Figure 5.

Dynamics of hinge bending visualized by smFRET

(A) Design of the hinge bending sensors.

(B) Example smFRET trace of sensor pair 1.

(C) FRET distributions of hinge bending (sensor pair 1). Shown are means ± SD (3 replicates). N, number of molecules analyzed.

(D) Transition density plots of sensor pair 1.

(E) Transition density plots of sensor pairs 2–5. Pooled data from 3 replicates (sensors 2 and 3) and 2 replicates (sensors 4 and 5).

(F) Rotary shadowed electron micrographs of cohesin^{FKBP-SCC1/SMC3-intFRB}. Top cartoon: design of cohesin^{FKBP-SCC1/SMC3-intFRB}. Bottom cartoons: observed conformations.

(G) Time-lapse recordings of loop extrusion events by cohesin^{FKBP-SCC1/SMC3-intFRB} in presence of DMSO or rapamycin. DNA was stained with Sytox Orange. Scale bar, 2 μm.

(H) Loop maintenance in presence of DMSO or rapamycin. Loops were first formed by cohesin^{FKBP-SCC1/SMC3-intFRB} (step 1) followed by flow-in of DMSO or rapamycin (step 2). Bottom: example stills of the three major fates of loops upon rapamycin flow-in. Scale bar, 2 μm.

(I) Loop extrusion frequencies of cohesin^SMC3-intFRB and cohesin^{FKBP-SCC1/SMC3-intFRB}. Shown are means ± SD and values of individual replicates.

(J) Fraction of loops formed before (1) and after (2) flow-in of DMSO or rapamycin. Shown are means ± SD (3 replicates).

See also Figure S5.