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Topic 3

Topic 3. Joachim Frank. Generation of initial models, and fitting methods. Initial models usually generated by hand, using O as stereo modeling environment Fitting methods: A) single component: rigid-body search using locally normalized CC B) multiple component flexible fitting:

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Topic 3

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  1. Topic 3 Joachim Frank

  2. Generation of initial models, and fitting methods • Initial models usually generated by hand, using O as stereo modeling environment • Fitting methods: A) single component: rigid-body search using locally normalized CC B) multiple component flexible fitting: 1) real-space refinement 2) NMFF (normal mode-based flexible fitting) 3) (when applicable) MD simulations with CC against observed density map J. Frank

  3. Example: NMFF of protein-conducting channel SecYEG channel bound to the translating ribosome: One model (front to front arrangement of SecYEG heterotrimers) explains densities for both inactive and active channels. Application of NMFF (normal mode-based flexible fitting). (see next slide) J. Frank Mitra et al., Nature, in press

  4. J. Frank Mitra et al., Nature, in press

  5. Quality of fitted model • Subjective criteria (i) the extent to which the model explains all observed density – is any density left unexplained? (ii) do any parts of the atomic model “stick out” of the observed density? (iii) for unreconciled multi-component docking: are different parts of the model in steric conflict? Can the conflict be removed by postulating conformational changes? • Objective/quantitative criteria: Real-space refinement and NMFF return measures for “goodness of fit”, from which the final quality can be gauged: Correlation coefficient, R-factor, and Error Residual (see examples in next slide) J. Frank

  6. Goodness of fit by real-space refinement, as a function of the number of pieces used In the flexible fitting of the ribosome. Goodness of fit criteria, and their sensitivity to shift of the model away from the “correct” position. J. Frank

  7. Validation requirements 1)Reproducibility of the fitting: Apply fitting procedure to two independent (not “halfset”) reconstructions, and compare the results using rmsd. 2) Reproducibility of conformational changes deduced: When real-space refinement is used on two density maps A and B, to obtain two models from which conformational changes are to be deduced, then we need a control reconstruction, A1, to which the same procedure is applied. Rmsd between the relevant components of the two structural models should be significantly larger (factor of 3) than the rmsd between A and A1. J. Frank

  8. Archiving of fitted models • Internal deposition (“Archive Database”) • Public deposition in the pdb, in conjunction with publications • We try to deposit the cryo-EM maps simultaneously in the 3DEM data base. (Ribosome complexes deposited by our group constitute the largest portion of the existing 3DEM data base.) J. Frank

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