Molecular Cell, Volume 46
Molecular Model of the Human 26S Proteasome
Paula C.A. da Fonseca, Jun He, and Edward P. Morris
Inventory of Supplemental Information
Figure S1 - Biochemical characterisation of the human 26S proteasome sample.
Related to Figure 1.
Figure S2 – Evaluation of cryo-electron microscope images of the human 26S
proteasome sample. Related to Figure 1.
Figure S3 - Estimation of resolution of the human 26S proteasome reconstruction.
Related to Figures 1 & 2.
Figure S4 - Rearrangement of 20S core subunits in the 26S proteasome. Related to
Figure S5 - The Rpt1-6 subcomplex. Related to Figure 3.
Figure S6 - Rpn1 and Rpn2. Related to Figure 4
Figure S7 – Structural coordinates for the PCI and MPN subunits of the 19S-RP.
Related to Figures 6 & 7.
Movie S1. Three-dimensional map of the human 26S proteasome in surface
representation rotating about its long axis. Related to Figure 1
Movie S2. Molecular model of the 26S proteasome. Related to Figure 7.
Supplemental Experimental Procedures
Figure S1 - Biochemical characterisation of the human 26S proteasome sample. (A)
SDS-PAGE gel showing the pattern of 26S proteasome subunits. (B) Native gel
electrophoresis. Left and centre panels show fluorescent images of gel stained for peptidase
activity with suc-LLVY-AMC, where the fluorescent signal is shown in dark against a light
background. Left panel shows activity for RP2-20S and RP-20S species (double capped and
single capped 26S proteasomes). In the centre panel activity is assessed in the presence of
0.02% SDS (w/v) and additionally shows activity for the small amount of uncapped 20S core
present in the 26S proteasome preparation. The fact that treatment with SDS only raises the
peptidase activity of uncapped 20S core present in the sample, but not that of the 26S
proteasome, indicates that the peptidase activity of the untreated 26S proteasome is intact.
Right panel is coomassie stained. The similar band intensities in the middle and right panels
indicate that the SDS treatment caused full 20S activation, with the faint bands for uncapped
20S core consistent with its low amount present in the analysed sample. (C) SDS-PAGE gel
immunoblotted to test for Rpn13. Lane 1: Cell lysate containing human Rpn13 expressed
with N-terminal double Step II tag. Lane 2: Hela cell extract (Abcam plc). Lane 3: Human
erythrocyte 26S proteasome preparation. No Rpn13 is detected in the Human erythrocyte
26S proteasome preparation. Lanes 1 and 2 serve as positive controls.
Figure S2 - Evaluation of cryo-electron microscope images of the human 26S
proteasome sample. (A) CCD recorded field of raw images in which side views of double-
capped 26S proteasome complexes are marked with red circles. (B,C) Rotational power
spectrum from A. (B) Greyscale representation where minima in fitted contrast transfer
function are marked as red arcs. (C) Graphical representation where minima in fitted
contrast transfer function are marked as vertical lines. Oscillations due to the contrast
transfer function extend beyond 7 Å.
Figure S3 – legend on next page
Figure S3 - Estimation of resolution of the human 26S proteasome reconstruction. (A)
Fourier shell correlation curve; ½ bit threshold curve (labelled) shown as solid line and 0.5
correlation coefficient threshold shown as dotted line giving rise to a resolution estimate of 7-
9 Å. (B-E) Comparison between the 26S proteasome structure (B,C – grey mesh with fitted
coordinates) and a model map calculated from the fitted 20S core low-pass filtered to 7 Å
(D,E – blue mesh with fitted coordinates). B & D are side view central sections. C & E are
top views sectioned at the interface between the α and β subunit rings.