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Researchers sought to determine the reasons for the loss of warm-threshold nucleation activity when cell-free ice nuclei are produced from ice nucleating bacteria. The sedimentation and filtration characteristics of cell-free nuclei demonstrated that the majority of such nuclei are less than 0.1 um in diameter. All cell-free ice nuclei examined could be immunosorbed by antisera directed against either an ice nucleation protein or the E. coli membrane; thus, these nuclei all appeared to contain both ice nucleation protein and E. coli membrane components. Immunofluorescent imaging of cell-free nuclei revealed only aggregates with diameters less than 0.2 um. Experiments with bacteria induced to express an ice nucleation protein indicate that formation of all nuclei begins with cooperative assembly of 3 to 5 molecules of ice nucleation protein. The data show that both size and structure of a nucleation site determine its nucleation threshold.