Extended Data Fig. 6: Recombinant THO–UAP56 complex cryo-EM image processing and reconstructions.
From: mRNA recognition and packaging by the human transcription–export complex
a. Three-dimensional image classification tree of the in vitro reconstituted THO–UAP56 cryo-EM data set27. The symmetry-expanded data set contained 314,583 high-quality particles. Classification and focused refinements in cryoSPARC63 yielded maps D (pink) and E (green) (see Methods for details). The percentage of THO–UAP56 dimer units contributing to each class is provided. The type of mask and overall resolution is indicated for each 3D refinement. b. Gold-standard Fourier shell correlation (FSC = 0.143) of the cryo-EM maps D and E. c. Orientation distribution plots for all particles contributing to cryo-EM maps D and E, visualized in cryoSPARC63. d. THO–UAP56 complex monomer A composite cryo-EM densities from front and left side views (maps D and E), colored by local resolution as determined by RELION 3.162,71. e. Representative regions of the newly determined THO–UAP56 cryo-EM densities (top) in comparison to previous data (bottom)27. The new densities are superimposed on the updated and refined THO–UAP56 coordinate model. Segments of THOC2 residues 316–330, residues 576–590, and THOC3 residues 163–170 are shown. f. A new model of the human THO–UAP56 complex. Newly modelled regions are shown in yellow, and contain segments of THOC1, THOC2, and THOC3. Regions with newly modelled sidechains are colored orange and are built on the previously available backbone models of THOC2 and THOC3. This updated model reveals new contacts among THOC1, −2, and, −3 subunits. The newly built THOC1 C-terminus meanders along the length of the THOC2 subunit ‘bow’, ‘MIF4G’, and ‘stern’ domains (Fig. 2e). The THOC1 C-terminal residues (458–528) were initially modelled using AlphaFold (Methods)84,85. The THOC2 ‘anchor’ forms a 5-helix bundle that packs against THOC5 helix α2 and THOC7 helices α2 and α3, and the THOC3 β-propeller blades 3 and 4 make a stabilizing contact with THOC2 ‘bow’ loop α17-α18 (Fig. 2e). Unchanged regions are colored grey and green and contain modelled backbones or sidechain, respectively.
