(a) Kymograph of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex after 750 mM NaCl buffer wash. (b) Distribution of background subtracted EMCCD counts of Scc1Halo-TMR-TEVcohesin complexes immobilized on the coverslip. Peaks at ~ 4 x 103 and ~ 8 x 103 EMCCD counts correspond to single fluorophores and a small fraction of double fluorophores respectively. n = 290 regions with fluorescent molecules, 228 regions with background. (c) Photobleaching kinetics of a single DNA-bound Scc1Halo-TMR-TEVcohesin complex. (d) Diffusion coefficients of Smc1/3 wild type or K38A ATP binding deficient 'KA' forms of Scc1Halo-TMR-TEV-cohesin in the presence or absence of ATP. Diffusion coefficients were calculated based on the linear fit of the average mean square displacement of ≥ 13 freely diffusing molecules per condition over a 1 second time period. 