Conformational dynamics are central to the function of many proteins, including the translational guanosine triphosphatase EF-Tu. AlphaFold2-based approaches such as AF-Cluster use sequence ensembles to sample alternative conformations, but most clustering analyses focus on predicted structures without examining the sequence and phylogenetic or ecological context of the multiple sequence alignments (MSAs) that generate them. Complex landscapes can also be poorly resolved by single-step clustering, because one global metric may conflate motions occurring at different architectural levels. Here, we address these limitations using a structure-guided strategy for EF-Tu. Query MSAs were clustered to generate diverse predicted structures, then sorted in 2 steps: first by the interdomain orientation of helix 183 to 199, capturing global open and closed forms, and then by switch I loop 54 to 58, resolving local variation at the guanosine triphosphate binding pocket. Comparing wild-type and D81 mutant queries showed that D81 substitution reshapes sequence retrieval and recruits a broader ecological range of organisms. Although D81A, D81F, and D81K retained ribosome-stimulated guanosine triphosphatase activity under the tested conditions, position 81 remained aspartate across virtually all retrieved sequences, indicating experimental substitutability but evolutionary invariance. AlphaFold3 modeling with ribosomal RNA components further suggested weakened Mg 2+ coordination in the guanosine diphosphate state and guanosine triphosphate-specific sarcin–ricin loop displacement, consistent with possible effects on nucleotide handling or downstream coupling rather than loss of hydrolysis. These findings establish mutant-seeded MSA perturbation as a strategy for uncovering hidden sequence, ecological, and conformational diversity in conserved proteins.