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. 2021 Oct 14;184(21):5448–5464.e22. doi: 10.1016/j.cell.2021.09.016

Figure S4.

Figure S4

Kinetics of coiled coil alignment visualized by smFRET, related to Figure 4

A.) Purity and labeling of the cohesin coiled coil sensor with donor and acceptor fluorophores. YBBR-tags were introduced into the ‘elbow’ region of the SMC1 and SMC3 coiled coils. This flexible region comprises residues 392-393 and 781-803 in SMC1 and 375-392 and 790-791 in SMC3. CY3: donor emission. CY5: acceptor emission.

B.) Coupled ATPase assay of wild-type and coiled coil sensor cohesin complexes (as described in Figure S3G).

C.) Quantification of ATPase rates as measured in B.) under different conditions. A 75 base pair DNA fragment was used as the DNA substrate. Shown are means ± SD and values of individual replicates.

D.) Example smFRET traces of coiled coil alignments in presence of ATP and NIPBL.

E.) Example smFRET traces of coiled coil alignments in the absence of ATP but in the presence of NIPBL.

F.) Transition density plots of wt cohesin, cohesinR711Q and cohesinEEEG in presence of ATP and in presence or absence of NIPBL. N: total number of molecules.

G.) FRET distributions of the coiled coil sensor in presence of ATP and NIPBL and in presence or absence of either 75 bp DNA or puc19 plasmid DNA. N: total number of molecules. Shown are means ± SD from three replicates (‘- DNA’ and ‘+ 75 bp DNA’) and two replicates (‘+ puc19’ DNA).

H.) Effect of high salt concentrations on the behavior of the coiled coil sensor. Cohesin was imaged in the presence of ATP and in the presence of 50 mM (blue bars) or 750 mM (red bars) potassium chloride (KCl). Shown are FRET distributions of the two conditions.

I.) Examples of Cornelia de Lange mutations found in the coiled coil domain of SMC1.

J.) Further HS-AFM stills of cohesinEEEG. Heads (he), hinge (hi) and STAG1 (S) are indicated.

K.) Frequencies of frames with aligned coiled coils for wt cohesin and cohesinEEEG (left) and frequencies of hinge bending (right), as observed by HS-AFM. Shown are the frequencies determined from movies of individual molecules. The number of molecules analyzed is indicated (n). Shown are values of individual molecules and violin plots of their distributions.