The formation of SOD1 filament is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), with numerous genetic variants identified in familial cases. This study reveals a novel cryo-EM structure of the wild-type SOD1 amyloid filament formed under physiological conditions. The difference in filament morphology compared to the previous structure is attributed to the metalation state of the SOD1 protein before filamentation. In contrast to the previous study that used metal-deprived apo- SOD1 protein, we utilized as-isolated wild-type SOD1, maintaining a high proportion of the enzymatically active holo-form. We found the Trp32 residue at the core of the SOD1 filament structure, where the Trp32 residue engages in crucial interactions with adjacent Lys91. The W32A mutation or the addition of Trp32-containing peptides modified the rate of filament formation in holo-SOD1. Additionally, the Cys111 residue located in a confined hydrophobic space within the filament undergoes bulkier modifications that impede filament assembly, emphasizing the role of the Cys111 in filament formation. Moreover, we revealed the cryo-EM structure of ALS-related and cysteine mutant SOD1 protein filaments. Comparative analysis of wild-type and mutant SOD1 structures elucidates the amyloid filament formation mechanism, emphasizing the significance of amino acid residue counts within the filament core for assembly energy requirements. These insights enhance our understanding of ALS pathogenesis by elucidating how genetic variations contribute to the development of potentially harmful filaments.