Subnanometer Resolutions Research Articles

Three-dimensional structures of Vibrio cholerae typing podophage VP1 in two states.

Lytic podophages (VP1-VP5) play crucial roles in subtyping Vibrio cholerae O1 biotype El Tor. However, until now no structures of these phages have been available, which hindered our understanding of the molecular mechanisms of infection and DNA release. Here, we determined the cryoelectron microscopy (cryo-EM) structures of mature and DNA-ejected VP1 structures at near-atomic and subnanometer resolutions, respectively. The VP1 head is composed of 415 copies of the major capsid protein gp7 and 11 turret-shaped spikes. The VP1 tail consists of an adapter, a nozzle, a slender ring, and a tail needle, and is flanked by three extended fibers I and six trimeric fibers II. Conformational changes of fiber II in DNA-ejected VP1 may cause the release of the tail needle and core proteins, forming an elongated tail channel. Our structures provide insights into the molecular mechanisms of infection and DNA release for podophages with a tail needle.

Coherent diffractive imaging with twisted X-rays: Principles, applications, and outlook

Recent technological breakthroughs in synchrotron and x-ray free electron laser facilities have revolutionized nanoscale structural and dynamic analyses in condensed matter systems. This review provides a comprehensive overview of the advancements in coherent scattering and diffractive imaging techniques, which are now at the forefront of exploring materials science complexities. These techniques, notably Bragg coherent diffractive imaging and x-ray photon correlation spectroscopy, x-ray magnetic dichroism, and x-ray correlation analysis leverage beam coherence to achieve volumetric three-dimensional imaging at unprecedented sub-nanometer resolutions and explore dynamic phenomena within sub-millisecond timeframes. Such capabilities are critical in understanding and developing advanced materials and technologies. Simultaneously, the emergence of chiral crystals—characterized by their unique absence of standard inversion, mirror, or other roto-inversion symmetries—presents both challenges and opportunities. These materials exhibit distinctive interactions with light, leading to phenomena such as molecular optical activity, chiral photonic waveguides, and valley-specific light emissions, which are pivotal in the burgeoning fields of photonic and spintronic devices. This review elucidates how novel x-ray probes can be leveraged to unravel these properties and their implications for future technological applications. A significant focus of this review is the exploration of new avenues in research, particularly the shift from conventional methods to more innovative approaches in studying these chiral materials. Inspired by structured optical beams, the potential of coherent scattering techniques utilizing twisted x-ray beams is examined. This promising direction not only offers higher spatial resolution but also opens the door to previously unattainable insights in materials science. By contextualizing these advancements within the broader scientific landscape and highlighting their practical applications, this review aims to chart a course for future research in this rapidly evolving field.

Thermal expansion in photo-assisted tunneling: Visible light versus free-space terahertz pulses

Photo-assisted tunneling in scanning tunneling microscopy has attracted considerable interest to combine sub-picosecond and sub-nanometer resolutions. The illumination of a junction with visible or infrared light, however, induces thermal expansion of the tip and the sample, which strongly affects the measurements. Employing free-space THz pulses instead of visible light has been proposed to solve these thermal issues while providing photo-induced currents of similar magnitude. Here we compared the impact of illuminating the same tunneling junction, reaching comparable response in the tunneling current, with red light and with THz radiations. Our data provide a clear and direct evidence of thermal expansion with red light-illumination, while such thermal effects are negligible with THz radiations.

Construction of nanoparticle-on-mirror nanocavities and their applications in plasmon-enhanced spectroscopy.

Plasmonic nanocavities exhibit exceptional capabilities in visualizing the internal structure of a single molecule at sub-nanometer resolution. Among these, an easily manufacturable nanoparticle-on-mirror (NPoM) nanocavity is a successful and powerful platform for demonstrating various optical phenomena. Exciting advances in surface-enhanced spectroscopy using NPoM nanocavities have been developed and explored, including enhanced Raman, fluorescence, phosphorescence, upconversion, etc. This perspective emphasizes the construction of NPoM nanocavities and their applications in achieving higher enhancement capabilities or spatial resolution in dark-field scattering spectroscopy and plasmon-enhanced spectroscopy. We describe a systematic framework that elucidates how to meet the requirements for studying light-matter interactions through the creation of well-designed NPoM nanocavities. Additionally, it provides an outlook on the challenges, future development directions, and practical applications in the field of plasmon-enhanced spectroscopy.

Cryo-EM structures of prefusion SIV envelope trimer.

Simian immunodeficiency viruses (SIVs) are lentiviruses that naturally infect non-human primates of African origin and seeded cross-species transmissions of HIV-1 and HIV-2. Here we report prefusion stabilization and cryo-EM structures of soluble envelope (Env) trimers from rhesus macaque SIV (SIVmac) in complex with neutralizing antibodies. These structures provide residue-level definition for SIV-specific disulfide-bonded variable loops (V1 and V2), which we used to delineate variable-loop coverage of the Env trimer. The defined variable loops enabled us to investigate assembled Env-glycan shields throughout SIV, which we found to comprise both N- and O-linked glycans, the latter emanating from V1 inserts, which bound the O-link-specific lectin jacalin. We also investigated in situ SIVmac-Env trimers on virions, determining cryo-electron tomography structures at subnanometer resolutions for an antibody-bound complex and a ligand-free state. Collectively, these structures define the prefusion-closed structure of the SIV-Env trimer and delineate variable-loop and glycan-shielding mechanisms of immune evasion conserved throughout SIV evolution.

Capturing actin assemblies in cells using in situ cryo-electron tomography

Actin contributes to an exceptionally wide range of cellular processes through the assembly and disassembly of highly dynamic and ordered structures. Visualizing these structures in cells can help us understand how the molecular players of the actin machinery work together to produce force-generating systems. In recent years, cryo-electron tomography (cryo-ET) has become the method of choice for structural analysis of the cell interior at the molecular scale. Here we review advances in cryo-ET workflows that have enabled this transformation, especially the automation of sample preparation procedures, data collection, and processing. We discuss new structural analyses of dynamic actin assemblies in cryo-preserved cells, which have provided mechanistic insights into actin assembly and function at the nanoscale. Finally, we highlight the latest visual proteomics studies of actin filaments and their interactors reaching sub-nanometer resolutions in cells.

Evaluation of SIF retrievals from narrow-band and sub-nanometer airborne hyperspectral imagers flown in tandem: Modelling and validation in the context of plant phenotyping

Solar-induced chlorophyll fluorescence (SIF) can be used as an indicator of crop photosynthetic activity and a proxy for vegetation stress in plant phenotyping and precision agriculture applications. SIF quantification is sensitive to the spectral resolution (SR), and its accurate retrieval requires sensors with sub-nanometer resolutions. However, for accurate SIF quantification from imaging sensors onboard airborne platforms, sub-nanometer imagers are costly and more difficult to operate than the commonly available narrow-band imagers (i.e., 4- to 6-nm bandwidths), which can also be installed on drones and lightweight aircraft. Although a few theoretical and experimental studies have evaluated narrow-band spectra for SIF quantification, there is a lack of research focused on comparing the effects of the SR on SIF from airborne hyperspectral imagers in practical applications. This study investigates the effects of SR and sensor altitude on SIF accuracy, comparing SIF quantified at the 760-nm O2-A band (SIF760) from two hyperspectral imagers with different spectral configurations (full width at half-maximum resolutions of 0.1–0.2 nm and 5.8 nm) flown in tandem on board an aircraft. SIF760 retrievals were compared from two different wheat and maize phenotyping trials grown under different nitrogen fertilizer application rates over the 2019–2021 growing seasons. SIF760 from the two sensors were correlated (R2 = 0.77–0.9, p < 0.01), with the narrow-band imager producing larger SIF760 estimates than the sub-nanometer imager (root mean square error (RMSE) 3.28–4.69 mW/m2/nm/sr). Ground-level SIF760 showed strong relationships with both sub-nanometer (R2 = 0.90, p < 0.001, RMSE = 0.07 mW/m2/nm/sr) and narrow-band (R2 = 0.88, p < 0.001, RMSE = 3.26 mW/m2/nm/sr) airborne retrievals. Simulation-based assessments of SIF760 for SRs ranging from 1 to 5.8 nm using the SCOPE model were consistent with experimental results showing significant relationships among SIF760 quantified at different SRs. Predictive algorithms of leaf nitrogen concentration using SIF760 from either the narrow-band or sub-nanometer sensor yielded similar performance, supporting the use of narrow-band resolution imagery for assessing the spatial variability of SIF in plant phenotyping, vegetation stress detection and precision agriculture contexts.

Structural basis for allosteric regulation of Human Topoisomerase II\u03b1

The human type IIA topoisomerases (Top2) are essential enzymes that regulate DNA topology and chromosome organization. The Topo IIα isoform is a prime target for antineoplastic compounds used in cancer therapy that form ternary cleavage complexes with the DNA. Despite extensive studies, structural information on this large dimeric assembly is limited to the catalytic domains, hindering the exploration of allosteric mechanism governing the enzyme activities and the contribution of its non-conserved C-terminal domain (CTD). Herein we present cryo-EM structures of the entire human Topo IIα nucleoprotein complex in different conformations solved at subnanometer resolutions (3.6–7.4 Å). Our data unveils the molecular determinants that fine tune the allosteric connections between the ATPase domain and the DNA binding/cleavage domain. Strikingly, the reconstruction of the DNA-binding/cleavage domain uncovers a linker leading to the CTD, which plays a critical role in modulating the enzyme’s activities and opens perspective for the analysis of post-translational modifications.

Molecular dynamics simulations of internal stress evolution in ultrathin amorphous carbon films subjected to thermal annealing

The evolution of internal stress in ultrathin amorphous carbon (a-C) films is a complex physical process that is difficult to experimentally analyze due to the very small film thickness (a few nanometers) and the lack of instruments that can perform spatiotemporal stress measurements at sub-nanometer resolutions. Even more challenging is the elucidation of the correlation between internal stress, film activated, and temperature. Molecular dynamics (MD) provides potent computational capability for tracking structural changes activated by stress and temperature at the atomic level. Consequently, the aim of this study was to perform a comprehensive MD analysis that elucidates the origin of internal stress in sub-2-nm-thick a-C films grown on single-crystal silicon under optimal deposition energy conditions and explore its dependence on prevalent structural features (e.g., hybridization state) and temperature. The physical mechanisms of a-C film growth and stress built-up under deposition conditions of energetic particle bombardment and stress relief due to thermal annealing are interpreted in the context of MD results. Simulations of film growth illuminate the correlation between film stress and energy of incident carbon atoms. A significant stress relief occurs mainly in the bulk layer of the multilayered a-C film structure at a critical annealing temperature, which continues to intensify with the further increase of temperature. Simulations of time-dependent variation of stress through the film thickness reveal that the stress relief is a very fast process that accelerates with the increase of temperature. The results of this study provide insight into the spatial and temporal variation of internal stress in ultrathin a-C films due to structure and temperature effects and the film stress-structure interdependence.

A multi-scale experimental study of crude oil-brine-rock interactions and wettability alteration during low-salinity waterflooding

In this study we present the results of a multi-scale experimental investigation of the mechanisms responsible for the increased oil recovery by low-salinity waterflooding (LSWF), i.e., low-salinity effect (LSE). Using core- and substrate-flooding experiments as well as imaging techniques at macro, micro, and nanometer scales, we probe the intricate crude oil-brine-rock interactions and the subtle pore-scale displacement events governing recovery enhancement during LSWF. We performed several LSWF and high-salinity waterflooding (HSWF) experiments on macro-scale (1 inch-diameter) reservoir sandstone core plugs while collecting effluent samples for further analysis.LSWF experiments exhibited a prolonged oil recovery response and notably higher oil recovery compared to HSWF. Imaging effluent samples, at sub-nanometer resolutions using transmission electron microscopy, demonstrated significant fines mobilization during LSWF. The occurrence of electric double layer expansion (DLE) as a source of fines mobilization during LSWF was confirmed by examining scanning electron microscopy images of the reservoir rock substrates before and after contacting the low-salinity brine. Moreover, geochemical analysis of the effluent samples showed evidence of proton excahnge and multi-component ion exchange (MIE) during LSWF. The mobilization of fine particles (due to DLE) and the ion exchange processes are believed to result in removal of oil molecules from the rock surfaces leading to an enhancement in oil recovery as well as alteration of wettability toward increased water-wetness. These observations were then linked to our findings from an earlier study during which LSWF and HSWF experiments were conducted on miniature (5 mm-diameter) core samples while the pore space was being imaged at micrometer resolution. In-situ oil/brine contact angles measured during flow tests conducted on the miniature core samples revealed a significant level of wettability alteration from weakly oil-wet toward increased water-wetness during LSWF. Analysis of fluid occupancy maps showed that this change in wettability lowers the threshold water pressure needed for water-displacing-oil events to take place in the pore elements, which in turn enhances displacement efficiency and oil recovery.

Conformational Landscape of the p28-Bound Human Proteasome Regulatory Particle

The proteasome holoenzyme is activated by its regulatory particle (RP) consisting of two subcomplexes, the lid and the base. A key event in base assembly is the formation of a heterohexameric ring of AAA-ATPases, which is guided by at least four RP assembly chaperones in mammals: PAAF1, p28/gankyrin, p27/PSMD9, and S5b. Using cryogenic electron microscopy, we analyzed the non-AAA structure of the p28-bound human RP at 4.5Å resolution and determined seven distinct conformations of the Rpn1-p28-AAA subcomplex within the p28-bound RP at subnanometer resolutions. Remarkably, the p28-bound AAA ring does not form a channel in the free RP and spontaneously samples multiple "open" and "closed" topologies at the Rpt2-Rpt6 and Rpt3-Rpt4 interfaces. Our analysis suggests that p28 assists the proteolytic core particle to select a specific conformation of the ATPase ring for RP engagement and is released in a shoehorn-like fashion in the last step of the chaperone-mediated proteasome assembly.

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

Cryo-Electron Tomography and Computer Simulations Reveal Distinct CheA Kinase Conformation in Bacterial Chemotaxis Signaling Receptor Complex

Improved sample preparation, data collection, and image processing pipelines have ushered in a new era in cryo-Electron Tomography (CryoET). Electron density maps of large, conformationally heterogeneous assemblies can be determined at subnanometer resolutions and coupled with high resolution structural information and large scale molecular dynamics simulation to gain atomistic understanding of large and complex cellular machinery that is otherwise unattainable. We use such an integrative approach to investigate the signal transduction in bacterial chemotaxis arrays - large transmembrane protein complex assemblies responsible for modulating the motility of bacteria. These arrays are responsible, in part for appropriately biasing the cell's inherent random walk, and also retaining the memory of recent states of the system. Despite recent advances, fundamental questions on how the components of the system work together to transduce the signal from membrane receptor to kinase molecule, and how the individual signaling unit works cohesively to achieve a large gain in signaling remain unanswered. We devised a novel in vitro reconstitution system to build extended signaling arrays on a lipid monolayer using purified protein components, providing an unprecedented number of signaling units in a thin layer (∼150-200nm) ideal for cryoET. The individual subvolumes from 3D tomograms are extracted, aligned, classified and averaged, resulting in a 3D density map of the basic chemotaxis signaling unit comprising the chemoreceptor, the histidine kinase CheA and the cofactor CheW. The density map reveals an unexpected asymmetry among three kinase CheA dimers, indicating distinct CheA conformations within the unit. Large scale all-atom molecular dynamics simulations further point to the specific side chain contacts that mediate the transition of CheA conformations, which we show, through biochemical assays, to fully abrogate chemotactic function if disrupted.

Structure Determination of Small Macromolecular Complexes by Cryo-Electron Microscopy

Application of emerging methods in single particle cryo-electron microscopy (cryo-EM) has led to the determination of the structures of a variety of macromolecular complexes at sub-nanometer resolutions. The overwhelming majority of these are large complexes, mostly with high symmetry. Extending cryo-EM methods to routinely determine the structures of small macromolecular complexes with sizes of < 500 kDa to sub-nanometer resolution still remains an exciting frontier in modern structural biology. Working with small protein complexes has certain inherent challenges such as conformational flexibility and beam induced movement during exposure to electrons which can significantly reduce resolution. Further, during image processing, errors can be introduced from model-bias, over-refinement, and inaccuracy in orientation determination. Although these errors are problematic for all reconstructions, they are particularly detrimental for structural analysis of complexes that are small and have either low or no internal symmetry. We show a step-wise systematic workflow for structure determination by cryo-EM at ∼ 6 A resolution of s-galactosidase, a 464 kDa protein, whose structure has previously been determined at atomic resolution by X-ray crystallography. Our approach employs a number of recent developments in cryo-EM technology that include the use of direct electron detectors, the ability to fractionate the dose to use only those portions of the exposure that are recorded before there is significant radiation damage, and the use of image processing procedures that can track and correct for the movement of single molecules during the course of the exposure.

Automated Workflows for Structure Determination by Single-Particle Cryo-EM at Sub-Nanometer Resolutions

The combination of hardware advances in transmission electron microscopes together with automated data collection and streamlined image processing routines have transformed the pace at which samples can be converted into 3D structures by single-particle cryo-EM. In this work, we present automated workflows for data collection and processing of large single-particle datasets. Efficient micrograph screening routines together with automatic particle picking strategies enable processing of entire datasets in high-throughput mode in a routine manner. We use this platform as a framework to perform comparative studies of the effects of different types of detector, acceleration voltages and different dose fractionation schemes on the resolution of reconstructions. We applied these workflows to multiple datasets of GroEL imaged under a variety of conditions showing that reconstructions at better than 7A resolution can be consistently achieved using this framework.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Spatial modeling for refining and predicting surface potential mapping with enhanced resolution

Quantitatively mapping surface properties with nanometer or even subnanometer resolutions is critical for advanced scanning probe microscopy (SPM) characterization. However, the characterization performance often suffers from noises and artifacts due to instrumentation or environmental limitations. In this paper, we proposed a novel statistical approach with bivariate spatial modeling to efficiently refine and predict surface property mapping. Scanning Kelvin probe microscopy (SKPM) was selected as a representative example to test our proposed method on lateral nanowire assemblies. We revealed that the proposed method can effectively retrieve the artifact-free surface potential distribution by automatically identifying topological artifacts from surface potential maps. Furthermore, the statistical model built upon low spatial resolution was successfully used to predict the potential values from higher-resolution topography data. Compared to conventional regression model, our model is able to predict the surface potential distribution from less raw data but yields much higher accuracy. Through this means, the spatial resolution of SKPM surface potential maps can be significantly improved. This statistics-enabled predictive method opens a new route toward high-precision and high-resolution SPM characterizations without the enhancement of instrumentation capabilities.

Effect of the Compatible Solute Ectoine on the Stability of the Membrane Proteins

Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.

Chemical mapping of mammalian cells by atom probe tomography.

In atom probe tomography (APT), a technique that has been used to determine 3D maps of ion compositions of metals and semiconductors at sub-nanometer resolutions, controlled emissions of ions can be induced from needle-shaped specimens in the vicinity of a strong electric field. Detection of these ions in the plane of a position sensitive detector provides two-dimensional compositional information while the sequence of ion arrival at the detector provides information in the third dimension. Here we explore the use of APT technology for imaging biological specimens. We demonstrate that it is possible to obtain 3D spatial distributions of cellular ions and metabolites from unstained, freeze-dried mammalian cells. Multiple peaks were reliably obtained in the mass spectrum from tips with diameters of ~50 nm and heights of ~200 nm, with mass-to-charge ratios (m/z) ranging from 1 to 80. Peaks at m/z 12, 23, 28 and 39, corresponding to carbon, sodium, carbonyl and potassium ions respectively, showed distinct patterns of spatial distribution within the cell. Our studies establish that APT could become a powerful tool for mapping the sub-cellular distribution of atomic species, such as labeled metabolites, at 3D spatial resolutions as high as ~1 nm.

Whole-Cell Imaging at Nanometer Resolutions Using Fast and Slow Focused Helium Ions

Observations of the interior structure of cells and subcellular organelles are important steps in unraveling organelle functions. Microscopy using helium ions can play a major role in both surface and subcellular imaging because it can provide subnanometer resolutions at the cell surface for slow helium ions, and fast helium ions can penetrate cells without a significant loss of resolution. Slow (e.g., 10–50 keV) helium ion beams can now be focused to subnanometer dimensions (∼0.25 nm), and keV helium ion microscopy can be used to image the surfaces of cells at high resolutions. Because of the ease of neutralizing the sample charge using a flood electron beam, surface charging effects are minimal and therefore cell surfaces can be imaged without the need for a conducting metallic coating. Fast (MeV) helium ions maintain a straight path as they pass through a cell. Along the ion trajectory, the helium ion undergoes multiple electron collisions, and for each collision a small amount of energy is lost to the scattered electron. By measuring the total energy loss of each MeV helium ion as it passes through the cell, we can construct an energy-loss image that is representative of the mass distribution of the cell. This work paves the way to use ions for whole-cell investigations at nanometer resolutions through structural, elemental (via nuclear elastic backscattering), and fluorescence (via ion induced fluorescence) imaging.

Mechanical Stabilization of Membrane Proteins using Compatible Solutes

Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. The single molecules without inherent symmetry can directly be monitored in their physiological conditions using atomic force microscopy (AFM). Recent developments in AFM enable us to go beyond the ensemble average and measure the behavior of individual molecules. In nature, compatible solutes (organic osmolytes) are used for protecting cells against high osmotic stress. They are compatible with cell metabolism even at molar concentrations. The influence of ectoine (1M), betaine (1M) and taurine (0.4M) on the mechanical properties of bacteriorhodopsin (BR) has been investigated by single molecule force spectroscopy. Unfolding experiments taking BR as a model system revealed that these compatible osmolytes increase the tendency of the polypeptide to coil, thereby decreasing its persistence length. This allows us to explain the mechanism of interaction between the unfolded polypeptide chain and the osmolyte. The osmolytes are expelled from the protein surface due to the increase in chemical potential of the stretched (denatured) state collapsing the protein into a more compact structure. These information and approaches provide basis for our further studies regarding the effects of compatible solutes on other membrane proteins of medical importance (particularly in case of liver diseases) which can directly resolve transient intermediate states and multiple reaction pathways, and thus are uniquely powerful in characterizing the complex dynamics of protein folding. Thus, this study is set to provide exciting possibilities in the field of drug development for liver diseases including in vitro rescue of the misfolded proteins and to directly analyze and correlate their structural and functional properties at the sub-molecular level.