Structural biology

Summary

Structural biology is a field that is many centuries old which, as defined by the Journal of Structural Biology, deals with structural analysis of living material (formed, composed of, and/or maintained and refined by living cells) at every level of organization. Early structural biologists throughout the 19th and early 20th centuries were primarily only able to study structures to the limit of the naked eye's visual acuity and through magnifying glasses and light microscopes. In the 20th century, a variety of experimental techniques were developed to examine the 3D structures of biological molecules. The most prominent techniques are X-ray crystallography, nuclear magnetic resonance, and electron microscopy. Through the discovery of X-rays and its applications to protein crystals, structural biology was revolutionized, as now scientists could obtain the three-dimensional structures of biological molecules in atomic detail. Likewise, NMR spectroscopy allowed information about protein

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https://en.wikipedia.org/wiki/Structural_biology

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Amplitude and phase reconstruction for Low-Energy Electron Holography of individual proteins

Hannah Julia Ochner

Single-molecule imaging methods are of importance in structural biology, and specifically in the imaging of proteins, since they can elucidate conformational variability and structural changes that might be lost in imaging methods relying on averaging processes. Low-energy electron holography (LEEH) is a promising technique for imaging individual proteins with negligible radiation damage, which can be combined with a sample preparation process by native electrospray ion beam deposition (native ES-IBD) ensuring the creation of chemically pure samples suitable for holographic imaging.The central step in the analysis of data measured by LEEH is the numerical reconstruction of the object from the experimentally acquired holograms.Full information about the imaged object consists of both amplitude and phase information imprinted on the scattered wave during the interaction of the electron beam with the molecule and encoded in the hologram created by the interference of the scattered wave and the transmitted incident wave. A propagation-based algorithm with the goal of reconstructing the wave field in the plane of the object is presented and applied to holograms of individual proteins prepared by ES-IBD and measured with a low-energy electron holography microscope. The information retrieved from the reconstruction of the amplitude distribution in the object plane is discussed by analysing holograms of antibodies, demonstrating that the inherent conformational variability of these molecules can be mapped by LEEH. The influence of the sample preparation process on the surface conformations is tracked by tuning the landing energy of the proteins during deposition.To complement the amplitude data, an iterative phase retrieval algorithm is implemented to reconstruct the phase distribution in the object plane along with the amplitude distribution. The algorithm's performance and robustness is thoroughly evaluated and simulations regarding multiple scattering effects and element-dependent variations in scattering strength are carried out to provide a reference for the interpretation of phase data retrieved from experimentally acquired holograms. The iterative phase retrieval algorithm is then applied to protein data, indicating that both molecular density and charges can be related to features in the phase reconstructions, while the presence of metals does not correlate with specific phase signals at the current resolution obtainable from the experimental data.Since proteins are inherently three-dimensional, approaches towards three-dimensional reconstruction schemes are discussed, which will be the focus of future work.

EPFL2022

Evaluation of oxidation-sensitive polymersomes as vaccine nanocarriers to enhance humoral responses against arenavirus envelope glycoproteins

Clara Galan Navarro

Vaccines currently represent one of the most effective means to control and prevent infectious diseases affecting humans and animals and have dramatically improved public health, quality of life, and life expectancy. Over the last years several conceptual and technological advances in the field of bioengineering and immunology have allowed the development of new vaccines designs. Expanding novel domains such as structural biology, human monoclonal antibody isolation, high throughput sequencing, and design of new nanocarrier delivery platforms have changed the landscape of vaccinology. These new vaccine formulations allow the design of tailor-made antigens and particulate vehicles which allow better and fine-tuned protective immune responses against known and newly emerging pathogens. Over the past decades emerging human pathogens with the potential to cause severe epidemics have become a major public health problem. The last Ebola and Zika virus epidemics demonstrated in a dramatic manner that there is an urgent need to develop prophylactic and therapeutic approaches against pathogenic emerging viruses. Most successful vaccines rely on attenuated live microorganisms to induce protective immunity. However, changed demographics and associated safety concerns makes the development of a safer alternative a priority. Subunit vaccines composed of microbial components and proteins have emerged as a suitable alternative, however they are generally less immunogenic. To address this problematic our laboratory works on the development of immunization platforms which allow 1) the development of safe recombinant subunit vaccines and 2) the rapid generation of specific monoclonal antibodies using biosynthetic immunogens without the need for prior isolation and culture of the agent. The approach evaluated in the present studies is based on a novel polymersome (PS) platform serving as nanocarrier for efficient antigen delivery and the induction of humoral immunity. The potential of PS was assessed in the context of two important human emerging pathogens form the arenavirus family that cause severe hemorrhagic fevers and of which there are neither FDA-approved vaccines not prophylactic treatments: Lassa virus (LASV), endemic in Western Africa, and Machupo virus (MACV), the causative agent of Bolivian hemorrhagic fever. In a first part, we evaluated the PS platformâs capacity to enhance humoral immunity against the envelope glycoprotein 1 of LASV, (LASV GP1) that is notorious for its weak immunogenicity and poor antibody response. Immunization of mice with adjuvanted PS (LASV GP1) enhanced the quality of the humoral response to LASV GP1, eliciting antibodies with higher binding affinity to virion GP1, increased levels of polyfunctional anti-viral CD4 T cells, and the frequency of IgG-secreting B cells. PS (LASV GP1) elicited a more diverse epitope repertoire of anti-viral IgG. In a second part, we employed the PS platform in combination with single cell B cell sorting and cloning of recombinant IgG to generate a first set of species-specific mAbs against MACV. These new mAbs show exquisite specificity and negligible cross-reactivity to closely related arenaviruses in relevant techniques making them a unique and powerful tool for research and diagnostics purposes.

EPFL2017

The true understanding of most cellular functions is only really achievable through the structural determination of their underlying macromolecular assemblies. However, their size, large number of individual components and metastable states make their elucidation by canonical structural biology techniques a vain effort in many situations. As a result, integrative approaches have been in high demand in the recent years. By combining any available data, both computational and experimental, integrative or hybrid modeling strategies have been able to tackle biomolecular structures otherwise intractable by any singular method alone. Over the past decade, structural biology has undergone a significant revolution in the form of cryo-electron microscopy (cryo-EM). With more than 12.000 models of biomolecular structures under its name, cryo-EM however still struggles to approach larger, flexible assemblies at atomic resolution. Instead, such assemblies now routinely fall in an intermediate resolution range, opening new avenues for the development of hybrid approaches primarily based on cryo-EM data. However, the large degrees of flexibility of such assemblies imposes the inclusion of dynamics in the modeling process. Unfortunately, this only makes the already challenging task of defining a scoring function even more difficult. In general, current integrative strategies are thus unable to approach flexible assemblies in a reliable manner. To tackle these issues, two new methods made available to the community are presented in this thesis in addition to their applications to a variety of systems. A new clustering analysis tool, CLoNe, is first introduced. As a significant upgrade to the recent Density Peaks algorithm, we show that CLoNe rivals and outperforms many state-of-the-art algorithms even beyond structural biology. Then, we show how CLoNe is able to extract a variety of information from structural ensembles in general or reduce large ensembles to key components, enabling their successful integration into hybrid modeling approaches. Second, the MaD software is presented. Taking inspiration from traditional computer vision concepts and methods, MaD bypasses the need of a traditional scoring function through the generation of local macromolecular feature descriptors. MaD takes full advantage of the ongoing cryo-EM resolution revolution by integrating local structural information from both cryo-EM data and existing atomic structures. Specifically, MaD is able to predict the quaternary structure of large assemblies regardless of symmetry and conformational variability. Finally, the MaD-CLoNe combination enabled the modeling of molecular chaperones with unprecedented ease. Chaperonins are of crucial importance to ensure proper protein folding and proteostasis. Perturbation of these processes are implicated in neurodegenerative diseases and cancer. In the specific case of the group II chaperonin Mm-Cpn, the use of MaD-CLoNe along with molecular dynamics simulations uncovered new structural models and insight into the chaperonin's functional pathway.

EPFL2021