Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation.

Protein Purification, ATPase and Luciferase Reactivation Assay Wild type Hsp104 was purified as described 56 . Hexameric Hsp104 (0.042 μM) was incubated with ATP (1 mM) at 25°C before monitoring the release of inorganic phosphate over 5 minutes using a malachite green kit (Innova). ATPase turnover was measured at a maximum rate of ~14/min, which corresponded to a functional, ATPase-active complex. Disaggregase function was measured by a luciferase reactivation assay as previously described 35 . Aggregated luciferase (50nM) was incubated with Hsp104 (0.167μM hexamer) in the presence of equimolar Hsc70 and Hdj2 (Enzo Life Sciences) plus ATP (5.1 mM) and an ATP regeneration system (1 mM creatine phosphate, 0.25 μ M creatine kinase) for 90 min at 25°C. Cryo Sample Preparation and Data Collection WT Hsp104 (8.5 mg/mL ) was incubated with AMP-PNP (5 mM ) for 20 minutes at 25°C. Samples were diluted to 0.7 mg/ml in 40 mM HEPES pH=7.5, 40 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, 5mM AMP-PNP and 3.5 μl was applied to plasma cleaned C-Flat 2/2 holey carbon grids (Protochips). Vitrification was performed using a Vitrobot (FEI Company) and samples were blotted for 1.5–2 seconds prior to plunge freezing in liquid ethane. Of note, during initial attempts at imaging Hsp104 routinely dissociated into monomers and significant optimization of buffer conditions, sample concentration and freezing conditions was required to achieve proper ice thickness and a homogeneous spread of hexameric particles. Furthermore, the presence of DDM detergent was tested to increase the angular distribution but resulted in dissociation of the hexamer at a variety of concentrations (data not shown). Samples were imaged using a Titan Krios TEM (FEI Inc.) operated at 300 kV. Images were recorded on a Gatan K2 Summit direct electron detector operated in counted mode at 50,000X nominal magnification corresponding to a calibrated to 1.00 Å/pixel. Dose fractionated imaging was performed by semi-automated collection methods using UCSF Image 4 57 with a defocus range of 1.5–3 um. Total exposure time was 8 seconds with 0.2 second frames with a cumulative dose of ~45 e − per Å 2 for 40 frames. Motion corrected frames were summed with the first two frames excluded, and the FFT was visually inspected for sufficient Thon rings prior to additional processing 58 .

Image Processing and 3D Refinement

All micrographs were CTF corrected using CTFFIND4 59 and poorly corrected micrographs were removed following visual inspection of the FFT and CTF estimation. An initial single particle dataset was achieved by manual particle picking using e2boxer (EMAN2) 60 , which yielded ~50,000 particles from 1731 micrographs. Well-populated reference-free 2D class averages, determined with Relion 61 , were used for templated automated particle picking with the Template Picker in Appion 62 to achieve a ~200,000 single particles dataset from 1930 micrographs. The total dataset of ~250,000 particles was initially sorted following 2D classification by removing particles images from poorly resolved class averages, resulting in a total dataset of ~190,000 particles. All subsequent 3D processing was performed using Relion 61 with no symmetry imposed based on the asymmetric arrangement identified in the 2D class averages ( Figure 1b ). A previously determined cryo-EM 3D reconstruction of ATP-Hsp104 (EMDB: 1600) 31 , low pass filtered to 50Å, served as an initial model for 3D classification and refinement. The 8 models generated from 3D classification appeared homogenous (data not shown), therefore, the full dataset was used for initial gold-standard 3D refinement. The resulting 3D model refined to an estimated resolution of 7.5 Å (data not shown). Z-score parameters based on this refinement, defined in Relion, were written for the data set and the dataset was trimmed to the highest Z-score for 160,000 particles. Additional 3D refinement was performed with this trimmed data set and resulted in a final model with an estimated resolution of 6.54 Å using the FSC = 0.143 FSC criterion ( Supplementary Fig 1e ). Additional trimming based on the Z-score showed no improvement and extensive 3D classification and refinement of individual classes as well as local refinement using 3D masking was tested and did not did not yield improvements in the map. Thus, the resolution is likely limited by flexibility of multiple regions in the complex ( Supplementary 1h ) and moderate preferred orientation ( Supplementary Fig. 1f ). For the final sharpened map, the “Post-processing” procedure was used to generate a soft mask for the two half maps prior to FSC estimation, which was determined to be 5.64 Å ( Supplementary Fig 1e ). Automated B-factor sharpening was carried on the combined map with an estimated -188 B-factor. The local resolution was estimated using ResMap on the unsharpened map 63 .

Modeling

This final sharpened map was used for all rigid body docking and flexible fitting of atomic structures. Rigid body fitting was performed with UCSF Chimera 64 . Initial comparisons were performed by fitting individual protomers with the protomers in T. thermophilus ClpB crystal structure (PBD_ID=1qvr), but resulted in a low cross correlation value and clear conformational differences were apparent. Individual subdomains (NTD, NBD1 large, NBD1 small, MD (residues 409–467), NBD2 large, NBD2 small) from the crystal structure were then docked as rigid bodies and resulted in an improved fit, however a number of AAA+ domain helices did not align with the density. Therefore, to more accurately interrupt the map, a homology model of Hsp104 (residues 6–857) was determined from the ClpB structure (residues 4–850) using SWISS_MODEL 65 followed by rigid body docking the subdomains. Flexible fitting of the protomer sub volumes and the entire hexamer map was then performed using phenix. real_space_refine 66 using backbone carbons of our homology model. The real space refinement was implemented at 6σ, resolution 6Å and the cross correlation improved from 0.72 to 0.9 for the complete map after refinement. Nucleotide density was seen for 10 of the nucleotide binding pockets, therefore this molecule was included in the model and docked based on the ClpB structure. Manual modeling of the tyrosine loop regions was performed in COOT 67 . Loop regions within the NBD1 domain (248–260,287–300) were modeled by backbone positioning within the electron density; residues 251–259, 292–297 were unable to be modeled in the density. Of note, a major shift was required to model the NBD2 tyrosine loop (656–669), which showed a distinct curve away from the flanking helices. Similarly, the most central portion of the loop lacked density and was not modeled (659–666). All images were generated using UCSF Chimera 64 . Accession codes The cryo-EM electron density map and molecular model have been deposited in the Electron Microscopy Data Bank (EMDB-3416).