase (Rca) is a critical AAA+ ATPase protein complex that remodels and promotes the Rubisco enzyme, a key player in photosynthetic performance and carbon fixation. The assembly and function of the Rca protein complex are regulated by a range of factors, including subunit concentration, nucleotide-binding states, thermal conditions, metal-ion coordination, and post-translational modifications, such as phosphorylation. Despite its importance in photosynthesis, the detailed molecular mechanisms underlying the regulation of plant Rca and how it activates Rubisco remain elusive. This project aims to bridge this knowledge gap by integrating sophisticated enzymology tools with single-molecule methods and high-resolution electron microscopy to elucidate the structure and function of plant Rca. Through these multiple approaches, we have systematically investigated how the activity of plant Rca is impacted by various factors, such as phosphorylation and metal-ion coordination. The Rca complex assembly/disassembly dynamics were captured using anti-Brownian electrokinetic (ABEL) trap-based measurements, providing unprecedented insight into its structural flexibility and diverse assembly states. Furthermore, the structural analysis of Rca through electron crystallography and single-particle cryogenic electron microscopy (cryo-EM) reveals novel assembly states of the spinach Rca, providing insight into the mechanistic action for Rubisco remodeling. By combining cutting-edge tools and approaches, this work uncovers critical aspects of Rca’s regulation and assembly, paving the way for a deeper understanding of its role in photosynthetic efficiency and the potential for enhancing carbon fixation in crops.
Quantification of Dynamic Scattering Effects in Molecular Crystals using Large Angle Rocking Beam Electron Diffraction
Electron crystallography provides a pathway to solve structure of small crystals (< 1um) in size, and thus overcomes difficult synthesis constraints involved in growing large crystals. Generally, electron diffraction data is collected in the form of integrated intensity using continuous rotation or by precession. The measured intensities in 3D are utilized for structure solution. Using this approach, structure solution of difficult crystals, such as small crystals of zeolites, metal-organic frameworks, molecular crystals, and proteins , can be solved by electron diffraction. However, electron structure solutions are regularly reported with higher Rvalues than x-ray or neutron diffraction. While similar structures are found despite the high R-values, the differences in the measure intensity and theory calculated intensity limit information that can be extracted by electron diffraction. Previous work demonstrated that including multiple-scattering effects significantly reduce the R-values . Thus, it is critical to be able to quantify the dynamical diffraction effects in molecular crystals.
Mapping Electron Beam-Induced Radiolytic Damage in Molecular Crystals
Every electron crystallography experiment is fundamentally constrained by radiation damage [1]. Despite its crucial importance, little is understood about the onset and progression of radiolysis in beam-sensitive molecular crystals [2]. Since the typical metric used for monitoring the degradation induced by radiolysis is the attenuation of Bragg reflections in electron diffraction patterns [3], the scope of previous analyses [2-6] has mostly been limited to indirect observation via the back focal plane. Here, a simultaneous visualization of the effects of electron beam-induced radiolysis—in both real space and reciprocal space—remains elusive.
Conditional diffusion machine-learning framework for mapping valence electron distribution from convergent beam electron diffraction
Quantitative convergent beam electron diffraction (CBED) enables determination of aspherical valence electron distributions through refinement of low-order structure factors, which are highly sensitive to chemical bonding and charge density variations. However, conventional quantitative CBED (QCBED) requires solving a highly nonlinear inverse problem with many coupled parameters, and computationally intensive dynamical diffraction calculations, making it time-consuming and difficult to apply to complex systems. More broadly, reconstructing charge density and orbital electron distribution from diffraction data has long been a central challenge in both x-ray and electron crystallography. Here, in this study, we introduce an artificial-intelligence (AI)-based framework that replaces traditional refinement with a data-driven inverse solver. Using a large synthetic CBED dataset generated by Bloch-wave simulations, we train a conditional diffusion model to directly infer crystal structural parameters and multipole density formalism parameters, and hence valence electron distributions, from CBED patterns alone. By learning from forward simulations across realistic parameter space, the model effectively solves the inverse problem. Compared with direct regression approaches, the diffusion-based framework provides posterior parameter distributions for rigorous uncertainty quantification while preserving quantitative fidelity and reducing analysis time by orders of magnitude. By eliminating the need for external single-crystal x-ray diffraction data and complex nonlinear refinement, this approach enables practical, high-throughput, and in situ quantitative CBED, enabling real-time mapping of valence electron distributions and their correlation with functional responses in quantum and energy materials.
Pyroxenes in Serra de Mage - Cooling history in comparison with Moama and Moore County
Thin sections and single grains of pyroxenes from the Serra de Mage feldspar cumulate eucrites were studied by X-ray crystallography, electron microprobe and optical techniques. It was concluded that the pyroxene crystallized as pigeonite. On cooling augite was exsolved along (001) and inverted to hypersthene, with exsolution of (100) augite from hypersthene during continued slow cooling. The estimated original bulk composition of the pigeonite pyroxene is Wo10En51Fs39. The compositional data, textural relations, and existence of P2 sub 1 ca hypersthene suggest very low cooling (about 0.0004 deg C/year) below 800 deg. The Serra de Mage augite lamellae were found to be as thick or thicker than those of Moore County and Moama meteorites.
Reaching the potential of electron diffraction
Microcrystal electron diffraction (MicroED) is an emerging structural technique in which submicron crystals are used to generate diffraction data for structural studies. Structures allow for the study of molecular-level architecture and drive hypotheses about modes of action, mechanisms, dynamics, and interactions with other molecules. Combining cryoelectron microscopy (cryo-EM) instrumentation with crystallographic techniques, MicroED has led to three-dimensional structural models of small molecules, peptides, and proteins and has generated tremendous interest due to its ability to use vanishingly small crystals. In this perspective, we describe the current state of the field for MicroED methodologies, including making and detecting crystals of the appropriate size for the technique, as well as ways to best handle and characterize these crystals. Our perspective provides insight into ways to unlock the full range of potential for MicroED to access previously intractable samples and describes areas of future development.
Transmission electron microscopy of three recrystallized al-al sub 2 o sub 3 sap-type alloys.
Transmission electron microscopic study of microstructure of three recrystallized aluminum- aluminum oxide SAP type alloys
Solving the “Coloring Problem” in InPd 3– x Ag x ( x = 0–0.7) by Phase Diagrams Modeling and Diffraction Experiments
Here, a series of InPd 3–x Ag x (x = 0–1) compositions were synthesized by conventional high-temperature synthesis, and as-synthesized samples were characterized by powder X-ray diffraction experiments. Up to x = 0.7, InPd 3–x Ag x adopts the ternary substitutional variant of the InPd 3 structure (TiAl 3 -type), when x > 0.7, elemental Ag starts to segregate along with the main phase. Accurate structural characterization in InPd 3–x Ag x faces a critical challenge due to the narrow X-ray scattering contrast among constituents In, Pd, and Ag and nearly identical neutron scattering lengths of Pd and Ag. To overcome this “coloring problem”, a combination of calculation of phase diagrams modeling (CALPHAD) and diffraction techniques (X-ray and neutron) was employed. In the compositional range 0 ≤ x ≤ 0.7, InPd 3–x Ag x presents a ternary variant of the TiAl 3 -type structure, where Ag atoms selectively substitute one (the 2b Wyckoff site) of the two Pd sites in InPd 3 . Notably, in contrast to the isologous InPd 3–x Cu x (x = 0–1) system, Ag substitution does not form an ordered VRh 2 Sn-type structure at the limiting composition. The distinct site preference in InPd 3–x Ag x is elucidated by charge population analysis, electronic structure calculations, and orbital-resolved chemical bonding investigations, and the extent of substitution is supported by formation free energy calculations.
Structural properties and recrystallization effects in ion beam modified B20-type FeGe films
Disordered iron germanium (FeGe) has recently garnered interest as a testbed for a variety of magnetic phenomena as well as for use in magnetic memory and logic applications. This is partially owing to its ability to host skyrmions and antiskyrmions—nanoscale whirlpools of magnetic moments that could serve as information carriers in spintronic devices. In particular, a tunable skyrmion–antiskyrmion system may be created through precise control of the defect landscape in B20-phase FeGe, motivating the development of methods to systematically tune disorder in this material and understand the ensuing structural properties. To this end, we investigate a route for modifying magnetic properties in FeGe. In particular, we irradiate epitaxial B20-phase FeGe films with 2.8 MeV Au 4+ ions, which creates a dispersion of amorphized regions that may preferentially host antiskyrmions at densities controlled by the irradiation fluence. To further tune the disorder landscape, we conduct a systematic electron diffraction study with in situ annealing, demonstrating the ability to recrystallize controllable fractions of the material at temperatures ranging from ~150 to 250°C. Finally, we describe the crystallization kinetics using the Johnson–Mehl–Avrami–Kolmogorov model, finding that the growth of crystalline grains is consistent with diffusion-controlled one-to-two dimensional growth with a decreasing nucleation rate.
Design of facilitated dissociation enables timing of cytokine signalling
Protein design has focused on the design of ground states, ensuring that they are sufficiently low energy to be highly populated. Designing the kinetics and dynamics of a system requires, in addition, the design of excited states that are traversed in transitions from one low-lying state to another. This is a challenging task because such states must be sufficiently strained to be poorly populated, but not so strained that they are not populated at all, and because protein design methods have focused on generating near-ideal structures. Here we describe a general approach for designing systems that use an induced-fit power stroke to generate a structurally frustrated and strained excited state, allosterically driving protein complex dissociation. X-ray crystallography, double electron–electron resonance spectroscopy and kinetic binding measurements show that incorporating excited states enables the design of effector-induced increases in dissociation rates as high as 5,700-fold. We highlight the power of this approach by designing rapid biosensors, kinetically controlled circuits and cytokine mimics that can be dissociated from their receptors within seconds, enabling dissection of the temporal dynamics of interleukin-2 signalling.
A parametric study of single-wall carbon nanotube growth by laser ablation
Results of a parametric study of carbon nanotube production by the double-pulse laser oven process are presented. The effect of various operating parameters on the production of single-wall carbon nanotubes (SWCNTs) is estimated by characterizing the nanotube material using analytical techniques, including scanning electron microscopy, transmission electron microscopy, thermo gravimetric analysis and Raman spectroscopy. The study included changing the sequence of the laser pulses, laser energy, pulse separation, type of buffer gas used, operating pressure, flow rate, inner tube diameter, as well as its material, and oven temperature. It was found that the material quality and quantity improve with deviation from normal operation parameters such as laser energy density higher than 1.5 J/cm2, pressure lower than 67 kPa, and flow rates higher than 100 sccm. Use of helium produced mainly small diameter tubes and a lower yield. The diameter of SWCNTs decreases with decreasing oven temperature and lower flow rates.
Structural and Mechanistic Advances in the Chemistry of Methyl-Coenzyme M Reductase (MCR)
Methane represents 34% of U.S. energy consumption and is a major greenhouse gas related to the global carbon cycle and energy production. However, current industrial practices significantly increase atmospheric methane levels, necessitating a deeper understanding of its biosynthesis and oxidation. Methyl-coenzyme M reductase (MCR) is central to biological methane metabolism, catalyzing the final step of methanogenesis and the first step in anaerobic methane oxidation. It is also a key target for strategies to capture and transform methane into value-added chemicals.The active site of MCR is a buried Ni-based cofactor only accessible by the substrates via a 50 Å long tunnel. Although the Ni(I) state is required to initiate catalysis, capturing this state remains a challenge for the current structural techniques. Recent advances in structural biology using X-ray Free-Electron Laser serial crystallography have provided insights into MCR's inactive Ni(II) state at room temperature and show promise for capturing its active Ni(I) form.Our team has established several critical aspects of the MCR mechanism using a combination of experimental and computational studies. MCR uses CH 3 -SCoM and CoBSH as substrates, producing methane and a disulfide product CoMSSCoB. Kinetic analysis showed that productive substrate binding requires CH 3 -SCoM to bind first, inducing conformational changes that optimize the active site for subsequent CoBSH binding. Following substrate binding, four proposed methane production/oxidation mechanisms were examined, establishing whether the reaction proceeds through an organometallic methyl-nickel(III), methyl anion ion, or methyl radical intermediate. Experimental measurements using CoBSH analogs successfully slowed the reaction, allowing for mechanistic insight that demonstrated the methyl radical pathway, where the initial interactions involve homolytic cleavage of the methyl-sulfur bond, generating a methyl radical that quickly abstracts the thiol hydrogen atom of CoBSH to form methane. Computational studies further confirmed that, compared to other mechanisms, the methyl radical mechanism is thermodynamically more favorable and accessible under physiological conditions.Spectroscopic and computational studies challenged the conventional understanding of substrate binding in MCR by proposing an alternative positioning of CH 3 -SCoM and CoMSSCoB in the active site pocket. The research suggested that CH 3 -SCoM (substrate) and CoMSSCoB (product) bind via their sulfonate groups to the Ni(I) center of cofactor F 430 . This binding allows for the reaction without substrate reorganization in the pocket but would require a long-range electron transfer. Altogether, the work summarized in this review reflects our current understanding of the enzyme's catalytic mechanism and structural dynamics. This is essential for developing efficient methane conversion technologies that could mitigate its environmental impact while harnessing energy-storage properties.
Structures of a synthetic antibody selected against and bound to the C‐terminal domain of Clostridium perfringens enterotoxin
Abstract Clostridium perfringensenterotoxin (CpE) causes cytotoxic gastrointestinal disease in mammalian epithelium by binding membrane protein receptors called claudins. Claudins direct the formation of cell/cell tight junctions through oligomerization and govern the transport of molecules between individual cells. CpE binds claudins through its C‐terminal domain (cCpE) and induces cytotoxicity through its N‐terminal domain. The non‐toxic cCpE is a useful tool to study claudins, tight junctions, and for translational applications, such as increasing the permeability of restrictive tissues like the blood–brain barrier or selective targeting of claudin overexpressing cancers. Conversely, there are no specialized molecular tools to study CpE or cCpE, or to modulate or inhibit their functions. We previously reported the development of synthetic antigen‐binding fragments (sFabs) that bind cCpE, and low‐resolution structures of them bound to claudin/cCpE complexes. Here, we determine high‐resolution structures of sFab COP‐2 bound to cCpE using X‐ray crystallography and cryogenic electron microscopy. The structures and biophysical findings provide the mechanism of COP‐2 binding to cCpE and the molecular determinants driving their interactions. These insights can advance the design of new antibody‐based tools from our COP‐2 scaffold to study or alter cCpE function and give rise to a “Trojan horse” strategy that exploits cCpE's tight junction barrier disrupting function to selectively deliver conjugated therapeutics through normally impermeable tissues.
Surface studies with (clean) supported metal particles and clusters
Small particles are ideal for combined studies with surface analytical and electron microscopy/diffraction methods. In this way crystallography, microstructure, morphology, physical stability, and electronic structure can be correlated directly with chemical reactivity. Various integrated experimental approaches for conducting model studies with UHV-evaporated particles have been successfully developed; they entail the use of TPD, AES, XPS, and work function measurements on one hand and standard TEM/TED, in-situ TEM/TED, and in-situ STED/TEM techniques on the other hand. The essential features of four particular experimental approaches are discussed and a selection of representative results is presented to illustrate the potential usefulness of such studies in the field of catalysis.
3D Nanocrystallography and the Imperfect Molecular Lattice
Crystallographic analysis relies on the scattering of quanta from arrays of atoms that populate a repeating lattice. While large crystals built of lattices that appear ideal are sought after by crystallographers, imperfections are the norm for molecular crystals. Additionally, advanced X-ray and electron diffraction techniques, used for crystallography, have opened the possibility of interrogating micro- and nanoscale crystals, with edges only millions or even thousands of molecules long. These crystals exist in a size regime that approximates the lower bounds for traditional models of crystal nonuniformity and imperfection. Accordingly, data generated by diffraction from both X-rays and electrons show increased complexity and are more challenging to conventionally model. New approaches in serial crystallography and spatially resolved electron diffraction mapping are changing this paradigm by better accounting for variability within and between crystals. The intersection of these methods presents an opportunity for a more comprehensive understanding of the structure and properties of nanocrystalline materials.
High voltage electron microscopy of lunar samples
Lunar pyroxenes from Apollo 11, 12, 14, and 15 were investigated. The iron-rich and magnesium-rich pyroxene specimens were crushed to a grain size of ca. 50 microns and studied by a combination of X-ray and electron diffraction, electron microscopy, 57 Fe Mossbauer spectroscopy and X-ray crystallography techniques. Highly ordered, uniform electron-dense bands, corresponding to exsolution lamellae, with average widths of ca. 230A to 1000A dependent on the source specimen were observed. These were?qr separated by wider, less-dense interband spacings with average widths of ca. 330A to 3100A. In heating experiments, splitting of the dense bands into finer structures, leading finally to obliteration of the exsolution lamellae was recorded. The extensive exsolution is evidence for significantly slower cooling rates, or possibly annealing, at temperatures in the subsolidus range, adding evidence that annealing of rock from the surface of the moon took place at ca. 600 C. Correlation of the band structure with magnetic ordering at low temperatures and iron clustering within the bands was studied.
Anti-Ebola virus mAb 3A6 protects highly viremic animals from fatal outcome via binding GP(1,2) in a position elevated from the virion membrane
Abstract Monoclonal antibodies (mAbs) against Ebola virus (EBOV) glycoprotein (GP 1,2 ) are the standard of care for Ebola virus disease (EVD). Anti-GP 1,2 mAbs targeting the stalk and membrane proximal external region (MPER) potently neutralize EBOV in vitro and are protective in a mouse model of EVD. However, their neutralization mechanism is poorly understood because they target a GP 1,2 epitope that has evaded structural characterization. Using X-ray crystallography and cryo-electron tomography of mAb 3A6 complexed with its stalk–MPER epitope, we reveal a previously undescribed mechanism in which 3A6 binds to a conformation of GP 1,2 that is lifted from the virion membrane. We further show that in both domestic guinea pig and rhesus monkey EVD models, 3A6 provides therapeutic benefit at high-viremia advanced disease stages and at the lowest dose yet demonstrated for any anti-EBOV mAb-based monotherapy. The findings reported here can guide design of next-generation highly potent anti-EBOV therapeutics and vaccines.