Three-dimensional quantitative structure-activity relationships (3D-QSAR) analyses are methods correlating a pharmacological property with a mathematical representation of a molecular property distribution around three-dimensional molecular models for a set of congeners. 3D-QSAR methods are known to be highly sensitive to ligand conformation and alignment method. The current study collects 32 unique positions of congeneric ligands co-crystallized with the binding domain of AMPA receptors and aligns them using protein coordinates. Thus, it allows for a unique opportunity to consider a ligands' orientation aligned by their mode of binding in a native molecular target. Comparative molecular field analysis (CoMFA) models were generated for this alignment and compared with the results of analogous modeling using standard structure-based alignment or obtained in docking simulations of the ligands' molecules. In comparison with classically derived models, the model based on X-ray crystallographic studies showed much better performance and statistical significance. Although the 3D-QSAR methods are mainly employed when crystallographic information is limited, the current study underscores the importance that the selection of inappropriate molecular conformations and alignment methods can lead to generation of erroneous models and false conclusions.