Com o crescimento populacional e aumento das atividades humanas, grandes quantidades de poluentes são despejadas diariamente no meio ambiente. Nos ecosssistemas aquáticos, organismos são expostos por longos períodos à baixas concentrações de contaminantes. Assim estudos que focam em novas metodologias que sejam rápidas e não invasivas, são necessários para a avaliação de efeitos subletais. Um exemplo é a espectroscopia no infravermelho com transformada de Fourier (FTIR). Esta técnica é capaz de detectar alterações macromoleculares em amostras biológicas de forma rápida e não destrutiva, sendo uma ferramenta atrativa para análises ecotoxicológicas. Assim, o objetivo deste estudo foi verificar a aplicabilidade do FTIR como metodologia alternativa, utilizando perfis de composição macromoleculares como indicadores de efeitos subletais, em organismos expostos a elementos-traço. Para tal, ostrácodes (Chlamydoteca sp.) foram expostos aos elementos Cu, Cd, Hg e Mn. As alterações em perfis biomoleculares foram avaliadas em ostrácodes expostos por 48h ao Mn. Os resultados revelaram uma correlação negativa entre concentração de exposição e proporção de ácidos graxos saturados e insaturados com relação à proteína em organismos expostos ao Mn e a mesma correlação foi observada para o conteúdo de ácidos nucléicos. Isto sugere que os organismos tiveram seu metabolismo lipídico e sua capacidade de produção de proteína celular alterados devido à exposição ao Mn. Ademais, foram realizados testes crônicos (9 dias) com ostrácodes expostos ao Cu, Cd, Hg e Mn. Foi observada a diminuição de lipídios saturados e insaturados em relação à proteína em organismos expostos ao Cd, sugerindo o uso de reservas lipídicas devido ao estresse fisiológico. Ostrácodes expostos ao Cu, tiveram o aumento dos ácidos nucléicos, no qual o DNA pode ter sido o componente dominante. Alterações significativas relacionadas ao conteúdo de carbonato de cálcio foram reveladas nos organismos expostos ao Cd e ao Cu, o que sugere mudanças na composição da carapaça e possível alteração na capacidade de muda e crescimento dos organismos. Portanto, este estudo permite concluir que a espectroscopia de FTIR é capaz de detectar indícios de efeitos subletais em organismos expostos a contaminantes e que as alterações de composição macromolecular são uma ferramenta complementar promissora para futuras avaliações ecotoxicológicas. / With population growth and increases in human activities, large amounts of pollutants are released daily into the environment. In aquatic ecosystems organisms are exposed for long periods at low concentrations of contaminants. Therefore, studies that focus on fast and non-invasive new methodologies are necessary for the assessment of sublethal effects in organisms. An example is the Fourier Transform Infrared Spectroscopy (FTIR). This technique can detect macromolecular changes of biological samples in a fast, inexpensive and non-destructive way, all of which lend to the attractiveness of this methodology for ecotoxicological analyzes. Thus, the aim of this study was to verify the applicability of FTIR as an alternative methodology, using the composition of macromolecular profiles as an indicator of sublethal effects in organisms exposed to trace elements. For this, ostracods (Chlamydoteca sp.) were exposed to the elements Cu, Cd, Hg and Mn. Changes in biomolecular profiles were evaluated in ostracods exposed for 48h to Mn. The results showed a negative correlation between the concentration of exposure and the proportion of saturated and unsaturated fatty acids for protein in organisms exposed to Mn and the same correlation was observed for the content of nucleic acids. This suggests that the organisms had their lipid metabolism and their cellular protein production capacity altered due to Mn exposure. In addition, four chronic tests (9 days) were performed on ostracods exposed to the trace elements Cu, Cd, Hg and Mn. A decrease in saturated and unsaturated lipids in relation to the protein was observed in organisms exposed to Cd, suggesting that the use of lipid reserves was due to the physiological stress. Ostracods exposed to Cu had an increase in nucleic acids, in which DNA may have been the dominant component. Significant changes related to the calcium carbonate content were revealed in the organisms exposed to Cd and Cu, which suggests changes in carapace composition and the possible alteration in the organism capacity of molting and growth. Therefore, this study concludes that FTIR spectroscopy can detect indications of sublethal effects in organisms exposed to contaminants and that changes in macromolecular composition are a promising complementary tool for future ecotoxicological assessments.

Macromolecular X-ray crystallography (MX) is currently the dominant technique for the structural eluci- dation of macromolecules at near atomic resolution. However, the progression and deleterious effects of radiation damage remains a major limiting factor in the success of diffraction data collection and subsequent structural solution at modern third generation synchrotron facilities. For experiments conducted at 100 K, protein specific damage to particular amino acids has been widely reported at doses of just several MGy, before any observable decay in average diffraction intensities. When undetected, such artefacts of X-ray irradiation can lead to significant modelling errors in protein structures, and ultimately the failure to derive the correct biological function from a model. It is thus vital to develop tools to help MX experimenters to detect and correct for such damage events. This thesis presents the development of an automated program, RIDL, which is designed to objectively quantify radiation-induced changes to electron density at individual atoms, based on F_obs,n − F_obs,1 Fourier difference maps between different dose states for a single crystal. The high-throughput RIDL program developed in this work provides the ability to systematically investigate a wide range of macromolecular systems. To date, damage to the broad class of nucleic acids and nucleoprotein complexes has remained largely uncharacterised, and it is unclear how radiation damage will disrupt the validity of such models derived from MX experiments. This thesis presents the first systematic investigations on a range of nucleic acid, protein-RNA and protein-DNA complex case studies. In general, it is concluded that nucleic acids are highly robust to radiation damage effects at 100K, relative to control protein counterparts across the tested systems. For protein crystals at 100K, cleavage of the phenolic C-O bond in tyrosine has disseminated through the MX radiation damage literature as a dominant specific damage event at 100K, despite the absence of any energetically favourable cleavage mechanism. To clarify the radiation susceptibility of tyrosine, this thesis presents a systematic investigation on radiation damage to tyrosine in a wide range of MX protein radiation damage series retrieved from the Protein Data Bank. It is concluded that the tyrosine C-O bond remains intact following X-ray irradiation, however the aromatic side-group can undergo radiation-induced displacement. This thesis also presents further applications of the RIDL program. A protocol is introduced to calculate explicit half-dose values for the electron density at individual atoms to decay to half of their initial value at zero absorbed dose. In addition, a methodology is developed to detect radiation-induced changes to electron density occurring over the course of the collection of a single MX dataset of diffraction images, all of which are required for structural solution. These protocols aim to advise experimenters of when previously-undetected site-specific damage effects may have corrupted the quality of their macromolecular model. Overall, the work in this thesis is highly applicable to both the future understanding of radiation damage in macromolecular structures, as well as of interest to the wider crystallographic community.

<div>X-ray crystallography is a foundation of the modern structural biology. Thus, refinement of crystallographic structures remains an important and actively pursued area of research. We have built a software solution for refinement of crystallographic protein structures using X-ray diffraction data in conjunction with state-of-the-art MD modeling setup. This solution was implemented on the platform of Amber 16 biomolecular simulation package, making use of graphical processing unit (GPU) computing. The proposed refinement protocol consists of a short MD simulation, which represents an entire crystal unit cell containing multiple protein molecules and interstitial solvent. The simulation is guided by crystallographic restraints based on experimental structure factors, as well as conventional force-field terms. We assessed the performance of this new protocol against various refinement procedures based on the Phenix engine, which represents the current industry standard. The evaluation was conducted on a set of 84 protein structures with different realizations of initial models; the main criterion of success was free R-factor, R_free. Initially, we performed the re-refinement of the models deposited in the PDB bank. We found that in 58% of all cases our protocol achieved better R_free than Phenix. As a next step, we conducted the refinement on three different sets of lower-quality models that were manufactured specifically to test the competing algorithms (average C^α RMSD from the target structures 0.75, 0.89, and 1.02 Å). In these tests, our protocol outperformed the refinement procedures available in Phenix in up to 89% of all cases. Aside from R-factors, we also compared geometric qualities of the models as measured by MolProbity scores. It was found that our protocol led to consistently better geometries in all of the refinement comparisons.</div><div>Recently, a number of attempts have been made to fully utilize the information encoded in protein diffraction data, including diffuse scattering, which is dependent on molecular dynamics in the crystal. To understand the nature of this dependence, we have chosen three different crystalline forms of ubiquitin. By post-processing the MD data, we separated the effects from different types of motion on the diffuse scattering profiles. This analysis failed to identify any features of the diffuse scattering profiles that could be uniquely linked to certain specific motional modes (e.g. small-amplitude rocking motion of protein molecules in the crystal lattice). However, we were able to confirm the previous experimental observations, made in the combined X-ray diffraction and NMR study, suggesting that the amount of motion in the specific crystal is reflected in the amplitude of diffuse scattering.</div>

The exeC gene, found in the gram-negative bacteria Aeromonas hydrophila codes for a 31 kDa, three domain, bitopic inner membrane protein. The components of the ExeC protein include an amino-terminal cytoplasmic domain, a trans-membrane helix and two periplasmic domains. The two periplasmic domains are involved in recognition and selection of protein substrates which are subsequently transported across the outer membrane and free of the cell. This study focuses exclusively on the two periplasmic domains referred to hereafter as the HR and the PDZ domains. Three constructs were used throughout the course of this study. Two of them were designed, cloned and expressed for this study. The third is a result of previous work. Two constructs contained both the HR and PDZ domains while the other consists of the amino-terminal periplasmic HR domain. Only one construct was used to grow single crystals for analysis by X-ray crystallography. Crystals comprised of the PDZ domain from a degraded construct grew in a hexagonal space group with a hexagonal bi-pyramidal morphology. Crystals diffracted anisotropically to a maximum resolutions of 2 Å along the c axis and 3 Å in the a/b plane. Anisotropy in combination with twinning drastically complicated structure solution. Efforts toward elucidating the crystal structure will be discussed.

Protein folding is the process during which an extended and unstructured polypeptide converts to its compact folded structure that is most often the functional state. The process has been characterized extensively in dilute buffer in-vitro during the last decades but the actual biological place for this process is the inside of living cells. The cytoplasm of a cell is filled with a plethora of different macromolecules that together occupy up to 40% of the total volume. This large amount of macromolecules restricts the available space to each individual molecule, which has been termed macromolecular crowding. Macromolecular crowding results in excluded volume effects and also increases chances for non-specific interactions. Macromolecular crowding should favor reactions that lead to a decrease in the total occupied volume by all molecules, such as folding reactions. Theoretical models have predicted that the stability of protein folded states should increase in presence of macromolecular crowding due to unfavorable effects on the extended unfolded state. To understand protein folding and function in living systems, we need to have a defined quantitative link between in-vitro dilute conditions (where most biophysical experiments are made) and in-vivo crowded conditions. An important question is thus how macromolecular crowding modifies the biophysical properties of a protein. The work underlying this thesis focused on how macromolecular crowding tunes protein equilibrium stability and kinetic folding processes. To mimic the crowded cellular environment, synthetic sugar-based polymers (Dextrans of different sizes and Ficoll 70) were used as crowding agents (crowders) in controlled in-vitro experiments. In contrast to previous studies which often have focused on one protein and one crowder at a time, the goal here was to make systematic analyses of how size, shape and concentration of the crowders affect both equilibrium and kinetic properties of structurally-different proteins. Three model proteins (cytochrome c, apoazurin and apoflavodoxin) were investigated under crowding by Ficoll 70 and different-size Dextrans, using various spectroscopic techniques such as far-UV circular dichroism and intrinsic tryptophan fluorescence. Thermodynamic models were applied to explain the experimental results. It was discovered that equilibrium stability of all three proteins increased in presence of crowding agents in a crowder concentration dependent manner. The stabilization effect was around 2-3 kJ/mol, larger for the various Dextrans than for Ficoll 70 at the same g/l, but independent of Dextran size (in the range 20 to 70 kDa). To further investigate the cause for the stabilization a theoretical crowding model was applied. In this model, Dextran and Ficoll were modeled as elongated rods and the protein was represented as a sphere, where the folded sphere representation was smaller than the unfolded sphere representation. It is notable that the observed stability changes could be reproduced by this model taking only steric interactions into account. This correlation showed that when using sugar-based crowding agents, excluded volume effects could be studied in isolation and there were no contributions from nonspecific interactions. Time-resolved experiments with apoazurin and apoflavodoxin revealed an increase in the folding rate constants while the unfolding rates were invariant in the presence of crowding agents. For apoflavodoxin and cytochrome c, the presence of crowding agents also altered the folding pathway such that it became more homogeneous (cytochrome c) and it gave less misfolding (apoflavodoxin). These results showed that macromolecular crowding restricts the conformational space of the unfolded polypeptide chain, makes the conformations more compact which, in turn, eliminates access to certain pathways. The results from kinetic and equilibrium measurements on three model proteins, together with available data from the literature, demonstrate that macromolecular crowding effects due to volume exclusion are in the order of a few kJ/mol. Considering the numerous concentration balances and cross-dependent reactions of the cellular machinery, small changes in energetics/kinetics of the magnitudes found here can still have dramatic consequences for cellular fitness. In fact local and transient changes in macromolecular crowding levels may be a way to tune biochemical reactions without invoking gene expression.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Note: Thesis now published in various journals, details in the Access Instructions file. / During the past almost three decades, conducting polymers have been the subject of intense scientific and industrial research and development worldwide. A general background of the fundamental principles and concepts of conducting polymers and their applications is presented in Chapter 1. In Chapter 2, the characterization of chemically synthesised emeraldine base polyaniline (EB-PANI), acetone-extracted to remove impurities, is examined. Spectroscopic studies PANI at different levels of oxidation are reported, and the structural changes in as-synthesised EB-PANI upon doping with HCl and iodine vapour are investigated. A maximum in the number of unpaired electron spins was found in 1M HCl doped PANI, corresponding to emeraldine salt PANI (ES-PANI), which indicates that bipolarons form at higher doping levels. Also, a maximum conductivity of 0.478 S cm-1 was observed for 1M HCl doped PANI, and no significant change in the conductivity was observed at higher doping levels. A new mechanism of iodine doping of EB-PANI was proposed, which occurs by the oxidation of the benzenoid diamine units, instead of the quinoid diimine units as previously proposed. In Chapter 3, the incorporation of planar Cu(II) complexes as dopants into ES-PANI was investigated by means of EPR and IR spectroscopy. A decrease in the intensity of the EPR signals from polarons in Cu(II) complex-doped PANI samples was observed, while the Cu(II) anion concentration increases and favors the formation of bipolarons in Cu(II) complex-doped PANI systems. The reactions of EB-PANI following different levels of reduction or HCl doping with CuCl2 aqueous solution are also investigated and new reaction mechanisms are proposed. The conductivities of the resulting samples have also been measured and discussed. The ability of aniline and PANI samples to act as antioxidants using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical method are investigated by means of UV-VIS, FTIR and EPR spectroscopy in Chapter 4. A study of the kinetics of the reaction between DPPH and aniline showed that the reaction is first order with respect to DPPH and approximately second order with respect to aniline. A further investigation of the mechanism of the aniline/DPPH reaction and products of the reaction (aniline oligomers and DPPHH) by magnetic resonance techniques is discussed in Chapter 5. This study has resulted in the identification for the first time of uncapped dimers and trimers of aniline oligomers in solution by EPR spectroscopy. In Chapter 6, for the first time, the applicability of various 13C and 15N solid-state nuclear magnetic resonance (SSNMR) techniques in the investigation of the changes in the structure of PANI that occur upon reaction with DPPH radicals was investigated and discussed. The results indicate partial oxidation of PANI samples upon reaction with DPPH, which is consistent with previously obtained FTIR and EPR data in Chapter 4. In Chapter 7, some further research directions and projects involving chemically synthesised PANI are briefly described.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Note: Thesis now published in various journals, details in the Access Instructions file. / During the past almost three decades, conducting polymers have been the subject of intense scientific and industrial research and development worldwide. A general background of the fundamental principles and concepts of conducting polymers and their applications is presented in Chapter 1. In Chapter 2, the characterization of chemically synthesised emeraldine base polyaniline (EB-PANI), acetone-extracted to remove impurities, is examined. Spectroscopic studies PANI at different levels of oxidation are reported, and the structural changes in as-synthesised EB-PANI upon doping with HCl and iodine vapour are investigated. A maximum in the number of unpaired electron spins was found in 1M HCl doped PANI, corresponding to emeraldine salt PANI (ES-PANI), which indicates that bipolarons form at higher doping levels. Also, a maximum conductivity of 0.478 S cm-1 was observed for 1M HCl doped PANI, and no significant change in the conductivity was observed at higher doping levels. A new mechanism of iodine doping of EB-PANI was proposed, which occurs by the oxidation of the benzenoid diamine units, instead of the quinoid diimine units as previously proposed. In Chapter 3, the incorporation of planar Cu(II) complexes as dopants into ES-PANI was investigated by means of EPR and IR spectroscopy. A decrease in the intensity of the EPR signals from polarons in Cu(II) complex-doped PANI samples was observed, while the Cu(II) anion concentration increases and favors the formation of bipolarons in Cu(II) complex-doped PANI systems. The reactions of EB-PANI following different levels of reduction or HCl doping with CuCl2 aqueous solution are also investigated and new reaction mechanisms are proposed. The conductivities of the resulting samples have also been measured and discussed. The ability of aniline and PANI samples to act as antioxidants using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical method are investigated by means of UV-VIS, FTIR and EPR spectroscopy in Chapter 4. A study of the kinetics of the reaction between DPPH and aniline showed that the reaction is first order with respect to DPPH and approximately second order with respect to aniline. A further investigation of the mechanism of the aniline/DPPH reaction and products of the reaction (aniline oligomers and DPPHH) by magnetic resonance techniques is discussed in Chapter 5. This study has resulted in the identification for the first time of uncapped dimers and trimers of aniline oligomers in solution by EPR spectroscopy. In Chapter 6, for the first time, the applicability of various 13C and 15N solid-state nuclear magnetic resonance (SSNMR) techniques in the investigation of the changes in the structure of PANI that occur upon reaction with DPPH radicals was investigated and discussed. The results indicate partial oxidation of PANI samples upon reaction with DPPH, which is consistent with previously obtained FTIR and EPR data in Chapter 4. In Chapter 7, some further research directions and projects involving chemically synthesised PANI are briefly described.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Note: Thesis now published in various journals, details in the Access Instructions file. / During the past almost three decades, conducting polymers have been the subject of intense scientific and industrial research and development worldwide. A general background of the fundamental principles and concepts of conducting polymers and their applications is presented in Chapter 1. In Chapter 2, the characterization of chemically synthesised emeraldine base polyaniline (EB-PANI), acetone-extracted to remove impurities, is examined. Spectroscopic studies PANI at different levels of oxidation are reported, and the structural changes in as-synthesised EB-PANI upon doping with HCl and iodine vapour are investigated. A maximum in the number of unpaired electron spins was found in 1M HCl doped PANI, corresponding to emeraldine salt PANI (ES-PANI), which indicates that bipolarons form at higher doping levels. Also, a maximum conductivity of 0.478 S cm-1 was observed for 1M HCl doped PANI, and no significant change in the conductivity was observed at higher doping levels. A new mechanism of iodine doping of EB-PANI was proposed, which occurs by the oxidation of the benzenoid diamine units, instead of the quinoid diimine units as previously proposed. In Chapter 3, the incorporation of planar Cu(II) complexes as dopants into ES-PANI was investigated by means of EPR and IR spectroscopy. A decrease in the intensity of the EPR signals from polarons in Cu(II) complex-doped PANI samples was observed, while the Cu(II) anion concentration increases and favors the formation of bipolarons in Cu(II) complex-doped PANI systems. The reactions of EB-PANI following different levels of reduction or HCl doping with CuCl2 aqueous solution are also investigated and new reaction mechanisms are proposed. The conductivities of the resulting samples have also been measured and discussed. The ability of aniline and PANI samples to act as antioxidants using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical method are investigated by means of UV-VIS, FTIR and EPR spectroscopy in Chapter 4. A study of the kinetics of the reaction between DPPH and aniline showed that the reaction is first order with respect to DPPH and approximately second order with respect to aniline. A further investigation of the mechanism of the aniline/DPPH reaction and products of the reaction (aniline oligomers and DPPHH) by magnetic resonance techniques is discussed in Chapter 5. This study has resulted in the identification for the first time of uncapped dimers and trimers of aniline oligomers in solution by EPR spectroscopy. In Chapter 6, for the first time, the applicability of various 13C and 15N solid-state nuclear magnetic resonance (SSNMR) techniques in the investigation of the changes in the structure of PANI that occur upon reaction with DPPH radicals was investigated and discussed. The results indicate partial oxidation of PANI samples upon reaction with DPPH, which is consistent with previously obtained FTIR and EPR data in Chapter 4. In Chapter 7, some further research directions and projects involving chemically synthesised PANI are briefly described.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Note: Thesis now published in various journals, details in the Access Instructions file. / During the past almost three decades, conducting polymers have been the subject of intense scientific and industrial research and development worldwide. A general background of the fundamental principles and concepts of conducting polymers and their applications is presented in Chapter 1. In Chapter 2, the characterization of chemically synthesised emeraldine base polyaniline (EB-PANI), acetone-extracted to remove impurities, is examined. Spectroscopic studies PANI at different levels of oxidation are reported, and the structural changes in as-synthesised EB-PANI upon doping with HCl and iodine vapour are investigated. A maximum in the number of unpaired electron spins was found in 1M HCl doped PANI, corresponding to emeraldine salt PANI (ES-PANI), which indicates that bipolarons form at higher doping levels. Also, a maximum conductivity of 0.478 S cm-1 was observed for 1M HCl doped PANI, and no significant change in the conductivity was observed at higher doping levels. A new mechanism of iodine doping of EB-PANI was proposed, which occurs by the oxidation of the benzenoid diamine units, instead of the quinoid diimine units as previously proposed. In Chapter 3, the incorporation of planar Cu(II) complexes as dopants into ES-PANI was investigated by means of EPR and IR spectroscopy. A decrease in the intensity of the EPR signals from polarons in Cu(II) complex-doped PANI samples was observed, while the Cu(II) anion concentration increases and favors the formation of bipolarons in Cu(II) complex-doped PANI systems. The reactions of EB-PANI following different levels of reduction or HCl doping with CuCl2 aqueous solution are also investigated and new reaction mechanisms are proposed. The conductivities of the resulting samples have also been measured and discussed. The ability of aniline and PANI samples to act as antioxidants using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical method are investigated by means of UV-VIS, FTIR and EPR spectroscopy in Chapter 4. A study of the kinetics of the reaction between DPPH and aniline showed that the reaction is first order with respect to DPPH and approximately second order with respect to aniline. A further investigation of the mechanism of the aniline/DPPH reaction and products of the reaction (aniline oligomers and DPPHH) by magnetic resonance techniques is discussed in Chapter 5. This study has resulted in the identification for the first time of uncapped dimers and trimers of aniline oligomers in solution by EPR spectroscopy. In Chapter 6, for the first time, the applicability of various 13C and 15N solid-state nuclear magnetic resonance (SSNMR) techniques in the investigation of the changes in the structure of PANI that occur upon reaction with DPPH radicals was investigated and discussed. The results indicate partial oxidation of PANI samples upon reaction with DPPH, which is consistent with previously obtained FTIR and EPR data in Chapter 4. In Chapter 7, some further research directions and projects involving chemically synthesised PANI are briefly described.

<p>  </p> <p>Proteins are the active biomolecules of the cell. They perform metabolic action, give the cell structure, protect the cell from antigens, give the cell motility, and much more. The function of proteins are intrinsically linked to their structures, so it is therefore necessary to characterize the structure of a protein to fully understand its function and operation. In this research the application of computational methods, primarily molecular dynamics, towards protein structure determination, refinement, and quality assessment were studied. I applied molecular dynamics techniques to four major projects; the determination of relative error of atomic models deposited with electron microscopy maps in the EMDB, solving and refining atomics structure models for the PhageG major capsid proteins, the elucidation of the structure the protein USP7 and the binding pose of a of a candidate therapeutic drug, and the determination of relative stability of candidate protein folds to distinguish near native models from not. Each year an increasing number of protein structures have been solved using electron microscopy (EM). The influx of solved structure has proven to be a boon to the community, but it is necessary to note that the quality EM maps vary substantially. To understand to what extent atomic structure models generated from EM matched their respective maps, two computational structure refinement methods were used to examine how much structures could be refined. The deviation from the starting structure by refinement, as well as the disagreement between refined models produced by the two computational methods, scaled inversely with both the global and local map resolutions. The results suggested that the observed discrepancy between the deposited maps and refined models is due to the lack of resolvable structural data present in EM maps at low to moderate resolutions, and therefore these annotations must be used with caution in further applications. I also successfully implemented molecular dynamics as a method for protein structure quality assessment. Proteins tend towards shapes which minimize their energy. Experimentally, the stability of a protein can be measured through several techniques, one such technique includes the controlled application of tension to proteins in an atomic force microscopy (AFM) framework.  This kind of tension-based approach is of interest as it probes the force required to unfold individual domains of a protein rather than a bulk characteristic like molting point or activity. It has been shown that key features observed in an AFM experiment can be well reproduced with molecular dynamics simulation, which has been applied to characterize the mechanisms of unfolding of proteins as well as ligand-protein interactions.  Steered molecular dynamics (SMD) was applied to pull and unfold proteins and determine the force required to unfold them. The relative force required to unfold different models with the same sequence was used to estimate relative model accuracy.  This follows from the hypothesis that the structural stability of a given model’s conformation would positively correlate with its accuracy, i.e. how close that model is to its native fold. It was found that near-native models could be successfully selected by comparing the forces required to unfold models, indicating that high unfolding forces indeed indicated high model stability, which in turn correlated with model accuracy. I also applied molecular dynamics-based approaches for refinement of protein structures that are determined from cryo-EM density maps. Computational approaches for protein structure refinement are often developed with the design aim of requiring a user input and experimental data. I modeled the atomic structure of the major capsid protein gp27 and the decoration protein gp26 of PhageG to a 6.1Å resolution electron microscopy map. PhageG modeling was done by mapping the sequences to a presumed homolog (Hk97), arranging the subunits into hexamers and trimmers as suggested by mass spectroscopy data, rigid docking to respective map segments, refinement against half maps using MDFF across a range of weights, and then finally refinement to the whole map using the optimized weight. I also modeled the atomic structure of the protein USP7 to an 8.2 Å resolution map. USP7 modeling was done by combining crystalized domains of the whole structure, rigidly docking the model to the EM map by hand, and then refining in a similar manner as PhageG, with the added approach of weight scaling to overcome local minima along the relaxation. The USP7 model was further validated by exhibiting a ligand-protein binding pose, determined by glide, which corresponded to enzymatic activity mutation assays. In summary I applied molecular dynamics, in conjunction with other computational methods, towards protein structure determination, refinement, and quality assessment.</p>