1：Experimental Design and Method Selection:

The study used optical tweezers to measure the displacements of single-headed cytoplasmic dynein interacting with microtubules under various ATP concentrations. The methodology included constructing single-headed dynein molecules, measuring FRET efficiencies, and analyzing the ATP-dependent displacement and binding times.

2：Sample Selection and Data Sources:

Single-headed dynein constructs (D384, D384GB, and mutants) were prepared and their interactions with microtubules were observed. Data were collected from FRET measurements, optical tweezer experiments, and biochemical assays.

3：List of Experimental Equipment and Materials:

Optical tweezers, spectrofluorometer (FP-6600, Jasco), electron microscope (Tecnai F20), and various biochemical reagents including ATP, ADP-Vi, and microtubules.

4：Experimental Procedures and Operational Workflow:

Dynein-coated beads were fixed on a glass chamber, and microtubules bound to trapped beads were brought into contact with dynein. Displacements and binding times were measured under different ATP concentrations. FRET efficiencies were measured to investigate linker swing.

5：Data Analysis Methods:

Displacements were analyzed using Gaussian distributions and cumulative distributions. Binding times were fit to exponential curves. ATP-dependent efficiencies and displacements were fit to sigmoidal and Michaelis-Menten equations, respectively.