Flexible_fitting_validation - I2PC/scipion-courses GitHub Wiki

Modeling workflow to refine and validate the initial model of the alpha subunit of the human haemoglobin according to the map asymmetric unit (lower part of the alpha subunit modeling workflow):

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1. Step 9a: Interactive flexible fitting or refinement with CCP4 Coot protocol (details in p. 52-61 and Appendix 8 of the modeling tutorial)

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Relevant information:

• To display a specific map and select its density level (Fig. 39 in p.55 of the modeling tutorial)
• To select a specific map for refinement (Fig. 39 in p.55 of the modeling tutorial)
• To assess the fitting of the atomic structure regarding a specific map (Fig. 39 in p.55 of the modeling tutorial)
• To remove the MET first residue of the atomic structure (Fig. 40 in p. 56 of the modeling tutorial)
• To edit the coot.ini file to perform a semi-automatic refinement (Fig. 41 in p. 57 of the modeling tutorial)
• To observe the rearrangement of the TYR residue 141 in the atomic structure (Fig. 42 in p. 58 of the modeling tutorial)
• To save the refined atomic structure in Scipion: Press key w

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2. Step 10a: Automatic flexible fitting or refinement with Phenix Real space refine protocol (details in p. 61-65 and Appendix 23 of the modeling tutorial)

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3. Step 10b: Automatic flexible fitting or refinement with CCP4 Refmac protocol (details in p. 71-74 and Appendix 9 of the modeling tutorial)

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4. Step 9c: Validation of the refined atomic structure with Phenix Validation cryoEM protocol (details in p. 80-82 and Appendix 22 of the modeling tutorial)

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5. Step 9d: Validation of the refined atomic structure regarding to the map asymmetric unit with Phenix EMRinger protocol (details in p. 76-78 and Appendix 20 of the modeling tutorial)

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Comparison of validation scores Compare the validation scores obtained in steps 9c, 9d, 10e, 10f, 10g and 10h with those score values obtained for the published human Hgb alpha subunit (PDB ID 5ni1). If you want to compute those values, follow this workflow (steps 18a, 21a, 22a, 22b, 23a and 23b; p. 77-81 of the modeling tutorial).

Try to complete the next validation table with the scores obtained in steps 9c, 9d, 10e, 10f, 10g and 10h. Is your model of the human Hgb alpha subunit better or worse than the published one?

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Once we have the refined atomic structure of the human Hgb alpha-subunit, the beta-subunit should being modeled in a similar way. Follow the next workflow steps and repeat the process to generate the structure of the human Hgb beta-subunit, highligthed in the general workflow. Take into account UniPROTKB sequence IDs of p. 37 and recommendations of p. 41-42 of the modeling tutorial and do not forget to validate your structure (validation steps are omitted in this schema).