1. Volume 6, Issue 6, Pages 1515-1521 (December 2000)
Visualization of Substrate Binding and Translocation by the ATP-Dependent Protease, ClpXP 
Joaquin Ortega, Satyendra K Singh, Takashi Ishikawa, Michael R Maurizi, Alasdair C Steven 
Molecular Cell 
Volume 6, Issue 6, Pages (December 2000)
DOI: /S (00)

2. Figure 1
Polylysine Treatment of Grids Increases the Number of Sideviews of ClpXP Complexes
Electron micrographs of negatively stained ClpXP–ATPγS complexes isolated by gel filtration and adsorbed to a carbon film (A), or a carbon film treated with 0.1% polylysine (B). Bottom, right inset, averaged sideview from the latter preparation (N = 588; resolution, 32 Å). ClpP, the central part of the complex, has an apparent width of ∼100 Å, which is somewhat larger than the known dimension of ∼90 Å (Wang et al. 1997), indicative of flattening as a consequence of negative staining and air drying.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2 Visualization of Binding of λ O Protein by ClpXP Complexes
(A) Ternary complexes were formed by adding ClpX (50 μg) to 50 μl of 50 mM Tris-HCl (pH 7.5), 0.2 M KCl, 10 mM MgCl2, and 2 mM ATPγS. After 5 min, 15 μg of λ O protein was added, and 5 min later followed by 30 μg of ClpPS111C or ClpP. The final volume was 100 μl. Complexes were purified by gel filtration on 0.3 × 20 cm Superdex 200 columns (Pharmacia) equilibrated in the same buffer with 1 mM ATPγS. The flow rate was 0.08 ml/min and fractions were collected at 1 min intervals. Aliquots were removed for analysis by SDS-PAGE and proteins were stained with Coomassie blue.
(B) Negatively stained electron micrographs of column-purified ClpXPS111C complexes in the presence of ATPγS with (left) and without (right) bound λ O. Arrowheads mark complexes in which the terminal density that denotes λ O binding is relatively conspicuous.
(C) Averaged images of (left) 2:1 complexes (i.e., two ClpX hexamers per ClpPS111C tetradecamer), and (right) 1:1 complexes. The λ O–bound complexes are shown at top, with the controls below. The failure of the stain to penetrate the central cavity of ClpP is evident, compared to complexes containing wild-type ClpP (cf. Figure 1B). Vertical arrows mark the λ O–associated densities. The protruding domains on the distal surface of ClpX are marked with horizontal arrowheads. This feature tends to be more evident in ClpPS111C-containing complexes than with wild-type ClpP, perhaps because the former molecules may be less susceptible to flattening. The terminal densities are also marked in the corresponding difference images (bottom panels). Each average image combined ∼500 particles, for resolutions of 30–32 Å.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3
Translocation of λ O Protein from Its Initial Binding Site into the Proteolytic Chamber of ClpXP
(A–D) The left panels show averaged images (resolution, 29–32 Å) of various ATPγS-stabilized complexes with (B–D) or without (A) bound λ O. The right panels show the same complexes 2 min after adding ATP in 4-fold molar excess.
(A) Control experiment of ClpXP in the absence of λ O: no substantial differences were seen between the two nucleotide states. The central dark feature represents the penetration of stain into the digestion chamber.
(B) After addition of ATP to complexes containing active ClpP, the terminal λ O–associated densities (arrows, left) essentially disappear.
(C) Upon addition of ATP to complexes containing inactivated ClpPin, the terminal densities are greatly diminished, and concomitantly the digestion chamber becomes no longer stain penetrable, reflecting accumulation of protein density.
(D) With complexes containing the mutant ClpXPS111C whose cavity is already occupied by propeptides, there is little diminution of the terminal density upon ATP addition.
(E) Model of the hexameric ring of ClpY ATPase domains (Bochtler et al. 2000), after removal of the I domains (cf. top left). The sites of two insertions in ClpX, Asp-317 and Ser-2, are assigned to opposite faces of the ring. The face with Asp-317 corresponds to the face of ClpY that binds to the protease, ClpQ (Ishikawa et al. 2000). If the corresponding surface of ClpX binds to ClpP, its N-terminal domain (inserted at Ser-2) is a more likely candidate for its distal-protruding, putatively substrate binding, domain.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Visualization of Internalized λ O Substrate in ClpPin Molecules
(A) ClpXPin–λ O complexes containing His6-tagged ClpPin were assembled in the presence of 1 mM ATPγS. After adding 8 mM ATP to allow translocation, the complexes were disrupted by dilution and removal of nucleotide and Mg2+, passed over metal ion affinity columns, and the bound proteins eluted. C, λ O reference; U, unbound and buffer washed; W, 10 mM imidazole washes; D, bound material eluted with 0.2 M imidazole.
(B) Negatively stained electron micrographs comparing (right panel) His6-ClpPin with translocated λ O and (left panel) control ClpPin. The control was isolated from ClpXPin–λ O.ATPγS complexes to which no ATP had been added, thus minimizing translocation. Top insets, averaged images comparing top views of the respective molecules, showing their 7-fold symmetry. Bottom insets, global averages of molecules in all viewing orientations. In both cases, the relative stain penetrability of the two populations is evident. At bottom are given the respective percentages of stain-occluded and stain-penetrated molecules.
Molecular Cell 2000 6, DOI: ( /S (00) )