Negative stain electron microscopy and single particle reconstruction

Purified chaperonin samples at a concentration of 0.2 mg/ml were applied to continuous carbon film with 400-mesh copper grids that were previously glow discharged for 30 seconds. Excess protein solution was wicked off with Whatman-1 filter paper and grids were stained with 2% methylamine tungstate and 2% uranyl acetate. The negative stain imaging was performed using a JEOL 3200FS transmission electron microscope operated at 300 kV and the images were collected between 0.3–2.5 μm under-focus and at a magnification of 138,000X using a Gatan UltraScan CCD camera. The pixel size calculated for the data set was 1.09 Å/pixel.

We collected data under the following conditions to capture the major nucleotide-dependent conformations within the hsp60/10 protein folding cycle. For the ATP-bound conformation (without hsp10), 2 mM ATP was added to hsp60. For the ATP-bound conformations in the presence of hsp10, 100 mM ATP was added to hsp60 and hsp10 (2:1 molar ratio). To obtain the ADP conformation, 2 mM Mg-ADP was mixed with hsp60 and hsp10 (1:1 molar ratio). The purified proteins with incubated with the nucleotides at room temperature for approximately five minutes before they were applied to the grids. A total of 583 particles were picked from the negative stained micrographs using EMAN2³⁰. CTF correction, reference-free class averaging, initial model building, and single particle reconstruction were all performed in EMAN2³¹. Visualization and figures were generated using UCSF’s Chimera³².

Due to the heterogeneity in the hsp60/10 ATP-conformation dataset, a slightly different approach was utilized to separate the particles corresponding to the bullet and football complexes. The reference-free class averages that corresponded to the side-views of the two complexes were separated and individually used to generate two low resolution initial models. The two models were then used as references for the EMAN2 version of multi-refine to separate the particles into two sub-sets. The total data-set contained 3203 particles that were split into sub-sets containing 1654 (bullet) and 1536 (football) particles. Once the particles were split, single refinements were performed for both data-sets. C7 symmetry was used for the bullet refinement due to the different conformational states of the two rings³³. The bullet reconstruction converged to a resolution of approximately 17 Å. D7 symmetry was used for the refinement of the football complex that converged to approximately 20 Å. The football complex generated in EMAN2 served as an initial model for additional refinements in RELION2 to reduce the amount of noise in the 3-D reconstruction^30,34. For the hsp60 D3G and V72I APO conformations, 3144 and 2491 particles were picked form the negative stained micrographs, respectively. CTF correction, reference-free class averaging, initial model building, and single particle reconstruction were all performed in EMAN2. D7 symmetry was applied for the refinement using Eman2.