Supplementary Figure 4: Cross-linking data are consistent with the cryo-EM structure of OCCM.
From: Structural basis of Mcm2–7 replicative helicase loading by ORC–Cdc6 and Cdt1
(a) Titration for cross-linking mass spectrometry. After a titration using a wide range of BS3 proportions, it was estimated that the best results were obtained between 1:5400 and 1:16000 protein-crosslinker ratios compared with the OCCM crosslinked with 2% glutaraldehyde. (b) Ten microliters of the final crosslinking reaction were run on a SDS PAGE before the cross-linking mass spectrometry analysis to confirm the homogeneity of the sample. (c) Atomic model of S. cerevisiae OCCM complex shown in surface view. The regions of Orc6 (grey), Mcm5 (yellow) and Orc2 (brown) that were not resolved in the atomic model, but covered with the cross-linking data are displayed as flat semi-transparent 2D surfaces delimited by a dashed line. The connector between the main body of Mcm3 and the Winged Helix Domain of the protein is presented in the same style. (d) A sketch of the OCCM structure in the same view as in (c). (e) Cross-links between ORC-Cdc6 and Cdt1-Mcm2-7 are shown as dashed green lines. (f) Histogram of alpha-carbon distances of observed cross-links as measured in the atomic model of the OCCM. Around 86% of the observed cross-links are within the allowed distance (less than 30 Å) while the rest can be explained due to the presence of flexible regions or large conformational changes in the complex.
