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Figure 2
(A) Electron micrographs of a negatively stained proteasome mixture showing the six different proteasome species: a 20S proteasome, proteasomes that are singly capped (19S−20S) and doubly capped (19S−20S−19S) with 19S complex, proteasomes that are singly capped (PA28−20S) and doubly capped (PA28−20S−PA28) with PA28 rings as well as a hybrid proteasome with a 19S complex and a PA28 ring bound to either end (19S−20S−PA28). For visualization of the different complexes, an excess of PA28 was used due to the weak interaction of PA28 rings with the 20S proteasome. Accordingly, our images showed many top views of unbound PA28 rings. As a result of the weak PA28−proteasome interaction, PA28 rings often dissociated from the 20S proteasome upon adsorption to the carbon film and could be seen as a top view adjacent to the proteasome (see, for example, the particle denoted PA28−20S−PA28). Such particles were excluded from the multireference alignment procedure and not used for the calculation of averages. (B) Known forms of the proteasome: gallery of negatively stained particles and the corresponding average images. (1) 20S proteasomes with two 19S caps (19S−20S−19S); (2) proteasomes with one 19S cap (19S−20S); (3) 20S proteasomes (20S); (4) proteasomes with one PA28 cap (20S−PA28); and (5) proteasomes with two PA28 caps (20S−PA28). The corresponding averages contain 106, 748, 164, 78 and 66 particles. The length of the side of the individual frames corresponds to 80 nm. (C) Hybrid proteasome complex (19S−20S−PA28): negatively stained particles and average image based upon 266 particles.
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