Simultaneous Recording of Remote Domain Dynamics in Membrane Proteins Using the Double-Labeled DXB/DXT Technique.

Protein dynamics play important roles in biological functions, which accompany allosteric structure changes. Diffracted X-ray blinking (DXB) uses monochromatic X-rays and nanocrystal probes. The intramolecular motion of target proteins is analyzed from the intensity changes in detector signals at the diffraction rings. In contrast, diffracted X-ray tracking (DXT) elucidates molecular dynamics by analyzing the trajectories of Laue spots. In this study, we have developed a dual-labeling technique for DXB and DXT, allowing the simultaneous observation of motions at different domains in proteins. We identified zinc oxide (ZnO) crystals as promising candidates for the second labeling probes due to their excellent diffraction patterns, high chemical stability, and favorable binding properties with proteins. The diffraction spots from the ZnO crystals are sufficiently separated from those of gold, enabling independent motion analysis at different domains. Dual-labeling DXB was employed for the motion analysis of the 5-HT2A receptor in living cells. Simultaneous motion recording of the N-terminus and the second extracellular loop demonstrated ligand-induced motion suppression at both domains. The dual-labeling DXT technique demonstrated a capsaicin-induced peak shift in the two-dimensional motion maps at the N-terminus of the TRPV1 protein, but the peak shift was not obvious in the C-terminus. The capsaicin-induced motion modulation was recovered by the addition of the competitive inhibitor AMG9810.

Real-time tilting and twisting motions of ligand-bound states of α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor is a member of the nicotinic acetylcholine receptor family and is composed of five α7 subunits arranged symmetrically around a central pore. It is localized in the central nervous system and immune cells and could be a target for treating Alzheimer's disease and schizophrenia. Acetylcholine is a ligand that opens the channel, although prolonged application rapidly decreases the response. Ivermectin was reported as one of the positive allosteric modulators, since the binding of Ivermectin to the channel enhances acetylcholine-evoked α7 currents. One research has suggested that tilting motions of the nicotinic acetylcholine receptor are responsible for channel opening and activation. To verify this hypothesis applies to α7 nicotinic acetylcholine receptor, we utilized a diffracted X-ray tracking method to monitor the stable twisting and tilting motion of nAChR α7 without a ligand, with acetylcholine, with Ivermectin, and with both of them. The results show that the α7 nicotinic acetylcholine receptor twists counterclockwise with the channel transiently opening, transitioning to a desensitized state in the presence of acetylcholine and clockwise without the channel opening in the presence of Ivermectin. We propose that the conformational transition of ACh-bound nAChR α7 may be due to the collective twisting of the five α7 subunits, resulting in the compression and movement, either downward or upward, of one or more subunits, thus manifesting tilting motions. These tilting motions possibly represent the transition from the resting state to channel opening and potentially to the desensitized state.

Preliminary studies of XANES and DFT calculation of Ru extraction by imino-diacetamide and related compounds.

We connected three research fields on Ru extraction, XANES, and DFT calculation and elucidate the sequence of distribution ratio (D) and their reactions. The magnitude order of the distribution ratio, D(Ru), from acids, HCl > H2SO4 > HNO3 > HClO4, by IDOA indicates to extract readily the stable Ru-Cl ions. The XANES signals, which suggests the electrical charge of Ru(III) extracted into the organic phase, supports the ion-pairing extraction of the anionic Ru-Cl complex with an extractant protonated. Ru(III) in other acids might be extracted by solvation of extractant, thus ion-pair extraction is stronger than solvation in Ru extraction. According to the D(Ru), the same extractant trend, NTAamide > MIDOA > IDOA, as the energy gap of HOMO and LUMO by DFT calculation is found, which suggests that DFT calculation can give the relative magnitude of each D(M) value when extractant and metal in an extraction are determined.

The Blinking of Small-Angle X-ray Scattering Reveals the Degradation Process of Protein Crystals at Microsecond Timescale.

X-ray crystallography has revolutionized our understanding of biological macromolecules by elucidating their three-dimensional structures. However, the use of X-rays in this technique raises concerns about potential damage to the protein crystals, which results in a quality degradation of the diffraction data even at very low temperatures. Since such damage can occur on the micro- to millisecond timescale, a development in its real-time measurement has been expected. Here, we introduce diffracted X-ray blinking (DXB), which was originally proposed as a method to analyze the intensity fluctuations of diffraction of crystalline particles, to small-angle X-ray scattering (SAXS) of a lysozyme single-crystal. This novel technique, called the small-angle X-ray blinking (SAXB) method, analyzes the fluctuation in SAXS intensity reflecting the domain fluctuation in the protein crystal caused by the X-ray irradiation, which could be correlated with the X-ray-induced damage on the crystal. There was no change in the protein crystal's domain dynamics between the first and second X-ray exposures at 95K, each of which lasted 0.7 s. On the other hand, its dynamics at 295K increased remarkably. The SAXB method further showed a dramatic increase in domain fluctuations with an increasing dose of X-ray radiation, indicating the significance of this method.

Time-Resolved X-ray Observation of Intracellular Crystallized Protein in Living Animal.

Understanding the cellular environment as molecular crowding that supports the structure-specific functional expression of biomolecules has recently attracted much attention. Time-resolved X-ray observations have the remarkable capability to capture the structural dynamics of biomolecules with subnanometre precision. Nevertheless, the measurement of the intracellular dynamics within live organisms remains a challenge. Here, we explore the potential of utilizing crystallized proteins that spontaneously form intracellular crystals to investigate their intracellular dynamics via time-resolved X-ray observations. We generated transgenic Caenorhabditis elegans specifically expressing the crystallized protein in cells and observed the formation of the protein aggregates within the animal cells. From the toxic-effect observations, the aggregates had minimal toxic effects on living animals. Fluorescence observations showed a significant suppression of the translational diffusion movements in molecules constituting the aggregates. Moreover, X-ray diffraction measurements provided diffraction signals originating from these molecules. We also observed the blinking behaviour of the diffraction spots, indicating the rotational motion of these crystals within the animal cells. A diffracted X-ray blinking (DXB) analysis estimated the rotational motion of the protein crystals on the subnanometre scale. Our results provide a time-resolved X-ray diffraction technique for the monitoring of intracellular dynamics.

Ubiquitination of Major Histocompatibility Complex II Changes Its Immunological Recognition Structure.

Ubiquitination is a process that dictates the lifespan of major histocompatibility complex class II (MHC II)/peptide complexes on antigen-presenting cells. This process is tightly controlled by the levels of ubiquitin ligases, and disruptions in the turnover of MHC II can lead to the improper development of CD4+ T cells within the thymus and hinder the formation of regulatory T cells in the peripheral tissue. To investigate the underlying mechanisms, we utilized dendritic cells lacking the Membrane-associated RING-CH (MARCH) I ubiquitin ligase. We discovered that the overexpression of MARCH I decreases the interaction with LAG-3. Moreover, the MHC II molecules tethered with ubiquitin also showed diminished binding to LAG-3. We employed Diffracted X-ray Blinking (DXB), a technique used for single-molecule X-ray imaging, to observe the protein movements on live cells in real time. Our observations indicated that the normal MHC II molecules moved more rapidly across the cell surface compared to those on the MARCH I-deficient dendritic cells or MHC II KR mutants, which is likely a result of ubiquitination. These findings suggest that the signaling from ubiquitinated MHC II to the T cell receptor differs from the non-ubiquitinated forms. It appears that ubiquitinated MHC II might not be quickly internalized, but rather presents antigens to the T cells, leading to a range of significant immunological responses.

Visualization of the Dynamics of Photoinduced Crawling Motion of 4-(Methylamino)Azobenzene Crystals via Diffracted X-ray Tracking.

The photoinduced crawling motion of crystals is a continuous motion that azobenzene molecular crystals exhibit under light irradiation. Such motion enables object manipulation at the microscale with a simple setup of fixed LED light sources. Transportation of nano-/micromaterials using photoinduced crawling motion has recently been reported. However, the details of the motion mechanism have not been revealed so far. Herein, we report visualization of the dynamics of fine particles in 4-(methylamino)azobenzene (4-MAAB) crystals under light irradiation via diffracted X-ray tracking (DXT). Continuously repeated melting and recrystallization of 4-MAAB crystals under light irradiation results in the flow of liquid 4-MAAB. Zinc oxide (ZnO) particles were introduced inside the 4-MAAB crystals to detect diffracted X-rays. The ZnO particles rotate with the flow of liquid 4-MAAB. By using white X-rays with a wide energy width, the rotation of each zinc oxide nanoparticle was detected as the movement of a bright spot in the X-ray diffraction pattern. It was clearly shown that the ZnO particles rotated increasingly as the irradiation light intensity increased. Furthermore, we also found anisotropy in the rotational direction of ZnO particles that occurred during the crawling motion of 4-MAAB crystals. It has become clear that the flow perpendicular to the supporting film of 4-MAAB crystals is enhanced inside the crystal during the crawling motion. DXT provides a unique means to elucidate the mechanism of photoinduced crawling motion of crystals.

Reproducibility of subscapularis muscle thickness measurement using ultrasound imaging. -Relationship between subscapularis muscle thickness and internal rotation torque of the shoulder joint.

BACKGROUND: Few studies have reported on the morphometry of the subscapularis muscle using ultrasound imaging (USI); and their reproducibility has not been verified. OBJECTIVES: This study aimed to clarify the relative and absolute reproducibility of USI measurements of subscapularis muscle thickness at rest and during isometric contraction as well as the degree of change in muscle thickness caused by the amount of internal rotational torque in the shoulder joint. DESIGN: Two-group repeated-measures study. METHODS: The subjects were the inferior fibers of the subscapularis muscle of 40 healthy adult males. Muscle thickness was measured at rest and at 10%-30% of the maximum isometric internal rotation torque. Intraclass correlation coefficients (ICC) and Brand Altman analysis were used for reproducibility measurement. The degree of change in muscle thickness at each torque was also calculated. RESULTS: Intra- and inter-rater ICCs (ranged from 0.69 to 0.91) were good. A proportional error was observed in intra-rater measurements. Both minimal detectable change 95 (ranged from 2.33 to 6.47) were high. The subscapularis muscle thickness was significantly increased at 10% torque (25.49 ± 3.80 mm), 20% torque (26.07 ± 3.90 mm), and 30% torque (25.96 ± 3.82 mm) as compared to that in resting conditions (24.53 ± 4.46 mm) (p < 0.05). CONCLUSION: The reproducibility and error of the subscapularis muscle thickness measurement using USI used in this study were clarified when repeated measurements were made in the same limb position and under the same probe installation conditions, suggesting that the contraction of the subscapularis muscle can be estimated by muscle thickness measurement.

The ice-binding site of antifreeze protein irreversibly binds to cell surface for its hypothermic protective function.

Antifreeze proteins (AFPs) are multifunctional polypeptides that adsorb onto ice crystals to inhibit their growth and onto cells to protect them from nonfreezing hypothermic damage. However, the mechanism by which AFP exerts its hypothermic cell protective (HCP) function remains uncertain. Here, we assessed the HCP function of three types of fish-derived AFPs (type I, II, and III AFPs) against human T-lymphoblastic lymphoma by measuring the survival rate (%) of the cells after preservation at 4 °C for 24 h. All AFPs improved the survival rate in a concentration-dependent manner, although the HCP efficiency was inferior for type III AFP compared to other AFPs. In addition, after point mutations were introduced into the ice-binding site (IBS) of a type III AFP, HCP activity was dramatically increased, suggesting that the IBS of AFP is involved in cell adsorption. Significantly, high HCP activity was observed for a mutant that exhibited poorer antifreeze activity, indicating that AFP exerts HCP- and ice-binding functions through a different mechanism. We next incubated the cells in an AFP-containing solution, replaced it with pure EC solution, and then preserved the cells, showing that no significant reduction in the cell survival rate occurred for type I and II AFPs even after replacement. Thus, these AFPs irreversibly bind to the cells at 4 °C, and only tightly adsorbed AFP molecules contribute towards the cell-protection function.

Diffracted X-ray Tracking for Observing the Internal Motions of Individual Protein Molecules and Its Extended Methodologies.

In 1998, the diffracted X-ray tracking (DXT) method pioneered the attainment of molecular dynamics measurements within individual molecules. This breakthrough revolutionized the field by enabling unprecedented insights into the complex workings of molecular systems. Similar to the single-molecule fluorescence labeling technique used in the visible range, DXT uses a labeling method and a pink beam to closely track the diffraction pattern emitted from the labeled gold nanocrystals. Moreover, by utilizing X-rays with extremely short wavelengths, DXT has achieved unparalleled accuracy and sensitivity, exceeding initial expectations. As a result, this remarkable advance has facilitated the search for internal dynamics within many protein molecules. DXT has recently achieved remarkable success in elucidating the internal dynamics of membrane proteins in living cell membranes. This breakthrough has not only expanded our knowledge of these important biomolecules but also has immense potential to advance our understanding of cellular processes in their native environment.

Molecular Dynamics Mappings of the CCT/TRiC Complex-Mediated Protein Folding Cycle Using Diffracted X-ray Tracking.

The CCT/TRiC complex is a type II chaperonin that undergoes ATP-driven conformational changes during its functional cycle. Structural studies have provided valuable insights into the mechanism of this process, but real-time dynamics analyses of mammalian type II chaperonins are still scarce. We used diffracted X-ray tracking (DXT) to investigate the intramolecular dynamics of the CCT complex. We focused on three surface-exposed loop regions of the CCT1 subunit: the loop regions of the equatorial domain (E domain), the E and intermediate domain (I domain) juncture near the ATP-binding region, and the apical domain (A domain). Our results showed that the CCT1 subunit predominantly displayed rotational motion, with larger mean square displacement (MSD) values for twist (χ) angles compared with tilt (θ) angles. Nucleotide binding had a significant impact on the dynamics. In the absence of nucleotides, the region between the E and I domain juncture could act as a pivotal axis, allowing for greater motion of the E domain and A domain. In the presence of nucleotides, the nucleotides could wedge into the ATP-binding region, weakening the role of the region between the E and I domain juncture as the rotational axis and causing the CCT complex to adopt a more compact structure. This led to less expanded MSD curves for the E domain and A domain compared with nucleotide-absent conditions. This change may help to stabilize the functional conformation during substrate binding. This study is the first to use DXT to probe the real-time molecular dynamics of mammalian type II chaperonins at the millisecond level. Our findings provide new insights into the complex dynamics of chaperonins and their role in the functional folding cycle.

Comparison of the Molecular Motility of Tubulin Dimeric Isoforms: Molecular Dynamics Simulations and Diffracted X-ray Tracking Study.

Tubulin has been recently reported to form a large family consisting of various gene isoforms; however, the differences in the molecular features of tubulin dimers composed of a combination of these isoforms remain unknown. Therefore, we attempted to elucidate the physical differences in the molecular motility of these tubulin dimers using the method of measurable pico-meter-scale molecular motility, diffracted X-ray tracking (DXT) analysis, regarding characteristic tubulin dimers, including neuronal TUBB3 and ubiquitous TUBB5. We first conducted a DXT analysis of neuronal (TUBB3-TUBA1A) and ubiquitous (TUBB5-TUBA1B) tubulin dimers and found that the molecular motility around the vertical axis of the neuronal tubulin dimer was lower than that of the ubiquitous tubulin dimer. The results of molecular dynamics (MD) simulation suggest that the difference in motility between the neuronal and ubiquitous tubulin dimers was probably caused by a change in the major contact of Gln245 in the T7 loop of TUBB from Glu11 in TUBA to Val353 in TUBB. The present study is the first report of a novel phenomenon in which the pico-meter-scale molecular motility between neuronal and ubiquitous tubulin dimers is different.

Real-Time Observation of Capsaicin-Induced Intracellular Domain Dynamics of TRPV1 Using the Diffracted X-ray Tracking Method.

The transient receptor potential vanilloid type 1 (TRPV1) is a multimodal receptor which responds to various stimuli, including capsaicin, protons, and heat. Recent advances in cryo-electron microscopy have revealed the structures of TRPV1. However, due to the large size of TRPV1 and its structural complexity, the detailed process of channel gating has not been well documented. In this study, we applied the diffracted X-ray tracking (DXT) technique to analyze the intracellular domain dynamics of the TRPV1 protein. DXT enables the capture of intramolecular motion through the analysis of trajectories of Laue spots generated from attached gold nanocrystals. Diffraction data were recorded at two different frame rates: 100 µs/frame and 12.5 ms/frame. The data from the 100 µs/frame recording were further divided into two groups based on the moving speed, using the lifetime filtering technique, and they were analyzed separately. Capsaicin increased the slope angle of the MSD curve of the C-terminus in 100 µs/frame recording, which accompanied a shifting of the rotational bias toward the counterclockwise direction, as viewed from the cytoplasmic side. This capsaicin-induced fluctuation was not observed in the 12.5 ms/frame recording, indicating that it is a high-frequency fluctuation. An intrinsiccounterclockwise twisting motion was observed in various speed components at the N-terminus, regardless of the capsaicin administration. Additionally, the competitive inhibitor AMG9810 induced a clockwise twisting motion, which is the opposite direction to capsaicin. These findings contribute to our understanding of the activation mechanisms of the TRPV1 channel.

Ligand-Dependent Intramolecular Motion of Native Nicotinic Acetylcholine Receptors Determined in Living Myotube Cells via Diffracted X-ray Tracking.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play an important role in signal transduction at the neuromuscular junction (NMJ). Movement of the nAChR extracellular domain following agonist binding induces conformational changes in the extracellular domain, which in turn affects the transmembrane domain and opens the ion channel. It is known that the surrounding environment, such as the presence of specific lipids and proteins, affects nAChR function. Diffracted X-ray tracking (DXT) facilitates measurement of the intermolecular motions of receptors on the cell membranes of living cells, including all the components involved in receptor function. In this study, the intramolecular motion of the extracellular domain of native nAChR proteins in living myotube cells was analyzed using DXT for the first time. We revealed that the motion of the extracellular domain in the presence of an agonist (e.g., carbamylcholine, CCh) was restricted by an antagonist (i.e., alpha-bungarotoxin, BGT).

Activity of deep trunk muscles for postural control to predictable and unpredictable sagittal plane perturbations: Electromyographic analysis.

BACKGROUND: The activity of deep trunk muscles (psoas major; PM, quadratus lumborum; QL, transverse abdominis; TrA, and lumbar multifidus; MF) in response to external perturbation is not clearly known. OBJECTIVE: This study aimed to record the onset and amount of activity of the deep trunk muscles during sagittal plane perturbations. METHODS: Fourteen healthy males participated in this study. The activity of the right deep trunk muscles was recorded using wire electrodes. In standing, the participants performed three tasks: a pendulum impacted from anterior with predictable and unpredictable and posterior with unpredictable. RESULTS: In predictable anterior perturbation, the TrA and PM demonstrated feedforward activation, while all deep trunk muscles demonstrated feedback activation in unpredictable anterior and posterior perturbations. In the anticipatory postural adjustment phase, the activity of the TrA was large in predictable anterior perturbation, while that of all deep trunk muscles was slight in other perturbations. In the compensatory postural adjustment phase, the activity of the PM, QL, and TrA in unpredictable anterior perturbation and those of the PM, QL, and MF in unpredictable posterior perturbation were large. CONCLUSIONS: These results showed that the onset and magnitude of deep trunk muscle activity changed depending on both predictable or unpredictable perturbation and the direction of perturbation.

Extraction of Rh(III) from hydrochloric acid by protonated NTAamide(C6) and analogous compounds and understanding of extraction equilibria by using UV spectroscopy and DFT calculations.

Extraction of Rh from hydrochloric acid is conducted using NTAamide(C6) (N,N,N´,N´,N´´,N´´-hexahexyl-nitrilotriacetamide) and other related compounds. We use the ion-pair extraction of anionic species of Rh-chloride and protonated extractant. Rh ions exist as Rh(Cl)n(H2O)6-n (n ≤ 5) and the tertiary nitrogen atom in an extractant are protonated to produce a quaternary amine in acidic condition. The D(Rh) values are changeable because the Rh-Cl-H2O complex forms from + 3 to - 2 valency. Rh-chloride ion with a peak of spectrum at 504 nm can be extracted effectively, where RhCl4(H2O)- and RhCl5(H2O)2- exist from Density functional theory calculation and UV spectrum. The maximum distribution ratio (D) of Rh(III) is 16, and 85 mM Rh can be extracted from 1 M HCl dissolving 96 mM, due to less third phase formation. Approximate 80% of Rh can be stripped by the water-soluble reagents having the activities of neutralization and solvation. The figure for the Graphical Index saved in the JPEG, PNG or TIFF format at 300 dpi should be pasted with the size adjusted to the frame below (5 cm long and 8 cm wide).

The effect of ice-binding proteins on the cryopreservation of Caenorhabditis elegans.

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth. IBPs have also been reported to stabilize cell membranes under non-freezing conditions. The effects of IBPs help to reduce cold- and freezing-induced damage to cells and tissues in cryopreservation. Here, we examined whether certain IBPs, namely, fish NfeIBP6 and NfeIBP8 and fungal AnpIBP1a N55D (AnpIBP), improve the recovery rate of the nematode Caenorhabditis elegans after a deep cryopreservation at -80°C. The expression of fungus-derived AnpIBP in C. elegans significantly improved its recovery rate. This result provides useful information to establish a cryopreservation technique for long-term storage using IBP molecules.

Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

Characterization of forehead blood flow bias on NIRS signals during neural activation with a verbal fluency task.

The major problem of near-infrared spectroscopy (NIRS) for brain activity measurement during verbal fluency task is the overlapping forehead scalp blood flow (FBF) on the target cerebral blood flow (CBF). There could be among-individual differences in the influence of FBF on CBF. We investigated effects of FBF on CBF by comparing signals obtained through a laser Doppler flowmeter (LDF) and NIRS using the modified Beer-Lambert Law (MBLL). Among 25 healthy individuals, 7 participants showed a strong correlation between LDF and NIRS signals (rs >0.500). There were no significant differences according to age or sex. Subsequently, we applied the hemodynamic separation method to the values calculated using the MBLL (Δ[oxy-Hb]M): to separate the concentration of oxygenated hemoglobin in the forehead (Δ[oxy-Hb]F) and cerebral cortex (Δ[oxy-Hb]C). First, we found that the influence of Δ[oxy-Hb]F on Δ[oxy-Hb]C in the high rs group was almost twice as large as that in the low rs group. Second, presence of sex and age differences in the influence of Δ[oxy-Hb]F on Δ[oxy-Hb]C were suggested. Based on the results, we discuss the factors affecting FBF and the resulting variations in NIRS signals.

Visualizing Intramolecular Dynamics of Membrane Proteins.

Membrane proteins play important roles in biological functions, with accompanying allosteric structure changes. Understanding intramolecular dynamics helps elucidate catalytic mechanisms and develop new drugs. In contrast to the various technologies for structural analysis, methods for analyzing intramolecular dynamics are limited. Single-molecule measurements using optical microscopy have been widely used for kinetic analysis. Recently, improvements in detectors and image analysis technology have made it possible to use single-molecule determination methods using X-rays and electron beams, such as diffracted X-ray tracking (DXT), X-ray free electron laser (XFEL) imaging, and cryo-electron microscopy (cryo-EM). High-speed atomic force microscopy (HS-AFM) is a scanning probe microscope that can capture the structural dynamics of biomolecules in real time at the single-molecule level. Time-resolved techniques also facilitate an understanding of real-time intramolecular processes during chemical reactions. In this review, recent advances in membrane protein dynamics visualization techniques were presented.