Computational protocol: Human nonsense mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex

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Protocol publication

[…] Electron microscopy of UPF2-eRF3 was performed using a JEOL-1230 Transmission Electron Microscope operated at 100 kV after the samples were stained using 1% uranyl formate. Images of single molecules were recorded automatically at a final magnification of 2.28 Å per pixel using the F416 CMOS camera from TVIPS under the control of the EM-TOOLs program. A total of 143366 images were automatically extracted from the micrographs and selected bad particles (noise and background) were identified and removed by 2D classification using RELION (), ending with a dataset containing 73057 images (Supplementary Figure S1). After a new round of 2D classification, we detected a number of images corresponding to UPF2 by comparing with images of purified UPF2. The remaining data set was then refined, assuming that contained mostly images corresponding to UPF2–eRF3. Several methods were used to generate a collection of initial templates for refinement, a phantom blob with the approximate size of the UPF2–eRF3 complex, a low pass filter version of UPF2 (), and a volume generated using reference-free averages of the complex and EMAN2 initial volume generator (command: e2initialmodel.py)() (Supplementary Figure S2). When starting refinement from a low pass filter version of UPF2 or a blob, first rounds of refinement used reference-free averages as input, since these have a better signal to noise ration, better suited to initiate refinement from references without much features. After several rounds, refinement was followed using single particles. Volumes converged to similar low-resolution solutions after some rounds of refinement, despite the bias introduced by the initial reference. Then, the preliminary model of UPF2–eRF3 obtained using EMAN2 initial volume generator (command: e2initialmodel.py)() was used as seed to search in 3D for images of free UPF2 using maximum-likelihood methods in RELION (). After this 3D classification, 19053 images of UPF2 were found to still be part of the dataset (Supplementary Figure S1). These were removed and the remaining 54004 images of the UPF2–eRF3 complex were refined until convergence using EMAN () and the initial template obtain from the EMAN2 initial volume generator. After refinement, the images processed showed a good distribution of Euler angles (Supplementary Figure S3). The resolution of the structure was estimated as 20.0 Å, using the Fourier Shell Correlation method and a 0.5 correlation coefficient (Supplementary Figure S3). Atomic structures of MIF4G domain 1 (MIF4G-1) and the tandem MIF4G2–3 of UPF2 (), and the model of eRF3a (residues 207–634) () were fitted within the EM structure of UPF2–eRF3 using UCSF Chimera () (cross-correlation = 0.9461, 0.8833 and 0.8124, respectively). We were unable to define the handedness of the final structure. One hand was used for representation, but the opposite hand cannot be discarded. […]

Pipeline specifications

Software tools RELION, EMAN, UCSF Chimera
Application cryo-EM
Organisms Homo sapiens
Diseases Dental Caries