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[…] sen. For 3D reconstruction, a total of 8,869 particle pairs were selected from both the untilted and 50° tilted images of cryo-negatively stained specimens using WEB. The selected particles were windowed into 128 × 128-pixel images, and the particles from the untilted specimens were classified into 10 classes. Particles from classes with well-defined features were combined (3 classes for conformation I with a total of 1,993 particles; 2 classes for conformation II with a total of 1,686 particles), and two 3D reconstructions were independently calculated with the particle images from the tilted specimens using the backprojection, backprojection refinement and angular refinement procedures in SPIDER. Two-fold symmetrization in the final angular refinement step improved the resolution and the features of the density map of conformer I but had the opposite effect for that of conformer II (). The symmetrized density map of conformation I was filtered to 32 Å, and the unsymmetrized density map of conformation II was filtered to 34 Å, the resolutions estimated using the Fourier Shell Correlation (FSC) = 0.5 criterion., A model of the full seven-protein TRAPPI complex was generated by aligning the structures of the two mammalian TRAPPI subcomplexes (PDB codes: 2J3T and 2J3W) with the co-crystal structure of Ypt1 bound to the yeast TRAPPI core (PDB code: 3CUE). The PDZ domain in the crystal structure of mammalian Trs23 was removed, as it is not present in the yeast ortholog. The resulting model was manually fitted into the TRAPPII EM density map and further refined using the UCSF Chimera package. All molecular graphics images were produced using Pymol (http://www.pymol.org/) and the UCSF Chimera package., Anti-CBP antibody (Open Biosystems, 0.5 mg ml−1) was added at 1:15 to 1:50 dilutions to purified TRAPPII (Trs120-TAP or Trs130-TAP) and incubated for 15 to 30 minutes at room temperature. The samples were negatively stained and imaged as described above. 909 and 836 labeled particles selected for (Trs120-TAP + anti-CBP) and (Trs130-TAP + anti-CBP), respectively, were subjected to alignment and classification, specifying 100 output classes. In the case of (Trs120-TAP + anti-CBP), 93 particles from the 10 best output classes were combined and subjected to a secon […]

Pipeline specifications

Software tools SPIDER, FSC, UCSF Chimera