Molecular dynamics of ZAP-70 mutants and generation of knock-in mice. (A and B) Representative snapshots of ZAP-70 tandem SH2 unit with indicated mutation. Overlay snapshot of ZAP-70 tandem SH2 unit before (red) and after (cyan) 15 ns of MD simulations (A). Snapshots of MD simulations showing interactions between ZAP-70 SH2 and CD3ζ pITAM chain (B). See also Videos 1, 2, 3, and 4. (C) Dissociation constants as given by the ratio of unbound to bound CD3ζ/ ZAP-70 conformations in Fig. 1 C. F-test for statistical analysis. (D) Knock-in mutagenesis strategy. The targeting vector with either W163A or H165A mutation and a neomycin resistance gene flanked by loxP sites was generated for homologous recombination to ZAP-70 locus. PGK neo was subsequently deleted by crossing to a Cre deleter strain. (E) Protein levels of ZAP-70 in T cells of ZAC, SKG, W163A, and WT BALB/c mice. (F) Incidence of arthritis after 10 wk of transferring either CD4⁺ or CD8⁺ T cells from ZAC, SKG, W163A, and WT BALB/c mice into RAG2^−/− mice (n = 5 each).