Protein folding intermediates on the dimensionality reduced landscape with UMAP and native contact likelihood.

To understand protein folding mechanisms from molecular dynamics (MD) simulations, it is important to explore not only folded/unfolded states but also representative intermediate structures on the conformational landscape. Here, we propose a novel approach to construct the landscape using the uniform manifold approximation and projection (UMAP) method, which reduces the dimensionality without losing data-point proximity. In the approach, native contact likelihood is used as feature variables rather than the conventional Cartesian coordinates or dihedral angles of protein structures. We tested the performance of UMAP for coarse-grained MD simulation trajectories of B1 domain in protein G and observed on-pathway transient structures and other metastable states on the UMAP conformational landscape. In contrast, these structures were not clearly distinguished on the dimensionality reduced landscape using principal component analysis or time-lagged independent component analysis. This approach is also useful to obtain dynamical information through Markov state modeling and would be applicable to large-scale conformational changes in many other biomacromolecules.

Domain Organization in Plant Blue-Light Receptor Phototropin2 of Arabidopsis thaliana Studied by Small-Angle X-ray Scattering.

Phototropin2 (phot2) is a blue-light (BL) receptor protein that regulates the BL-dependent activities of plants for efficient photosynthesis. Phot2 is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) to absorb BL, and a kinase domain. Photo-activated LOV domains, especially LOV2, play a major role in photo-dependent increase in the phosphorylation activity of the kinase domain. The atomic details of the overall structure of phot2 and the intramolecular mechanism to convert BL energy to a phosphorylation signal remain unknown. We performed structural studies on the LOV fragments LOV1, LOV2, LOV2-linker, and LOV2-kinase, and full-length phot2, using small-angle X-ray scattering (SAXS). The aim of the study was to understand structural changes under BL irradiation and discuss the molecular mechanism that enhance the phosphorylation activity under BL. SAXS is a suitable technique for visualizing molecular structures of proteins in solution at low resolution and is advantageous for monitoring their structural changes in the presence of external physical and/or chemical stimuli. Structural parameters and molecular models of the recombinant specimens were obtained from SAXS profiles in the dark, under BL irradiation, and after dark reversion. LOV1, LOV2, and LOV2-linker fragments displayed minimal structural changes. However, BL-induced rearrangements of functional domains were noted for LOV2-kinase and full-length phot2. Based on the molecular model together with the absorption measurements and biochemical assays, we discuss the intramolecular interactions and domain motions necessary for BL-enhanced phosphorylation activity of phot2.

Blue light-excited LOV1 and LOV2 domains cooperatively regulate the kinase activity of full-length phototropin2 from Arabidopsis.

Phototropin2 (phot2) is a blue-light (BL) receptor that regulates BL-dependent activities for efficient photosynthesis in plants. phot2 comprises two BL-receiving light-oxygen-voltage-sensing domains (LOV1 and LOV2) and a kinase domain. BL-excited LOV2 is thought to be primarily responsible for the BL-dependent activation of the kinase. However, the molecular mechanisms by which small BL-induced conformational changes in the LOV2 domain are transmitted to the kinase remain unclear. Here, we used full-length wild-type and mutant phot2 proteins from Arabidopsis to study their molecular properties in the dark and under BL irradiation. Phosphorylation assays and absorption measurements indicated that the LOV1 domain assists the thermal relaxation of BL-excited LOV2 and vice versa. Using small-angle X-ray scattering and electron microscopy, we observed that phot2 forms a dimer and has a rod shape with a maximum length of 188 Å and a radius of gyration of 44 Å. Under BL, phot2 displayed large conformational changes that bent the rod shape. By superimposing the crystal structures of the LOV1 dimer, LOV2, and a homology model of the kinase to the observed changes, we inferred that the BL-dependent change consisted of positional shifts of both LOV2 and the kinase relative to LOV1. Furthermore, phot2 mutants lacking the photocycle in LOV1 or LOV2 still exhibited conformational changes under BL, suggesting that LOV1 and LOV2 cooperatively contribute to the conformational changes that activate the kinase. These results suggest that BL-activated LOV1 contributes to the kinase activity of phot2. We discuss the possible intramolecular interactions and signaling mechanisms in phot2.

Classification of ab initio models of proteins restored from small-angle X-ray scattering.

In structure analyses of proteins in solution by using small-angle X-ray scattering (SAXS), the molecular models are restored by using ab initio molecular modeling algorithms. There can be variation among restored models owing to the loss of phase information in the scattering profiles, averaging with regard to the orientation of proteins against the direction of the incident X-ray beam, and also conformational fluctuations. In many cases, a representative molecular model is obtained by averaging models restored in a number of ab initio calculations, which possibly provide nonrealistic models inconsistent with the biological and structural information about the target protein. Here, a protocol for classifying predicted models by multivariate analysis to select probable and realistic models is proposed. In the protocol, each structure model is represented as a point in a hyper-dimensional space describing the shape of the model. Principal component analysis followed by the clustering method is applied to visualize the distribution of the points in the hyper-dimensional space. Then, the classification provides an opportunity to exclude nonrealistic models. The feasibility of the protocol was examined through the application to the SAXS profiles of four proteins.

Diffraction apparatus and procedure in tomography X-ray diffraction imaging for biological cells at cryogenic temperature using synchrotron X-ray radiation.

X-ray diffraction imaging is a technique for visualizing the structure of biological cells. In X-ray diffraction imaging experiments using synchrotron radiation, cryogenic conditions are necessary in order to reduce radiation damage in the biological cells. Frozen-hydrated biological specimens kept at cryogenic temperatures are also free from drying and bubbling, which occurs in wet specimens under vacuum conditions. In a previous study, the diffraction apparatus KOTOBUKI-1 [Nakasako et al. (2013), Rev. Sci. Instrum. 84, 093705] was constructed for X-ray diffraction imaging at cryogenic temperatures by utilizing a cryogenic pot, which is a cooling device developed in low-temperature physics. In this study a new cryogenic pot, suitable for tomography experiments, has been developed. The pot can rotate a biological cell over an angular range of ±170° against the direction of the incident X-ray beam. Herein, the details and the performance of the pot and miscellaneous devices are reported, along with established experimental procedures including specimen preparation. The apparatus has been used in tomography experiments for visualizing the three-dimensional structure of a Cyanidioschyzon merolae cell with an approximate size of 5 µm at a resolution of 136 nm. Based on the experimental results, the necessary improvements for future experiments and the resolution limit achievable under experimental conditions within a maximum tolerable dose are discussed.

Blue Light-excited Light-Oxygen-Voltage-sensing Domain 2 (LOV2) Triggers a Rearrangement of the Kinase Domain to Induce Phosphorylation Activity in Arabidopsis Phototropin1.

Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS). The fragment existed as a dimer and displayed photoreversible SAXS changes reflected in the radii of gyration of 42.9 Å in the dark and 48.8 Å under BL irradiation. In the dark, the molecular shape reconstructed from the SAXS profiles appeared as two bean-shaped lobes in a twisted arrangement that was 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape under BL became slightly elongated from that in the dark. By fitting the crystal structure of the LOV2 dimer and a homology model of the kinase domain to their inferred shapes, the BL-dependent change could be interpreted as the positional shift in the kinase domain relative to that of the LOV2 dimer. In addition, we found that lysine 475, a functionally important residue, in the N-terminal region of LOV2 plays a critical role in transmitting the structural changes in LOV2 to the kinase domain. The interface between the domains is critical for signaling, suitably changing the structure to activate the kinase in response to conformational changes in the adjoining LOV2.

Specimen preparation for cryogenic coherent X-ray diffraction imaging of biological cells and cellular organelles by using the X-ray free-electron laser at SACLA.

Coherent X-ray diffraction imaging (CXDI) allows internal structures of biological cells and cellular organelles to be analyzed. CXDI experiments have been conducted at 66 K for frozen-hydrated biological specimens at the SPring-8 Angstrom Compact Free-Electron Laser facility (SACLA). In these cryogenic CXDI experiments using X-ray free-electron laser (XFEL) pulses, specimen particles dispersed on thin membranes of specimen disks are transferred into the vacuum chamber of a diffraction apparatus. Because focused single XFEL pulses destroy specimen particles at the atomic level, diffraction patterns are collected through raster scanning the specimen disks to provide fresh specimen particles in the irradiation area. The efficiency of diffraction data collection in cryogenic experiments depends on the quality of the prepared specimens. Here, detailed procedures for preparing frozen-hydrated biological specimens, particularly thin membranes and devices developed in our laboratory, are reported. In addition, the quality of the frozen-hydrated specimens are evaluated by analyzing the characteristics of the collected diffraction patterns. Based on the experimental results, the internal structures of the frozen-hydrated specimens and the future development for efficient diffraction data collection are discussed.

CryoEM-sampling of metastable conformations appearing in cofactor-ligand association and catalysis of glutamate dehydrogenase.

Kinetic aspects of enzymatic reactions are described by equations based on the Michaelis-Menten theory for the initial stage. However, the kinetic parameters provide little information on the atomic mechanism of the reaction. In this study, we analyzed structures of glutamate dehydrogenase in the initial and steady stages of the reaction using cryoEM at near-atomic resolution. In the initial stage, four metastable conformations displayed different domain motions and cofactor/ligand association modes. The most striking finding was that the enzyme-cofactor-substrate complex, treated as a single state in the enzyme kinetic theory, comprised at least three different metastable conformations. In the steady stage, seven conformations, including derivatives from the four conformations in the initial stage, made the reaction pathway complicated. Based on the visualized conformations, we discussed stage-dependent pathways to illustrate the dynamics of the enzyme in action.

Prediction of hydrophilic and hydrophobic hydration structure of protein by neural network optimized using experimental data.

The hydration structures of proteins, which are necessary for their folding, stability, and functions, were visualized using X-ray and neutron crystallography and transmission electron microscopy. However, complete visualization of hydration structures over the entire protein surface remains difficult. To compensate for this incompleteness, we developed a three-dimensional convolutional neural network to predict the probability distribution of hydration water molecules on the hydrophilic and hydrophobic surfaces, and in the cavities of proteins. The neural network was optimized using the distribution patterns of protein atoms around the hydration water molecules identified in the high-resolution X-ray crystal structures. We examined the feasibility of the neural network using water sites in the protein crystal structures that were not included in the datasets. The predicted distribution covered most of the experimentally identified hydration sites, with local maxima appearing in their vicinity. This computational approach will help to highlight the relevance of hydration structures to the biological functions and dynamics of proteins.

Coenzyme-binding pathway on glutamate dehydrogenase suggested from multiple-binding sites visualized by cryo-electron microscopy.

The structure of hexameric glutamate dehydrogenase (GDH) in the presence of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP) was visualized using cryogenic transmission electron microscopy to investigate the ligand-binding pathways to the active site of the enzyme. Each subunit of GDH comprises one hexamer-forming core domain and one nucleotide-binding domain (NAD domain), which spontaneously opens and closes the active-site cleft situated between the two domains. In the presence of NADP, the potential map of GDH hexamer, assuming D3 symmetry, was determined at a resolution of 2.4 Å, but the NAD domain was blurred due to the conformational variety. After focused classification with respect to the NAD domain, the potential maps interpreted as NADP molecules appeared at five different sites in the active-site cleft. The subunits associated with NADP molecules were close to one of the four metastable conformations in the unliganded state. Three of the five binding sites suggested a pathway of NADP molecules to approach the active-site cleft for initiating the enzymatic reaction. The other two binding modes may rarely appear in the presence of glutamate, as demonstrated by the reaction kinetics. Based on the visualized structures and the results from the enzymatic kinetics, we discussed the binding modes of NADP to GDH in the absence and presence of glutamate.

Ultrastructure and fractal property of chromosomes in close-to-native yeast nuclei visualized using X-ray laser diffraction.

Genome compaction and activity in the nucleus depend on spatiotemporal changes in the organization of chromatins in chromosomes. However, the direct imaging of the chromosome structures in the nuclei has been difficult and challenging. Herein, we directly visualized the structure of chromosomes in frozen-hydrated nuclei of budding yeast in the interphase using X-ray laser diffraction. The reconstructed projection electron density maps revealed inhomogeneous distributions of chromosomes, such as a 300 nm assembly and fibrous substructures in the elliptic-circular shaped nuclei of approximately 800 nm. In addition, from the diffraction patterns, we confirmed the absence of regular arrangements of chromosomes and chromatins with 400-20 nm spacing, and demonstrated that chromosomes were composed of self-similarly assembled substructural domains with an average radius of gyration of 58 nm and smooth surfaces. Based on these analyses, we constructed putative models to discuss the organization of 16 chromosomes, carrying DNA of 4.1 mm in 800 nm ellipsoid of the nucleus at the interphase. We anticipate the structural parameters on the fractal property of chromosomes and the experimental images to be a starting point for constructing more sophisticated 3D structural models of the nucleus.

Red light-induced structure changes in phytochrome A from Pisum sativum.

Phytochrome A (phyA) is a photoreceptor protein of plants that regulates the red/far-red light photomorphogenic responses of plants essential for growth and development. PhyA, composed of approximately 1100 amino acid residues, folds into photosensory and output signaling modules. The photosensory module covalently binds phytochromobilin as a chromophore for photoreversible interconversion between inactive red light-absorbing (Pr) and active far-red light-absorbing (Pfr) forms to act as a light-driven phosphorylation enzyme. To understand the molecular mechanism in the initial process of photomorphogenic response, we studied the molecular structures of large phyA (LphyA) from Pisum sativum, which lacks the 52 residues in the N-terminal, by small-angle X-ray scattering combined with multivariate analyses applied to molecular models predicted from the scattering profiles. According to our analyses, Pr was in a dimer and had a four-leaf shape, and the subunit was approximated as a bent rod of 175 × 50 Å. The scattering profile of Pfr was calculated from that recorded for a mixture of Pr and Pfr under red-light irradiation by using their population determined from the absorption spectrum. The Pfr dimer exhibited a butterfly shape composed of subunits with a straight rod of 175 × 50 Å. The shape differences between Pr and Pfr indicated conformational changes in the Pr/Pfr interconversion which would be essential to the interaction with protein molecules involved in transcriptional control.

Common architectures in cyanobacteria Prochlorococcus cells visualized by X-ray diffraction imaging using X-ray free electron laser.

Visualization of intracellular structures and their spatial organization inside cells without any modification is essential to understand the mechanisms underlying the biological functions of cells. Here, we investigated the intracellular structure of cyanobacteria Prochlorococcus in the interphase by X-ray diffraction imaging using X-ray free-electron laser. A number of diffraction patterns from single cells smaller than 1 µm in size were collected with high signal-to-noise ratio with a resolution of up to 30 nm. From diffraction patterns, a set of electron density maps projected along the direction of the incident X-ray were retrieved with high reliability. The most characteristic structure found to be common among the cells was a C-shaped arrangement of 100-nm sized high-density spots, which surrounded a low-density area of 100 nm. Furthermore, a three-dimensional map reconstructed from the projection maps of individual cells was non-uniform, indicating the presence of common structures among cyanobacteria cells in the interphase. By referring to the fluorescent images for distributions of thylakoid membranes, nucleoids, and carboxysomes, we inferred and represented their spatial arrangements in the three-dimensional map. The arrangement allowed us to discuss the relevance of the intracellular organization to the biological functions of cyanobacteria.

Assessment of Force Field Accuracy Using Cryogenic Electron Microscopy Data of Hyper-thermostable Glutamate Dehydrogenase.

Molecular dynamics (MD) simulations in biophysically relevant time scales of microseconds is a powerful tool for studying biomolecular processes, but results often display force field dependency. Therefore, assessment of force field accuracy using experimental data of biomolecules in solution is essential for simulation studies. Here, we propose the use of structural models obtained via cryo-electron microscopy (cryoEM), which provides biomolecular structures in vitreous ice mimicking the environment in solution. The accuracy of the AMBER (ff99SB-ILDN-NMR, ff14SB, ff15ipq, and ff15FB) and CHARMM (CHARMM22 and CHARMM36m) force fields was assessed by comparing their MD trajectories with the cryoEM data of thermostable hexameric glutamate dehydrogenase (GDH), which included a cryoEM map at a resolution of approximately 3 Å and structure models of subunits reflecting metastable conformations in domain motion occurring in GDH. In the assessment, we validated the force fields with respect to the reproducibility and stability of secondary structures and intersubunit interactions in the cryoEM data. Furthermore, we evaluated the force fields regarding the reproducibility of the energy landscape in the domain motion expected from the cryoEM data. As a result, among the six force fields, ff15FB and ff99SB-ILDN-NMR displayed good agreement with the experiment. The present study demonstrated the advantages of the high-resolution cryoEM map and suggested the optimal force field to reproduce experimentally observed protein structures.

Energy landscape of domain motion in glutamate dehydrogenase deduced from cryo-electron microscopy.

Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo-electron microscopy and proposed a novel method to construct free-energy landscape of protein conformations. Each subunit of the homo-hexameric enzyme comprises nucleotide-binding domain (NAD domain) and hexamer-forming core domain. A large active-site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three-dimensional map reconstructed from all cryo-electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200-ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free-energy landscape in domain motion. In the open/close domain motion, two free-energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free-energy landscape were discussed. DATABASE: The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the four conformations were deposited to the Protein Data Bank (PDB) as accession codes 6JN9 (open), 6JNA (half-open1), 6JNC (half-open2), and 6JND (closed), respectively.

Molecular shape under far-red light and red light-induced association of Arabidopsis phytochrome B.

Phytochrome B (phyB) is a plant photoreceptor protein that regulates various photomorphogenic responses to optimize plant growth and development. PhyB exists in two photoconvertible forms: a red light-absorbing (Pr) and a far-red light-absorbing (Pfr) form. Therefore, to understand the mechanism of phototransformation, the structural characterization of full-length phyB in these two forms is necessary. Here, we report the molecular structure of Arabidopsis thaliana phyB in Pr form and the molecular properties of the Pfr form determined by small-angle X-ray scattering coupled with size-exclusion chromatography. In solution, the Pr form associated as a dimer with a radius of gyration of 50 Å. The molecular shape was a crossed shape, in which the orientation of the photosensory modules differed from that in the crystal structure of dimeric photosensory module. PhyB exhibited structural reversibility in the Pfr-to-Pr phototransformation and thermal reversion from Pfr to Pr in the dark. In addition, Pfr only exhibited nonspecific association, which distinguished molecular properties of Pfr form from those of the inactive Pr form.

Methods and application of coherent X-ray diffraction imaging of noncrystalline particles.

Microscopic imaging techniques have been developed to visualize events occurring in biological cells. Coherent X-ray diffraction imaging is one of the techniques applicable to structural analyses of cells and organelles, which have never been crystallized. In the experiment, a single noncrystalline particle is illuminated by an X-ray beam with almost complete spatial coherence. The structure of the particle projected along the direction of the beam is, in principle, retrieved from a finely recorded diffraction pattern alone by using iterative phase-retrieval algorithms. Here, we describe fundamental theory and experimental methods of coherent X-ray diffraction imaging and the recent application in structural studies of noncrystalline specimens by using X-rays available at Super Photon Ring of 8-Gev and SPring-8 Angstrom Compact Free Electron Laser in Japan.