Understanding the structures of biological macromolecules is highly important as they are closely associated with cellular functionalities. Comprehending the precise organization actin filaments is crucial because they form the dynamic cytoskeleton, which offers structural support to cells and connects the cell's interior with its surroundings. However, determining the precise organization of actin filaments is challenging due to the poor quality of cryo-electron tomography (cryo-ET) images, which suffer from low signal-to-noise (SNR) ratios and the presence of missing wedge, as well as diverse shape characteristics of actin filaments. To address these formidable challenges, the primary component of this dissertation focuses on developing sophisticated computational techniques for tracing actin filaments. In particular, three novel methodologies have been developed: i) BundleTrac, for tracing bundle-like actin filaments found in Stereocilium, ii) Spaghetti Tracer, for tracing filaments that move individually with loosely cohesive movements, and iii) Struwwel Tracer, for tracing randomly orientated actin filaments in the actin network. The second component of the dissertation introduces a convolutional neural network (CNN) based segmentation model to determine the location of protein secondary structures, such as helices and beta-sheets, in medium-resolution (5-10 Angstrom) 3-dimensional cryo-electron microscopy (cryo-EM) images. This methodology later evolved into a tool named DeepSSETracer. The final component of the dissertation presents a novel algorithm, cylindrical fit measure, to estimate image structure match at helix regions in medium-resolution cryo-EM images. Overall, my dissertation has made significant contributions to addressing critical research challenges in structural biology by introducing various computational methods and tools.