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1
Vuillemot, Rémi, Mohamad Harastani, Ilyes Hamitouche, and Slavica Jonic. "MDSPACE and MDTOMO Software for Extracting Continuous Conformational Landscapes from Datasets of Single Particle Images and Subtomograms Based on Molecular Dynamics Simulations: Latest Developments in ContinuousFlex Software Package." International Journal of Molecular Sciences 25, no. 1 (2023): 20. http://dx.doi.org/10.3390/ijms25010020.
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Cryo electron microscopy (cryo-EM) instrumentation allows obtaining 3D reconstruction of the structure of biomolecular complexes in vitro (purified complexes studied by single particle analysis) and in situ (complexes studied in cells by cryo electron tomography). Standard cryo-EM approaches allow high-resolution reconstruction of only a few conformational states of a molecular complex, as they rely on data classification into a given number of classes to increase the resolution of the reconstruction from the most populated classes while discarding all other classes. Such discrete classification approaches result in a partial picture of the full conformational variability of the complex, due to continuous conformational transitions with many, uncountable intermediate states. In this article, we present the software with a user-friendly graphical interface for running two recently introduced methods, namely, MDSPACE and MDTOMO, to obtain continuous conformational landscapes of biomolecules by analyzing in vitro and in situ cryo-EM data (single particle images and subtomograms) based on molecular dynamics simulations of an available atomic model of one of the conformations. The MDSPACE and MDTOMO software is part of the open-source ContinuousFlex software package (starting from version 3.4.2 of ContinuousFlex), which can be run as a plugin of the Scipion software package (version 3.1 and later), broadly used in the cryo-EM field.
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Luchinat, Claudio. "Exploring the conformational heterogeneity of biomolecules: theory and experiments." Physical Chemistry Chemical Physics 18, no. 8 (2016): 5684–85. http://dx.doi.org/10.1039/c6cp90029a.
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This themed collection reports on recent progress in the investigation of the conformational variability of biomolecules (proteins and nucleic acids), both from an experimental and theoretical point of view.
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DeVore, Kira, and Po-Lin Chiu. "Probing Structural Perturbation of Biomolecules by Extracting Cryo-EM Data Heterogeneity." Biomolecules 12, no. 5 (2022): 628. http://dx.doi.org/10.3390/biom12050628.
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Single-particle cryogenic electron microscopy (cryo-EM) has become an indispensable tool to probe high-resolution structural detail of biomolecules. It enables direct visualization of the biomolecules and opens a possibility for averaging molecular images to reconstruct a three-dimensional Coulomb potential density map. Newly developed algorithms for data analysis allow for the extraction of structural heterogeneity from a massive and low signal-to-noise-ratio (SNR) cryo-EM dataset, expanding our understanding of multiple conformational states, or further implications in dynamics, of the target biomolecule. This review provides an overview that briefly describes the workflow of single-particle cryo-EM, including imaging and data processing, and new methods developed for analyzing the data heterogeneity to understand the structural variability of biomolecules.
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Sorzano, C. O. S., A. Jiménez, J. Mota, et al. "Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy." Acta Crystallographica Section F Structural Biology Communications 75, no. 1 (2019): 19–32. http://dx.doi.org/10.1107/s2053230x18015108.
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Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed.
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Ma, Shaoqing, Zhiwei Li, Shixiang Gong, Chengbiao Lu, Xiaoli Li, and Yingwei Li. "High Frequency Electromagnetic Radiation Stimulates Neuronal Growth and Hippocampal Synaptic Transmission." Brain Sciences 13, no. 4 (2023): 686. http://dx.doi.org/10.3390/brainsci13040686.
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Terahertz waves lie within the rotation and oscillation energy levels of biomolecules, and can directly couple with biomolecules to excite nonlinear resonance effects, thus causing conformational or configuration changes in biomolecules. Based on this mechanism, we investigated the effect pattern of 0.138 THz radiation on the dynamic growth of neurons and synaptic transmission efficiency, while explaining the phenomenon at a more microscopic level. We found that cumulative 0.138 THz radiation not only did not cause neuronal death, but that it promoted the dynamic growth of neuronal cytosol and protrusions. Additionally, there was a cumulative effect of terahertz radiation on the promotion of neuronal growth. Furthermore, in electrophysiological terms, 0.138 THz waves improved synaptic transmission efficiency in the hippocampal CA1 region, and this was a slow and continuous process. This is consistent with the morphological results. This phenomenon can continue for more than 10 min after terahertz radiation ends, and these phenomena were associated with an increase in dendritic spine density. In summary, our study shows that 0.138 THz waves can modulate dynamic neuronal growth and synaptic transmission. Therefore, 0.138 terahertz waves may become a novel neuromodulation technique for modulating neuron structure and function.
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Valimehr, Sepideh, Rémi Vuillemot, Mohsen Kazemi, Slavica Jonic, and Isabelle Rouiller. "Analysis of the Conformational Landscape of the N-Domains of the AAA ATPase p97: Disentangling the Continuous Conformational Variability in Partially Symmetrical Complexes." International Journal of Molecular Sciences 25, no. 6 (2024): 3371. http://dx.doi.org/10.3390/ijms25063371.
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Single-particle cryo-electron microscopy (cryo-EM) has been shown to be effective in defining the structure of macromolecules, including protein complexes. Complexes adopt different conformations and compositions to perform their biological functions. In cryo-EM, the protein complexes are observed in solution, enabling the recording of images of the protein in multiple conformations. Various methods exist for capturing the conformational variability through analysis of cryo-EM data. Here, we analyzed the conformational variability in the hexameric AAA + ATPase p97, a complex with a six-fold rotational symmetric core surrounded by six flexible N-domains. We compared the performance of discrete classification methods with our recently developed method, MDSPACE, which uses 3D-to-2D flexible fitting of an atomic structure to images based on molecular dynamics (MD) simulations. Our analysis detected a novel conformation adopted by approximately 2% of the particles in the dataset and determined that the N-domains of p97 sway by up to 60° around a central position. This study demonstrates the application of MDSPACE in analyzing the continuous conformational changes in partially symmetrical protein complexes, systems notoriously difficult to analyze due to the alignment errors caused by their partial symmetry.
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Pancera, S. M., H. Gliemann, D. F. S. Petri, and T. Schimmel. "Adsorption Behaviour of Creatine Phosphokinase onto Silicon Wafers: Comparison between Ellipsometric and Atomic Force Microscopy Data." Microscopy and Microanalysis 11, S03 (2005): 56–60. http://dx.doi.org/10.1017/s1431927605050889.
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Protein adsorption plays a major role in a variety of important technological and biological processes [1-2] and the understanding of the fundamental factors that determine protein adsorption are imperative to the development of biocompatible materials and biotechnological devices [3-4] as for example biosensors [5]. The adsorption of proteins on surfaces is a complex process. Due to the large size and different shapes of these adsorbing particles, the interactions between the adsorbed proteins on the surface can be strongly influentiated by the fact that the particles may undergo conformational changes upon adsorption [6-7]. In a previous work the adsorption behaviour of creatine phosphokinase (CPK) onto hydrophilic (silicon wafers and amino-terminated surfaces) and hydrophobic (Polystyrene, PS, coated wafers) substrates was investigated by means of null-ellipsometry and contact angle measurements [8]. This previous ellipsometric study led to a model, where CPK adsorption takes place in four stages: (i) a diffusive one, where all the arriving biomolecules are immediately adsorbed; (ii) the arriving biomolecules might stick on the latter and afterward diffuse to the free sites on the substrate, followed by conformational changes [6-7], (iii) formation of a monolayer and (iv) continuous and irreversible adsorption. A multilayer system might be formed, as well as aggregation processes might play a role at this stage. In this work Atomic Force Microscopy (AFM) measurements under water were done in order to confirm this four steps model and to observe changes in the film topography and homogeneity along the adsorption process. The thickness of the adsorbed CPK biofilm obtained by ellipsometry was also compared with that obtained by the wet AFM method.
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Harastani, Mohamad, Mikhail Eltsov, Amélie Leforestier, and Slavica Jonic. "TomoFlow: Analysis of Continuous Conformational Variability of Macromolecules in Cryogenic Subtomograms based on 3D Dense Optical Flow." Journal of Molecular Biology 434, no. 2 (2022): 167381. http://dx.doi.org/10.1016/j.jmb.2021.167381.
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Wang, Chenzheng, Yuexia Lin, Devin Bougie, and Richard E. Gillilan. "Predicting data quality in biological X-ray solution scattering." Acta Crystallographica Section D Structural Biology 74, no. 8 (2018): 727–38. http://dx.doi.org/10.1107/s2059798318005004.
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Biological small-angle X-ray solution scattering (BioSAXS) is now widely used to gain information on biomolecules in the solution state. Often, however, it is not obvious in advance whether a particular sample will scatter strongly enough to give useful data to draw conclusions under practically achievable solution conditions. Conformational changes that appear to be large may not always produce scattering curves that are distinguishable from each other at realistic concentrations and exposure times. Emerging technologies such as time-resolved SAXS (TR-SAXS) pose additional challenges owing to small beams and short sample path lengths. Beamline optics vary in brilliance and degree of background scatter, and major upgrades and improvements to sources promise to expand the reach of these methods. Computations are developed to estimate BioSAXS sample intensity at a more detailed level than previous approaches, taking into account flux, energy, sample thickness, window material, instrumental background, detector efficiency, solution conditions and other parameters. The results are validated with calibrated experiments using standard proteins on four different beamlines with various fluxes, energies and configurations. The ability of BioSAXS to statistically distinguish a variety of conformational movements under continuous-flow time-resolved conditions is then computed on a set of matched structure pairs drawn from the Database of Macromolecular Motions (http://molmovdb.org). The feasibility of experiments is ranked according to sample consumption, a quantity that varies by over two orders of magnitude for the set of structures. In addition to photon flux, the calculations suggest that window scattering and choice of wavelength are also important factors given the short sample path lengths common in such setups.
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Mora-Navarro, Camilo, Mario E. Garcia, Prottasha Sarker, et al. "Monitoring decellularization via absorbance spectroscopy during the derivation of extracellular matrix scaffolds." Biomedical Materials 17, no. 1 (2021): 015008. http://dx.doi.org/10.1088/1748-605x/ac361f.
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Abstract Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g. DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the standardization, cost-effectiveness, and scale-up process. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization systems to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor’s effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allow for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulation in vitro suggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.
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Rakhimov, Adil, Miras Zekebayev, Aray Kuatbek, and Vyacheslav Dyachkov. "Comparison of dosimetry parameters of three-dimensional conformational radiation therapy and volumetric modulated arc therapy on the example of treatment of a patient with salivary gland adenocarcinoma." Oncologia i radiologia Kazakhstana 59, no. 1 (2021): 25–30. http://dx.doi.org/10.52532/2521-6414-2021-1-59-25-30.
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Relevance: An optimal choice of radiation therapy
 method is the main prerequisite for successful completion
 of treatment. Continuous analysis of radiation therapy
 methods’ advantages and comparing their parameters
 and dose load in typical cases will serve to increase the
 treatment efficacy and reduce the unavoidable load on
 critical organs.
 Purpose: To check the plans of treatment by volumetric modulated arc therapy (VMAT) and three-dimensional conformal radiation therapy (3D-CRT) methods, make their comparison, and identify the advantages using an example of a treatment design for a patient treated at the Daily Radiotherapy Hospital of the Kazakh Institute of Oncology and Radiology (Almaty, Kazakhstan).
 Results: The presented illustrations of the process
 planning and the numerical analysis of the dose load on
 critical organs show VMAT advantages for radiation therapy of salivary gland cancer: optimal coverage, sparing
 load on critical organs and healthy tissues, gradient decay
 at the borders of the neoplasm.
 Conclusion: The variability of radiation therapy methods ensures the most acceptable coverage that keeps the
 dose load within international standards’ ranges. Mastering these methods will improve the effectiveness of radiation therapy for various cancer cases.
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Tissino, Erika, Tamara Bittolo, Dania Benedetti, et al. "The VLA-4 Integrin Is Constitutively Activated in a Subset of CD49d-Expressing CLL: A Relationship with the Autonomous BCR-Mediated Signaling?" Blood 132, Supplement 1 (2018): 5531. http://dx.doi.org/10.1182/blood-2018-99-111450.
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Abstract Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of tumor cell-microenvironment interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its specific ligands. In this context, VLA-4 needs to be maintained in an activated state by a continuous stream of inside-out signals from the BCR, which can be triggered by canonical antigens as well as in an autonomous antigen-independent manner, a BCR-mediated signaling recently emphasized to specifically occur in CLL cells. Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this still not described feature with the presence of signals from the BCR continuously activating VLA-4. Methods. Expression of the integrin alpha (CD49c, CD49d, CD49e) and beta1 (CD29) chains was analyzed by flow cytometry. The VLA-4 activation state was investigated by flow cytometry using the conformational sensitive anti-CD29 monoclonal antibody HUTS21, employed in conjunction with sources of VLA-4 ligands: either autologous plasma-containing fibronectin (FN) and soluble (s) VCAM-1 or increasing concentrations of exogenously added LDV-containing peptides as a VLA-4 specific ligand. In detail: i) HUTS21 expression was analyzed using whole blood (WB) samples from 727 CLL patients. Negative controls were obtained after plasma depletion through washing of WB samples; ii) the VLA-4 activation state expressed as receptor occupancy (RO) (J Biol Chem, 284,14337, 2009) was analyzed in sequential (t=0, day14, day 30) frozen/thawed samples from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=16) and from a clinical trial (NCT02827617, n=14). BCR engagement was performed using goat F(ab′)2 anti-human IgM. ELISA assays were used to quantify FN and sVCAM-1 in plasma samples. Results. According to the 30% cutoff, CD49d was expressed in 351/727 (48%), while CD29 was expressed in 676/727 (93%), and a strong correlation between CD49d and CD29 was observed (rho=0.7, p<0.0001). Among CD49d positive CLL cases, 81/351 (23%) expressed a constitutively activated form of VLA-4 identified by >20% (arbitrary cut-off) HUTS21 expression. Notably, HUTS21 positive expression was not circumscribed to VLA-4 expressing CLL, being also detected in 64/375 (17%) CD49d-, CD49c+ and/or CD49e+ CLL, thus envisioning a role of other integrins (CD49c and CD49e) in CLL cell interactions in tissues sites. HUTS21 expression did not correlate with a definite cytogenetic group, IGHV mutational status or with a specific IGHV family and stereotype. Depletion of the plasma component from the WB samples before HUTS21 staining significantly reduced the proportion of HUTS21 positive cells compared with those measured in WB samples (p=0.001, n=11), suggesting the need of plasma-borne ligands for HUTS21 epitope exposure. Consistently, both FN (mean 350 μg/ml) and sVCAM-1 (mean 4.8 μg/ml) were detected in the plasma of CLL cases, irrespective to HUTS21 positivity. According to these observations, variable constitutive VLA-4 activation states were observed in CLL cells collected at the pre-treatment stage from patients undergoing IB treatment (VLA-4 RO ranging from 0.22 to 0.40); these values significantly decreased in CLL cells collected at day 14 (p=0.03) and day 30 (p=0.02) on IB, suggesting an IB-dependent impairment of antigen-independent (autonomous) BCR signals. The variability of the constitutive VLA-4 activation levels observed at pre-treatment was paralleled by variable VLA-4 activation levels upon BCR triggering (VLA-4 RO ranging from 0.56 to 0.65). Of note, an inverse correlation between the VLA-4 constitutive level and the extent of anti-IgM induced-VLA-4 activation was found (n=19, rho=-0.5, p=0.0093), implying a competition between antigen-independent and antigen-driven BCR signalling. Conclusion. The presence of a constitutively activated form of VLA-4 is observed in a subset of CLL which might be connected to continuous VLA-4 inside-out stimulation derived from an autonomous BCR signaling, which is currently under evaluation. Disclosures Zaja: Takeda: Honoraria; Sandoz: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria.
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Harastani, Mohamad, Mikhail Eltsov, Amélie Leforestier, and Slavica Jonic. "HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes." Frontiers in Molecular Biosciences 8 (May 19, 2021). http://dx.doi.org/10.3389/fmolb.2021.663121.
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Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (in situ). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. We present HEMNMA-3D, which is, to the best of our knowledge, the first method for analyzing cryo electron subtomograms in terms of continuous conformational changes of complexes. HEMNMA-3D uses a combination of elastic and rigid-body 3D-to-3D iterative alignments of a flexible 3D reference (atomic structure or electron microscopy density map) to match the conformation, orientation, and position of the complex in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal Mode Analysis of the 3D reference) and experimental, subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge, due to the limited tilt angle of cryo-ET. The conformational parameters (amplitudes of normal modes) of the complexes in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (typically, 2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into the dynamics of the complexes, by calculating 3D averages of subtomograms with similar conformations from selected (densest) regions and by recording movies of the 3D reference's displacement along selected trajectories through the densest regions. We describe HEMNMA-3D and show its validation using synthetic datasets. We apply HEMNMA-3D to an experimental dataset describing in situ nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).
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Dsouza, Raison, Ghoncheh Mashayekhi, Roshanak Etemadpour, Peter Schwander, and Abbas Ourmazd. "Energy landscapes from cryo-EM snapshots: a benchmarking study." Scientific Reports 13, no. 1 (2023). http://dx.doi.org/10.1038/s41598-023-28401-w.
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AbstractBiomolecules undergo continuous conformational motions, a subset of which are functionally relevant. Understanding, and ultimately controlling biomolecular function are predicated on the ability to map continuous conformational motions, and identify the functionally relevant conformational trajectories. For equilibrium and near-equilibrium processes, function proceeds along minimum-energy pathways on one or more energy landscapes, because higher-energy conformations are only weakly occupied. With the growing interest in identifying functional trajectories, the need for reliable mapping of energy landscapes has become paramount. In response, various data-analytical tools for determining structural variability are emerging. A key question concerns the veracity with which each data-analytical tool can extract functionally relevant conformational trajectories from a collection of single-particle cryo-EM snapshots. Using synthetic data as an independently known ground truth, we benchmark the ability of four leading algorithms to determine biomolecular energy landscapes and identify the functionally relevant conformational paths on these landscapes. Such benchmarking is essential for systematic progress toward atomic-level movies of continuous biomolecular function.
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Dashti, Ali, Ghoncheh Mashayekhi, Mrinal Shekhar, et al. "Retrieving functional pathways of biomolecules from single-particle snapshots." Nature Communications 11, no. 1 (2020). http://dx.doi.org/10.1038/s41467-020-18403-x.
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Abstract A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, we present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Our approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations.
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Nierzwicki, Łukasz, and Giulia Palermo. "Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules." Frontiers in Molecular Biosciences 8 (April 5, 2021). http://dx.doi.org/10.3389/fmolb.2021.641208.
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Single-particle cryogenic electron microscopy (cryo-EM) has revolutionized the field of the structural biology, providing an access to the atomic resolution structures of large biomolecular complexes in their near-native environment. Today’s cryo-EM maps can frequently reach the atomic-level resolution, while often containing a range of resolutions, with conformationally variable regions obtained at 6 Å or worse. Low resolution density maps obtained for protein flexible domains, as well as the ensemble of coexisting conformational states arising from cryo-EM, poses new challenges and opportunities for Molecular Dynamics (MD) simulations. With the ability to describe the biomolecular dynamics at the atomic level, MD can extend the capabilities of cryo-EM, capturing the conformational variability and predicting biologically relevant short-lived conformational states. Here, we report about the state-of-the-art MD procedures that are currently used to refine, reconstruct and interpret cryo-EM maps. We show the capability of MD to predict short-lived conformational states, finding remarkable confirmation by cryo-EM structures subsequently solved. This has been the case of the CRISPR-Cas9 genome editing machinery, whose catalytically active structure has been predicted through both long-time scale MD and enhanced sampling techniques 2 years earlier than cryo-EM. In summary, this contribution remarks the ability of MD to complement cryo-EM, describing conformational landscapes and relating structural transitions to function, ultimately discerning relevant short-lived conformational states and providing mechanistic knowledge of biological function.
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Bogetti, Xiaowei, and Sunil Kumar Saxena. "Integrating Electron Paramagnetic Resonance Spectroscopy and Computational Modeling to Measure Protein Structure and Dynamics." ChemPlusChem, October 6, 2023. http://dx.doi.org/10.1002/cplu.202300506.
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Electron paramagnetic resonance (EPR) has become a powerful probe of conformational heterogeneity and dynamics of biomolecules. In this review, we discuss different computational modeling techniques that enrich the interpretation of EPR measurements of dynamics or distance restraints. A variety of spin labels are surveyed to provide a background for the discussion of modeling tools. Molecular dynamics (MD) simulations of models containing spin labels provide dynamical properties of biomolecules and their labels. These simulations can be used to predict EPR spectra, sample stable conformations and sample rotameric preferences of label sidechains. For molecular motions longer than milliseconds, enhanced sampling strategies and de novo prediction software incorporating or validated by EPR measurements are able to efficiently refine or predict protein conformations, respectively. To sample large‐amplitude conformational transition, a coarse‐grained or an atomistic weighted ensemble (WE) strategy can be guided with EPR insights. Looking forward, we anticipate an integrative strategy for efficient sampling of alternate conformations by de novo predictions, followed by validations by systematic EPR measurements and MD simulations. Continuous pathways between alternate states can be further sampled by WE‐MD including all intermediate states.
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Vuillemot, Rémi, Isabelle Rouiller, and Slavica Jonić. "MDTOMO method for continuous conformational variability analysis in cryo electron subtomograms based on molecular dynamics simulations." Scientific Reports 13, no. 1 (2023). http://dx.doi.org/10.1038/s41598-023-37037-9.
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AbstractCryo electron tomography (cryo-ET) allows observing macromolecular complexes in their native environment. The common routine of subtomogram averaging (STA) allows obtaining the three-dimensional (3D) structure of abundant macromolecular complexes, and can be coupled with discrete classification to reveal conformational heterogeneity of the sample. However, the number of complexes extracted from cryo-ET data is usually small, which restricts the discrete-classification results to a small number of enough populated states and, thus, results in a largely incomplete conformational landscape. Alternative approaches are currently being investigated to explore the continuity of the conformational landscapes that in situ cryo-ET studies could provide. In this article, we present MDTOMO, a method for analyzing continuous conformational variability in cryo-ET subtomograms based on Molecular Dynamics (MD) simulations. MDTOMO allows obtaining an atomic-scale model of conformational variability and the corresponding free-energy landscape, from a given set of cryo-ET subtomograms. The article presents the performance of MDTOMO on a synthetic ABC exporter dataset and an in situ SARS-CoV-2 spike dataset. MDTOMO allows analyzing dynamic properties of molecular complexes to understand their biological functions, which could also be useful for structure-based drug discovery.
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Giraldo-Barreto, Julian, Sebastian Ortiz, Erik H. Thiede, et al. "A Bayesian approach to extracting free-energy profiles from cryo-electron microscopy experiments." Scientific Reports 11, no. 1 (2021). http://dx.doi.org/10.1038/s41598-021-92621-1.
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AbstractCryo-electron microscopy (cryo-EM) extracts single-particle density projections of individual biomolecules. Although cryo-EM is widely used for 3D reconstruction, due to its single-particle nature it has the potential to provide information about a biomolecule’s conformational variability and underlying free-energy landscape. However, treating cryo-EM as a single-molecule technique is challenging because of the low signal-to-noise ratio (SNR) in individual particles. In this work, we propose the cryo-BIFE method (cryo-EM Bayesian Inference of Free-Energy profiles), which uses a path collective variable to extract free-energy profiles and their uncertainties from cryo-EM images. We test the framework on several synthetic systems where the imaging parameters and conditions were controlled. We found that for realistic cryo-EM environments and relevant biomolecular systems, it is possible to recover the underlying free energy, with the pose accuracy and SNR as crucial determinants. We then use the method to study the conformational transitions of a calcium-activated channel with real cryo-EM particles. Interestingly, we recover not only the most probable conformation (used to generate a high-resolution reconstruction of the calcium-bound state) but also a metastable state that corresponds to the calcium-unbound conformation. As expected for turnover transitions within the same sample, the activation barriers are on the order of $$k_BT$$ k B T . We expect our tool for extracting free-energy profiles from cryo-EM images to enable more complete characterization of the thermodynamic ensemble of biomolecules.
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Zhang, Jingxing, Rundong Wu, Yancong Feng, et al. "Optically Tunable Multistable Liquid Crystal Grating for Anti‐Counterfeiting through Multilayer Continuous Phase Analysis." Advanced Optical Materials, March 27, 2024. http://dx.doi.org/10.1002/adom.202302423.
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AbstractOptically tunable liquid‐crystal (LC) gratings have remarkable applications in the field of image analysis for anti‐counterfeiting. However, existing tunable LC gratings suffer from limited adjustable ranges and weak stability. The spacing variability and accuracy of the grating cannot satisfy the demands of continuous analysis. This study presents a novel approach to anti‐counterfeiting through multilayer continuous phase analysis (MCPA) based on the continuous multistable states of the LC grating. This optically tunable LC grating featuring continuous multistable states is successfully prepared using a novel optically tunable chiral dopant, tetra(n‐hex‐azo), which exhibits an exceptionally slow conformational relaxation rate (half‐life: 78.02 h). By exposing the LC grating to ultraviolet irradiation with an intensity of 0.1054 mW cm−2, the fringe spacing can be continuously and widely tuned between 0 and 250 µm, which enhances the ultraslow conformational relaxation of tetra(n‐hex‐azo). Four representative spacings (50, 55, 60, and 65 µm) are selected to construct four‐layer MCPA gratings, enabling simultaneous decoding of a four‐layer latent image for the first time. The mechanism of the MCPA grating is investigated through control experiments and theoretical simulations. This study demonstrates a facile, precise, and efficient approach for complex information encryption and anti‐counterfeiting.
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Punjani, Ali, and David J. Fleet. "3DFlex: determining structure and motion of flexible proteins from cryo-EM." Nature Methods, May 11, 2023. http://dx.doi.org/10.1038/s41592-023-01853-8.
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AbstractModeling flexible macromolecules is one of the foremost challenges in single-particle cryogenic-electron microscopy (cryo-EM), with the potential to illuminate fundamental questions in structural biology. We introduce Three-Dimensional Flexible Refinement (3DFlex), a motion-based neural network model for continuous molecular heterogeneity for cryo-EM data. 3DFlex exploits knowledge that conformational variability of a protein is often the result of physical processes that transport density over space and tend to preserve local geometry. From two-dimensional image data, 3DFlex enables the determination of high-resolution 3D density, and provides an explicit model of a flexible protein’s motion over its conformational landscape. Experimentally, for large molecular machines (tri-snRNP spliceosome complex, translocating ribosome) and small flexible proteins (TRPV1 ion channel, αVβ8 integrin, SARS-CoV-2 spike), 3DFlex learns nonrigid molecular motions while resolving details of moving secondary structure elements. 3DFlex can improve 3D density resolution beyond the limits of existing methods because particle images contribute coherent signal over the conformational landscape.
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Harastani, Mohamad, Rémi Vuillemot, Ilyes Hamitouche, Nima Barati Moghadam, and Slavica Jonic. "ContinuousFlex: Software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy and tomography data." Journal of Structural Biology, October 2022, 107906. http://dx.doi.org/10.1016/j.jsb.2022.107906.
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Ribeiro, I., F. Pires, C. R. C. Calado, and M. Gallard. Molina. "P–769 FTIR spectroscopy analysis reveals differences between human embryo culture media composition by type of formulation and by manufacturer." Human Reproduction 36, Supplement_1 (2021). http://dx.doi.org/10.1093/humrep/deab130.768.
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Abstract Study question Can we detect the variation in different commercial human embryo culture media composition by Fourier-transform infrared (FTIR) spectroscopic analysis? Summary answer The spectra reveals distint features that allow distinguishing between continuous and sequential media, as well as between manufacturers of the same media type. What is known already: Manufacturers do not fully disclose commercially available culture media formulations. For this reason, it is important to gain insight into de differences between the available formulations, in order to understand how they might be linked with efficacy and safety ART parameters. Fourier Transform Infra-Red (FTIR) can be used for this purpose. Study design, size, duration Culture media samples (1 mL) were collected from local IVF laboratories, and stored frozen at –20 °C. Five repeats of 25 µL aliquots from the samples were analysed using FTIR spectroscopy to acquire the whole molecular fingerprint of each culture media. Participants/materials, setting, methods Three continuous (SAGE 1-STEP, Origio, G-TL, Vitrolife, and GERI, Genea) and four sequential (G1 PLUS, Vitrolife; Sequential Cleav with phenolred, Origio; G2 PLUS, Vitrolife and Sequential Blast with phenolred, Origio) media were analysed. Different pre-processing methods (atmospheric and baseline correction, normalizations, and derivatives) were carried out to minimize physical artefacts while highlighting chemical features. To compare the spectra of different media, multivariate analysis, as principal component analysis (PCA) and hierarchical cluster analysis (HCA) were employed. Main results and the role of chance The whole molecular fingerprint of all media analysed showed a similar pattern, revealing that, overall, the composition is very similar. However, PCA and HCA analysis revealed that significant differences exist, both between media type (continuous vs. sequential), and between different manufacturers within the same media type. Average linkage clustering using Spearman’s rank correlation confirms the similarities between the continuous and the sequential formulations. An analysis focusing the fingerprint region of the spectra (900 – 1700 cm^–1), also revealed variability between manufacturer, between media type (continuous vs. sequential) and of stage-specific media (cleavage vs. blastocyst). For instance, GERI media visually appeared to have distinct peaks compared to all other media, which was confirmed later through multivariate statistical analysis. Limitations, reasons for caution FTIR spectroscopy does not allow for a direct identification of the analytes present in the culture media, we can only infer the functional groups, but that are common on diverse biomolecules. Wider implications of the findings: FTIR analysis reveals differences between different media, such as cleavage and blastocyst specific, sequential and continuous, or manufacturer’s formulations. The increased resolution of the FTIR profile proves to be a powerful tool for analysing human embryo media, and could be used to establish correlations with media clinical performance and safety. Trial registration number Not applicable
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Reynolds, Matthew J., Carla Hachicho, Ayala G. Carl, Rui Gong, and Gregory M. Alushin. "Bending forces and nucleotide state jointly regulate F-actin structure." Nature, October 26, 2022. http://dx.doi.org/10.1038/s41586-022-05366-w.
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AbstractATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation1–3. In turn, force4,5 and actin filament (F-actin) nucleotide state6 regulate actin dynamics by tuning F-actin’s engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP–F-actin and ADP-Pi–F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP–F-actin and ADP-Pi–F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation.
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Holguin, Bianka A., Zacariah L. Hildenbrand, and Ricardo A. Bernal. "Insights Into the Role of Heat Shock Protein 27 in the Development of Neurodegeneration." Frontiers in Molecular Neuroscience 15 (March 30, 2022). http://dx.doi.org/10.3389/fnmol.2022.868089.
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Small heat shock protein 27 is a critically important chaperone, that plays a key role in several essential and varied physiological processes. These include thermotolerance, apoptosis, cytoskeletal dynamics, cell differentiation, protein folding, among others. Despite its relatively small size and intrinsically disordered termini, it forms large and polydisperse oligomers that are in equilibrium with dimers. This equilibrium is driven by transient interactions between the N-terminal region, the α-crystallin domain, and the C-terminal region. The continuous redistribution of binding partners results in a conformationally dynamic protein that allows it to adapt to different functions where substrate capture is required. However, the intrinsic disorder of the amino and carboxy terminal regions and subsequent conformational variability has made structural investigations challenging. Because heat shock protein 27 is critical for so many key cellular functions, it is not surprising that it also has been linked to human disease. Charcot-Marie-Tooth and distal hereditary motor neuropathy are examples of neurodegenerative disorders that arise from single point mutations in heat shock protein 27. The development of possible treatments, however, depends on our understanding of its normal function at the molecular level so we might be able to understand how mutations manifest as disease. This review will summarize recent reports describing investigations into the structurally elusive regions of Hsp27. Recent insights begin to provide the required context to explain the relationship between a mutation and the resulting loss or gain of function that leads to Charcot-Marie Tooth disease and distal hereditary motor neuropathy.