PMC

(a) Top and side views of the the three-dimensional reconstruction of the human Rvb1/Rvb2 complex obtained from negatively stained electron micrographs. The images were prepared from EMDB ID: 1317.
(b) Two-dimensional averages of the yeast Rvb1/Rvb2 complex in the presence of three different nucleotide states.
(c) Surface rendering representation of the three-dimensional reconstruction of the yeast Rvb1/Rvb2 complex obtained by cryo-electron microscopy. A top and a side view are shown. The asterisks indicate the projected densities from the bottom ring into the equatorial domain and the location of DI, DII and DIII is indicated. The images were prepared from EMDB ID: 2865.

Side, top and bottom views of the EM structures of the three histidine tagged Rvb1/Rvb2 complexes. EM maps are shown using a surface rendering representation using a threshold value representing approximately 100% of their estimated mass (~600 kDa). The three layers of density in which the EM maps are divided are indicated in one of the side view. The arrow in the same map indicates one of the densities projecting from the top ring and that run through the middle layer in an oblique fashion.

(a) Purified untagged and N-terminal His₆-tagged fusion versions of the Rvb1 and Rvb2 proteins were resolved in a 12% SDS-PAGE and stained by Coomassie brilliant blue. Proteins containing a N-terminal His₆-tag are denoted with “H-” label before the name of the protein. (b) Untagged (Rvb1/Rvb2), singly (H-Rvb1/Rvb2 and Rvb1/H-Rvb2) and doubly (H-Rvb1/H-Rvb2) tagged complexes were assembled in the presence of 1.5 mM ADP and resolved in a BN PAGE gel and stained with silver staining. The untagged and the histidine tagged versions of the Rvb1 and Rvb2 proteins were also resolved in these gels. The bands corresponding to the dodecamers (12-mer), hexamers (6-mer) and monomers (1-mer) are indicated.

(a) Linear diagram (left) of the human Rvb1 protein indicating the location of the AAA+ core domains and globular part of the insertion domain. The linker regions between the two domains are indicated. Residues delimitating all these domains are also indicated in the diagram. In the right panel, the X-ray structure of the human Rvb1 monomer is shown as a ribbon representation. The domains of the protein are color coded as in the linear diagram. (b) Top view (left) and side view (right) of the hexameric ring formed by the human Rvb1 protein as a ribbon representation color coded as in (A) except for one of the monomers that is colored in green. The AAA+ core domains, globular parts of the insertion domains in the hexameric ring as well as the N-terminal and C-terminal ends of the monomer in green. Image in (a) and (b) were prepared from PDB file 2C9O.

(a) Elution profile of the untagged, singly and doubly histidine tagged Rvb1 and Rvb2 complexes from a Superdex-200 column. In all cases the size exclusion chromatography column was equilibrated with buffer containing 1.5 mM ADP. Proteins containing a N-terminal His₆-tag are denoted with “H-” label before the name of the protein. The arrows in the elution profiles indicate the expected elution volume for the dodecamer (9.8 mL), hexamer (11.3 mL) and monomer (15.2 mL). Samples were withdrawn from the fractions between 10–12 mL, resolved in a 12% SDS-PAGE gel and stained with Coomassie brilliant blue. (b) Representative negative staining electron micrographs of purified untagged, singly and doubly histidine tagged Rvb1/Rvb2 complexes obtained from fractions between 10–12 mL from the elution profiles in panel A. (c) Two-dimensional averages of untagged, singly and doubly histidine tagged Rvb1/Rvb2 complexes obtained from particle images selected from electron micrographs obtained from fractions between 10–12 mL from the elution profiles in panel a.

(a) Singly and doubly histidine tagged Rvb1/Rvb2 complexes were purified by size exclusion chromatography. A 50 μL sample of the fraction containing the complexes was treated with 5 μL of a 6 mg/mL stock of tobacco etch virus (TEV) protease to remove the histidine tags. A volume 7 μL of the reaction containing TEV treated and untreated complexes were mixed with equal volume of 2× concentrated loading buffer and resolved in a 12% SDS-PAGE and stained by Coomassie brilliant blue. Proteins containing a N-terminal His₆-tag are denoted with “H-” label before the name of the protein. Arrows in the right side of the gel indicate the identity of the protein bands. (b) A volume of 15μL of each reaction containing TEV treated or untreated Rvb1/Rvb2 complex was mixed with 5 μL of loading dye, resolved in a BN PAGE gel and stained with silver staining. The bands corresponding to the dodecamers (12-mer), hexamers (6-mer) and monomers (1-mer) are indicated.

(a) Cartoon representing the R2TP complex purified from Saccharomyces cerevisiae containing a chromosomal copy of PHI1-3FLAG replacing the wild type gene. The four proteins components of the complex and the location of the FLAG tag are indicated. The cartoon also represents the dissociation of the Tha1 and Phi1-FLAG proteins from the in vivo assembled Rvb1/Rvb2 complex upon incubation with ATP. (b) A volume of 6μL of purified R2TP and in vivo assembled Rvb1/Rvb2 complexes were loaded in a 12% SDS-PAGE gel and stained by silver staining. The identity of the bands representing the Pih1, Tah1 and the two Rvb proteins is indicated. (c) The two same samples (15μL each) were mixed with 5 μL of loading dye, resolved in a BN PAGE gel and stained by silver staining. The bands corresponding to the hexameric complexes (6-mer) are indicated. (d) Two-dimensional average of endogenous Rvb1/Rvb2 complex obtained from negatively stained particle images. The image on the left represents the two-dimensional average and the one on the right represent its 6-fold symmetrized version.

(a) The panel shows the fitting of the AAA+ core domain and globular part of the insertion domains of the crystal structure of human Rvb1 into the three-dimensional reconstruction of the His-Rvb1/Rvb2 complex. The AAA+ and insertion domains are shown as a ribbon representation color coded as in and prepared from PDB file 2C9O. The EM density map of the His-Rvb1/Rvb2 complex is shown as a mesh. (b) Layout of the two Rvb hexamers in the histidine tagged Rvb1/Rvb2 complexes. The left column shows the DII-DII, AAA-AAA and AAA-DII generated models for the dodecameric Rvb1/Rvb2 complex indicating three possible configurations of the two hexameric rings. The models were generated by fitting the crystal structure of human Rvb1 into the three-dimensional reconstruction of the His-Rvb1/Rvb2 complex in different orientations. The AAA+ and insertion domains are shown as a ribbon representation color coded as in . The second column from the left shows the surface rendering representation of the density maps generated from the models fitted into the density map of the three dimensional reconstruction of the His-Rvb1/Rvb2 complex represented as a mesh. The third column from the left shows the calculated side view projections from the models and the right column displays an average of experimental projections from the His-Rvb1/Rvb2 complex obtained from negatively stained electron micrographs.