Fig. 4: Motors containing inactive subunits exhibit slower start times, lower packaging speeds, and more pausing.

a Schematic of the optical trapping assay to measure the packaging dynamics of a single T4 motor. A multi-stream laminar flow chamber (left) is used to manipulate samples and buffer. A bead coated with anti-T4 antibodies is captured in one trap in a flow stream containing ATP (green stream; step ①). Another bead conjugated with arrested T4 capsid–gp17-DNA motors is captured in a second optical trap in the stream containing non-hydrolyzable analog ATP-γS (red stream; step ②). The motor bound to DNA in the presence of ATP-γS cannot initiate packaging due to a lack of energy source. To form a tether between the bead pair, the beads are moved close for 1 s and separated apart to detect binding of capsid to antibody (step ③). After a constant force (F = 5 pN) load is applied to the tether, the traps are moved into ATP and packaging is detected as the decrease of DNA extension when DNA is translocated into the capsid by the motor (step ④). b Extension of unpackaged DNA vs. time, under constant force load. Packaging activity is observed within seconds of entering the ATP stream. Representative packaging trajectories with WT (blue) and ISD motors (orange) are shown. c Distribution of the start times of WT (blue, n = 44) and ISD motors (orange, n = 62), defined as the dwell time between entry into the ATP stream and the start of packaging. d Distribution of mean pause-free packaging velocity derived from each packaging trace. ISD motors (orange) show a lower packaging velocity than WT motors (blue). e Histogram of the logarithm of pause frequency, defined as the number of pauses per kb packaged. ISD motors (orange) exhibit more pauses than WT motors (blue) on average. Motors with long start times (black stars; c–e) have a lower packaging velocity and higher pause frequency. The number of trajectories used is indicated by n = #.