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[…] All imaging data was collected using automated Leginon imaging software (). Images of negatively stained samples of wild-type and mutant lid complexes were acquired on a Tecnai Spirit LaB6 electron microscope operating at 120 keV, with a random defocus range of -0.5 μm to -1.5 μm and an electron dose of 20 e-/Å2. 331 images were acquired for wild-type lid, 433 images for the Rpn5 (H282A/K283A) double-mutant, 412 images for the Rpn8 (K86A/K88A) double mutant, 181 for the Rpn5 (N275A) mutant, and 204 for the Rpn5 (Y273A) mutant. Images were collected at a nominal magnification of 52,000 X on an F416 CMOS 4K X 4K camera (TVIPS) with a pixel size of 2.05 Å/pixel at the sample level.Imaging of frozen hydrated samples was performed using a Titan Krios electron microscope operating at 300 keV, with a defocus range of -1.5 μm to -3.5 μm. A Gatan K2 Summit was used for counting individual electron events at a dose rate of 9.9e-/pixel/s, using an exposure of 7.6 s consisting of 38 frames at 200 ms/frame. This resulted in a total electron dose of 43.8 e-/Å2, accounting for coincidence loss. A total of 3,432 images of wild-type lid were acquired at a nominal magnification of 22,500X, yielding a pixel size of 0.655 Å/pixel at the sample level when collected in super-resolution mode. [...] All image preprocessing was performed using the Appion image-processing pipeline (). The contrast transfer function (CTF) was estimated using CTFFIND3 (). For negative stain data, particles were selected using a difference of gaussians (DoG) picking algorithm (), and only micrographs having an overall CTF confidence of greater than 80% were used for subsequent processing. The phases of the micrograph images were corrected according to the estimated CTF, and the particles were extracted using a box size of 160 pixels, and pixel values were capped at 4.5 sigma above or below the mean. Boxed particles were binned by a factor of 2 for processing. Reference-free 2D class averages of the extracted particles were determined through five rounds of iterative multivariate statistical analysis and multi-reference alignment (). The results of the 2D analysis were used to remove damaged, aggregated, or falsely selected particles from the dataset used for 3D analysis.All 3D analysis was performed with RELION v1.31 (). Using a previously determined reconstruction of the wild type yeast proteasome lid as an initial model (EMD-1993) (), a 3D refinement of 17,680 particles wild-type lid complex provided a reconstruction at 19.6 Å resolution, according to a Fourier Shell Correlation at 0.143 of two independently determined half-maps. This volume was used as the initial model for all 3D analysis of the mutant lid datasets. 3D classification was performed on each of the negative stain mutant lid datasets, and only 3D classes exhibiting well-ordered structural details were selected and combined within each dataset for 3D refinement. 22,103 particles of the Rpn5 (H282A/K283A) mutant yielded a 25.2 Å reconstruction; 11,185 particles of the Rpn8 (K86A/K88A) mutant yielded a 27.3 Å reconstruction; 25,429 particles of the Rpn5 (N275A) mutant yielded a 23.4 Å reconstruction, and 44,272 particles of the Rpn5 (Y273A) mutant yielded a 21.8 Å reconstruction (). UCSF Chimera () was used to dock the atomic model model of the lid into the density. [...] For cryo-EM image preprocessing, the super-resolution images were binned by a factor of two in reciprocal space, and motion-corrected using MotionCorr (). The aligned frames were summed and used for all subsequent processing steps. The CTF was estimated using CTFFIND3 (), and only micrographs having a CTF confidence value that was greater than 50% at 4Å resolution were used for further processing (), resulting in a dataset of 3,365 micrographs. Particles were manually selected from the first 100 images, and the results of reference-free 2D analysis were used as templates for particle selection using FindEM (). A random subset of 50,000 particles were extracted from the micrographs with a box size of 256 and used for reference-free 2D analysis in order to rapidly assess the quality of particle selection (). Very few classes corresponding to damaged or aggregated particles were observed; so all particles were used for single particle analysis in RELION.A total of 254,112 particles were extracted from the micrographs using a box size of 288 pixels, binned by a factor of 4, and classified into 8 3D classes over the course of 22 iterations in RELION. The particles from the 4 classes that showed evidence of conformational and compositional stability were selected from this initial classification, providing a total of 139,561 particles. The x and y coordinates corresponding to these particles were adjusted according to the final translational alignments from the 3D classification, and the centered particle coordinates were used to extract an unbinned particle dataset for 3D refinement in RELION.3D refinement using the default RELION parameters yielded a 4.4 Å resolution structure after 22 iterations. These aligned particle parameters were used for the RELION ‘particle polishing’ method. Individual particle motion trajectories were estimated using a running average window of 7 frames and particle translations were limited using a prior with a standard deviation of 1. Particle movements were fit to a linear trajectory using a running average window of 7 frames, with an inter-particle distance contribution value set to 300 pixels. Per-frame B-factors and intercepts were estimated by comparing the reconstructed half-maps from individual frames to the full-frame half maps, and the spatial frequency contribution from each frame weighted according (). A new stack of particles was generated from the translationally aligned particles extracted from the weighted frames, which provided a reconstruction at 4.1 Å resolution.Due to the possibility that the flexible N-terminal domains of the PCI subunits were negatively influencing the particle alignment, a soft-edged 3D mask encompassing the PCI-domains, the helical bundle, and the MPN domains was generated (blue mask shown in ) and used for 3D classification of the particles into 3 classes. This 3D classification was performed using the alignments from the 3D refinement, without further alignment of particles. One of the 3D classes resulting from this analysis clearly exhibited higher resolution details than the other two, and the 109,396 particles contained in this class were further refined (in the absence of a mask) to achieve a 3.6 Å structure. The same soft-edged 3D mask that was used for the previous 3D classification was then used for continued 3D refinement, which improved the structural details of the region contained within this mask, and increased the resolution to 3.5 Å resolution. [...] Modeling and visualization of the lid was performed in COOT () using mostly the cryo-EM map that had been generated using a soft mask encompassing the PCI domains and the C-terminal helical bundle (deposited as EMD-6479), as this is the highest resolved region, and cross-validated using the unmasked map. Available structures and homology models generated using Modeller v9.15 () were initially fit into the unmasked cryo-EM map using Chimera (). These included: 1) the crystal structure of Drosophila melanogaster Rpn6 (residues 50–390) homolog (PDB ID: 3TXN) (); 2) the crystal structure of the Saccharomyces cerevisiae Rpn11-Rpn8 heterodimer (residues 24–220 and 10–280, respectively; PDB ID: 4O8X) (); 3) the NMR structures of the N-terminal (residues 4–140 (PDB ID: 2MQW) and C-terminal (residues 184–353 (PDB ID: 2MRI)) domains of Saccharomyces cerevisiae Rpn9 (); and 4) the N-terminal domain of Schizosaccharomyces pombe Rpn12 homolog (residues 6–200, PDB ID: 4B0Z) (). The most N-terminal helices of Rpn5 and Rpn6 were not modeled due to the limited resolution of these regions. Placement of the N-terminal helices of Rpn3 was possible, however the absolute sequence register could not be assigned and these helices were modeled as polyalanine.Following each round of real space refinement in Phenix v1.10 (), 100 models were generated in Rosetta (), clustered, and scored. The top scoring structures were then used for the next round of manual model building and an aggregate model was used for refinement in Phenix. For the final round of refinement, the SHAKE protocol in Phenix was used to displace all atoms of the top 5 scoring models by 0.5 Å before refinement against one of the unmasked half-maps. An ensemble of these 5 models have been deposited in the PDB under ID: 3JCK. [...] To visualize conformational changes undergone by the lid complex upon incorporation into the 26S proteasome, we first rigid-body fit individual components of the atomic model of our isolated lid (6 PCI domains, 6 N-terminal extensions, the MPN heterodimer, and the helical bundle) onto the pseudo-atomic model of the engaged lid (PDB-ID: 4CR2) () using the ‘MatchMaker’ tool in Chimera. These overlaid models were then docked into the EM density of the 26S holoenzyme in the S1 state (). Overall, the secondary structure organization of the atomic models matched with high fidelity, although the register of the C-terminal helices of Rpn11 and the N-terminal helices of Rpn9 of the incorporated lid model were modified to correspond to the isolated lid model. The domain movements were visualized using the ‘morph conformations’ tool in UCSF Chimera. The motion of Rpn6 was evaluated using the software DynDom (). […]

Pipeline specifications

Software tools Leginon, Appion, CTFFIND, RELION, UCSF Chimera, MotionCorr, FindEM, Coot, MODELLER, PHENIX, DynDom
Applications cryo-EM, Protein structure analysis
Chemicals Adenosine Triphosphate