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[…] Crystals of Sre1-CTD (residues 628-876, C644S/C672S) were grown at 18 °C by the hanging drop vapor diffusion method mixing 1 μl protein (at a concentration of ∼30 mg/ml) with 1 μl reservoir solution containing 0.1 M BTP (pH 7.0), 1.5 M NaNO3. Crystals appeared in 1-2 days and grew to full size in 1 week. Se-Met-labeled protein was crystallized similarly. Both native and Se-Met crystals were soaked in mother liquor containing 20% ethylene glycol before flash-freezing in a cold nitrogen stream at 100 K.Both the native- and SeMet-derived data were collected at SSRF beamline BL17U. All data sets were integrated and scaled using HKL2000. Further processing was carried out using programs from CCP4 suite. Data collection statistics are summarized in . The phase was solved by single anomalous diffraction (SAD) of Se-Met. Then a crude helical model was manually built in the program Coot. Using this partial model as input, the identified Se atom positions were refined and phases were recalculated using the SAD experimental phasing module of the program Phaser. With the improved map, the molecular boundary was unambiguously defined and two molecules were found in an asymmetry unit. Sequence assignment was aided with the selenium sites in the anomalous difference Fourier map. The model was further rebuilt with Coot and refined with Phenix. [...] Samples of WT or GraFixed Sre1-CTD/Scp1-CTD complex (4 μl) were applied to glow-discharged continuous carbon-coated grids (Zhongjingkeyi Technology) and were stained with 2% uranyl acetate for 1 min. The negative-stained grids were imaged on a FEI Tecnai T12 microscope at 120 kV with a 4k × 4k charge-coupled device camera (UltraScan 4000, Gatan). For each sample, ∼100 images (for the GraFixed sample, untilted and 40° tilt-pair micrographs were collected) were collected and about 20 000 particles were semi-automatically picked to perform 2D classification with EMAN2. Then a cylinder model (60 pixels × 30 pixels × 30 pixels) was built and used as an initial model to refine the bin2 particles from the GraFixed sample with Spider. After several iterative refinements, the model was convergent and was used as an initial model for the cryo-EM data processing. [...] Aliquots (4 μl) of Grafixed Sre1-CTD/Scp1-WD40 complex at a concentration of ∼20 mg/ml, which had been supplemented with 0.01% DDM were placed onto glow-discharged holey carbon grids (Cu R 1.2/1.3 200 mesh, Quantifoil). Grids were blotted for 2.5 s and flash frozen in liquid ethane using an FEI Vitrobot. Images were taken on FEI Titan Krios electron microscope operating at 300 kV with a nominal magnification of 22 500×. Images were recorded by a K2 Summit detector (Gatan) with the super-resolution mode, and binned to a pixel size of 1.32 Å. Defocus values varied from 1.5 to 2.5 μm. Each image was dose-fractionated to 32 frames with a dose rate of ∼8.2 counts/sec/physical-pixel (∼4.7 e−/sec/Å2), total exposure time of 8 and 0.25 s per frame. UCSFImage4 was used for all data collection (developed by XL). [...] A total of 1 064 cryo-EM micrographs were collected. All images were aligned and summed using whole-image motion correction. The defocus value of each image was determined by CTFFIND3. Auto-picking was performed using the reference-based particle picking subroutine in RELION and the templates for particle auto-picking were obtained from the 2D class averages calculated from ∼5 000 manually picked particles. After manual check of the auto-picking results, 306 675 particles were extracted. Reference-free 2D classification was applied to remove ice and contaminants yielding 274 341 good particles. After two rounds of 3D classification, 157 243 particles were selected and 3D reconstruction with D2 symmetry was preformed resulting in a resolution of 7.3 Å. Then soft mask was performed and the reported resolution was increased to 5.4 Å. All reported resolutions are based on the gold-standard FSC = 0.143. All processions were performed in RELION. [...] Given the availability of a high-quality crystal structure of Scp1-WD40 (PDB ID: 4YHC), which fitted very cleanly into the correct hand of the 5.4 Å density map, we docked four Scp1-WD40 molecules into the peripheral donuts first. Residues 1 055-1 086 of the Scp1-WD40, which were not resolved in the crystal structure, were also not modeled into the map. Then, two Sre1-CTD dimeric crystal structures were docked into the central moiety. The αI-αIII helices of Sre1-CTD were removed in COOT as there was no matched density in the cryo-EM map. Besides, a poly-A helices was built to the C-terminus of Sre1-CTD in COOT as clear density of a helix was seen in the cryo-EM map. Initial rigid-body fitting of all models was carried out using UCSF Chimera. To correct the geometry of the final models and to remove steric clashes, the model was refined in PHENIX by using phenix.real_space_refine and manually checked in COOT. […]

Pipeline specifications

Software tools CCP4, Coot, PHENIX, EMAN, SPIDER, UCSFImage, CTFFIND, RELION, UCSF Chimera
Organisms Schizosaccharomyces pombe
Chemicals Oxygen