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Na⁺ is the most abundant ion in living organisms and plays essential roles in regulating nutrient uptake, muscle contraction, and neurotransmission. The identification of Na⁺ in protein structures is crucial for gaining a deeper understanding of protein function in a physiological context. LeuT, a bacterial homolog of the neurotransmitter:sodium symporter family, uses the Na⁺ gradient to power the uptake of amino acids into cells and has been used as a paradigm for the study of Na⁺-dependent transport systems. We have devised a low-energy multi-crystal approach for characterizing low-Z (Z ≤ 20) anomalous scattering ions such as Na⁺, Mg²⁺, K⁺, and Ca²⁺ by combining Bijvoet-difference Fourier syntheses for ion detection and f” refinements for ion speciation. Using the approach, we experimentally identify two Na⁺ bound near the central leucine binding site in LeuT. Using LeuT microcrystals, we also demonstrate that Na⁺ may be depleted to study conformational changes in the LeuT transport cycle.