Flexibility analysis of the five loading intermediates. The atomic models are colored by their respective local B factors in ChimeraX. Rad24-RFC is omitted for clarity. Mec3 and Ddc1 lining the DNA gate are partially mobile (yellow) in the open gate and the partially open gate in steps 1–4, but they become stable (blue) in the closed gate in step 5. Consistent with the assigned temporal sequence, the chamber DNA is absent in step 1, is present but flexibly bound (red) in step 2, and becomes more stably bound (yellow) in step 3. The partial gate closure in step 4 destabilizes the chamber DNA (red), likely due to the perturbation by Mec3 movement. The chamber DNA is better stabilized in the 9-1-1 gate fully closed step 5. The 9-1-1 gate size is labeled. Four nucleotides and three nucleotides near the top 5' junction and the bottom 3' junction, respectively, are stabilized in the Rad24-RFC chamber. The dashed line represents three disordered nucleotides in the gap region. Because the Rad24-RFC A gate is open in all structures, ssDNA longer than 10 nt in the gap region can be easily accommodated by looping outward through the A gate.
