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<p dir="ltr">In life sciences, the determination of macromolecular structures and their functions, particularly proteins and protein complexes, is of paramount importance, as these molecules play critical roles within cells. The specific physical interactions of macromolecules govern molecular and cellular functions, making the 3D structure elucidation of these entities essential for comprehending the mechanisms underlying life processes, diseases, and drug discovery. Cryo-electron microscopy (cryo-EM) has emerged as a promising experimental technique for obtaining 3D macromolecular structures. In the course of my research, I proposed CryoREAD, an innovative AI-based method for <i>de nov</i>o DNA/RNA structure modeling. This novel approach represents the first fully automated solution for DNA/RNA structure modeling from cryo-EM maps at near-atomic resolution. However, as the resolution decreases, structure modeling becomes significantly more challenging. To address this challenge, I introduced Emap2sec+, a 3D deep convolutional neural network designed to identify protein secondary structures, RNA, and DNA information from cryo-EM maps at intermediate resolutions ranging from 5-10 Å. Additionally, I presented Alpha-EM-Multimer, a groundbreaking method for automatically building full protein complexes from cryo-EM maps at intermediate resolution. Alpha-EM-Multimer employs a diffusion model to trace the protein backbone and subsequently fits the AlphaFold predicted single-chain structure to construct the complete protein complex. Notably, this method stands as the first to enable the modeling of protein complexes with more than 10,000 residues for cryo-EM maps at intermediate resolution, achieving an average TM-Score of predicted protein complexes above 0.8, which closely approximates the native structure. Furthermore, I addressed the recognition of local structural errors in predicted and experimental protein structures by proposing DAQ, an evaluation approach for experimental protein structure quality that utilizes detection probabilities derived from cryo-EM maps via a pretrained multi-task neural network. In the pursuit of evaluating protein complexes generated through computational methods, I developed GNN-DOVE and DOVE, leveraging convolutional neural networks and graph neural networks to assess the accuracy of predicted protein complex structures. These advancements in cryo-EM-based structural modeling and evaluation methodologies hold significant promise for advancing our understanding of complex macromolecular systems and their biological implications.</p>

In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.

In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.

In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.

In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.

In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.

In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.

In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.

In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.