Korneev MJ , Lakämper S , Schmidt CF .

	Fig. 1. Motor attachment, experimental setup and bead traces. a–d, f Possible Eg5-motor attachments to beads (silica, 0.5 μm diameter) via the genetically encoded N-terminal His-tag of Eg5. a All four motor domains are bound, preventing motility. b One motor domain is unbound, likely allowing only non-processive motility. c Two motor domains are free, one at each end, likely allowing only non-processive motility. d One motor domain is bound, leaving a dimeric motor end free to interact with the microtubule. e Traces of bead motility generated by individual Eg5 (green) and Kinesin-1 (grey) motors. The averaged (15-point) and median-filtered (0.3 s (Eg5) and 0.05 s (Kinesin-1) sliding windows; rank 10) signal is overlaid in red over both traces. The trap stiffnesses were 0.03 pN/nm (Kinesin-1) and 0.013 pN/nm (Eg5). f One dimer is bound, one dimer is free; sketch of a silica-sphere with motor held in the laser-trap, such that it interacts with a surface-attached microtubule track
	Fig. 2. Motor-bead attachment. Fractions of beads not interacting with a microtubule over 2 min (grey), moving (red), attached for less than 2 min (without motion) (purple), irreversibly stuck (blue). a Constant high average number of antibodies per bead (6,950), variation of motor concentration. b Constant concentration of motors in solution (corresponding to 2,410 per bead), variation of antibody density in order to obtain optimal motor/antibody-ratio (r-value) to favor species d and f (Fig. 1). c Systematic reduction of the absolute number of motor/antibody-complexes at constant r-values between 1 and 2 revealed a clear concentration dependence of motile events. Data were collected typically over periods of minutes. The number of beads that was used at each concentration is shown under the histograms
	Fig. 3. Steps of Kinesin-1/control. Kinesin-1 motility using the same attachment chemistry to immobilize individual motors on 0.5 μm silica spheres. Stepwise displacement (uncalibrated axis) is evident before (grey) and after filtering the data (red, 4,096 Hz sampling rate, 15-point averaging and median-filtering: 0.05 s sliding window, rank 10)
	Fig. 4. Single-step analysis and quantification of release force. a Example traces selected from independent experiments displaying stepwise displacements of the beads in the laser-trap driven by individual Eg5 motors [grey, unfiltered data; red, averaged (15-point) and median-filtered data (0.3 s sliding window, rank 10)]. The trap stiffnesses were: trace I: 0.014; II: 0.021; III, IV: 0.029; V: 0.042 pN/nm. b PDF of 10 traces including the ones shown in a to detect steps. Double Gaussian fit to this distribution yields a mean step size of 6.5 ± 0.2 nm (mean ± SEM). c Histogram analysis of detachment forces observed for single Kinesin-1- (black) and Eg5-motors (red). d Histograms of relative motor speeds directly preceding detachment: we scored the speeds of beads driven by Kinesin-1 (black) and Eg5 (red) in time interval before detachment (between arrows in a and Fig. 3 corresponding to about 10 motor steps in both cases) relative to the unloaded motors speeds. e Systematic variation of the trap stiffness revealed a hyperbolic relation to the obtained displacement (green circles). Error bars denote SEM, the number of evaluated events varied between 5 < N < 73. The resulting detachment forces (product of displacement and trap stiffness) are nearly constant (purple triangles)

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