.. _`app:solutions`: Answers to Questions ==================== .. _`question1`: - | **Question 1**: :ref:`How to identify templates of the target sequence ` | Method: X_Ray diffraction. | Resolution: 2.5 Å | Chains: 2; A (:math:`\alpha` chain) and B (:math:`\beta` chain) .. _`question2`: - | **Question 2**: :ref:`PHENIX real space refine ` | ``NOTE:`` Actual values depend on previous fitting and they may differ from the ones shown in this appendix. | *CC(mask)* value: 0.778 | Residue with lower correlation value: 142 ARG (Misfit at the end of the chain) | Correlation value: 0.186172531458 | Second residue with lower correlation value: 1 MET (Post-translation processing) | Correlation value: 0.348504275208 | Correlation value of *HEME* group: 0.81328813 (To get this value, Select Residue Type (Other) and Show CC below (0.9 or 1.0)). .. _`question3`: - | **Question 3**: :ref:`PHENIX real space refine ` | ``NOTE:`` Actual values depend on previous fitting and they may differ from the ones shown in this appendix. | *CC(mask)* value has improved to 0.787. | A 142 ARG correlation has improved to 0.4282267700789. | *HEME* group correlation has not improved (0.81007005253). .. _`question4`: - | **Question 4**: :ref:`PHENIX real space refine ` | ``NOTE:`` Actual values depend on previous fitting and they may differ from the ones shown in this appendix. | *CC(mask)* value has improved to 0.805. | A 142 ARG correlation has improved to 0.474205806292. | *HEME* group correlation has also improved to 0.821341112742. .. _`question5`: - | **Question 5**: :ref:`CCP4 Refmac ` | ``NOTE:`` Actual values depend on previous fitting and they may differ from the ones shown in this appendix. .. container:: :name: table:refmac_question_6 .. table:: :math:`Refmac` results: ================ ======= ====== Statistic Initial Final ================ ======= ====== *R factor* 0.3865 0.3441 *Rms BondLength* 0.0142 0.0165 *Rms BondAngle* 2.0081 1.9696 *Rms ChirVolume* 0.1401 0.0844 ================ ======= ====== The improvement is quite remarkable. .. _`question6`: - | **Question 6**: :ref:`CCP4 Refmac ` .. container:: :name: table:refmac_question_5 .. table:: :math:`Refmac` results: ================ ======= ====== Statistic Initial Final ================ ======= ====== *R factor* 0.3506 0.3488 *Rms BondLength* 0.0137 0.0150 *Rms BondAngle* 1.6843 1.8655 *Rms ChirVolume* 0.0783 0.0783 ================ ======= ====== Why: Because the starting values were already very good. .. _`question7`: - | **Question 7**: :ref:`CCP4 Refmac ` | ``NOTE:`` Actual values depend on previous fitting and they may differ from the ones shown in this appendix. .. container:: :name: table:refmac_question_7 .. table:: :*Refmac* results. *RSRAC* stands for *Real Space Refine* after *Coot*. ================ ======= ====== ======== ====== *Refmac* *RSRAC* *Coot* *Statistic* Initial Final Initial Final ================ ======= ====== ======== ====== *R factor* 0.4869 0.4855 0.4971 0.4825 *Rms BondLength* 0.0176 0.0212 0.0136 0.0193 *Rms BondAngle* 1.9186 2.3549 1.8053 0.2382 *Rms ChirVolume* 0.1112 0.1055 0.1470 0.1043 ================ ======= ====== ======== ====== | Starting and final *Rfactor* values seem to be worse when we do not generate a mask volume around the atomic structure when *Refmac* runs both after *Coot + Real Space Refine* (compare with (Table 4) and after *Coot* (compare with Table 3). Whitout using a delimiting mask, the whole volume is considered, even if the structure fits to a small part of the volume. The use of mask is thus especially indicated when map and model show different sizes. However, no differences are detected when the volume generated by the **extract unit cell** protocol or normalized volume generated by *Coot* are used (data not shown). .. _`question8`: - | **Question 8**: :ref:`EMRinger ` | ``NOTE:`` Actual values depend on previous fitting and they may differ from the ones shown in this appendix. | *EMRinger score* after *ChimeraX rigid fit*: 3.86. | *EMRinger score* after *Coot*: 2.37; Manual refinement depends on each user and in this case, for instance, we did not pay attention to rotamers. | *EMRinger score* after *PHENIX real space refine* after *Coot*: 5.38 | *EMRinger score* after *Refmac* after *Coot*: 2.87; With *Refmac* parameters used, the improvement got with *PHENIX real space refine* after *Coot* is clearly higher than the improvement got with *Refmac* after *Coot*. | *EMRinger score* after *Refmac* after *PHENIX real space refine* after *Coot*: 5.34; *Refmac* does not improve very much the result (because it was already good). With the *EMRinger* statistic, we can say that the modeling workflow is helpful to get a quite good model. .. _`question9`: - | **Question 9**: :ref:`Validation CryoEM ` | ``NOTE:`` Actual values depend on previous fitting and they may differ from the ones shown in this appendix. .. container:: :name: table:refmac_question_9 .. table:: Validation statistics of human *metHgb* :math:`\alpha` subunit *model*. *RSRAC* stands for *Real Space Refine* after *Coot*. *Rama* stands for Ramachandran. +----------+----------+----------+--------+--------+--------+--------+ | | | | | | | | | | | |*PHENIX*|*Refmac*|*Refmac*| | |Statistic |*ChimeraX*| *Coot* |*RSRAC* |after |after | *5NI1* | | | | | |*Coot* |*RSRAC* | | +----------+----------+----------+--------+--------+--------+--------+ |CC(mask) | 0.569 | 0.725 | 0.787 | 0.803 | 0.801 | 0.843 | +----------+----------+----------+--------+--------+--------+--------+ |*EMRinger*| 3.86 | 2.37 | 5.38 | 2.87 | 5.34 | 3.98 | |*score* | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *RMS* | 0.0188 | 0.0183 | 0.0090 | 0.020 | 0.0191 | 0.0126 | | (Bonds) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *RMS* | 2.41 | 2.02 | 1.30 | 1.94 | 1.84 | 1.43 | | (Angles) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Rama | 97.14 | 97.12 | 95.68 | 97.12 | 95.68 | 94.24 | | favored* | | | | | | | | (%) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Rama | 2.15 | 2.88 | 4.32 | 2.88 | 4.32 | 5.76 | | allowed* | | | | | | | | (%) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Rama | 0.71 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | | outliers*| | | | | | | | (%) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Rotamer | 1.75 | 24.78 | 0.00 | 23.01 | 1.77 | 0.88 | | outliers*| | | | | | | | (%) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Clash | 70.34 | 26.24 | 1.81 | 21.73 | 1.36 | 2.26 | | score* | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Overall | 2.66 | 3.12 | 1.24 | 3.02 | 1.35 | 1.39 | | score* | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ |C | 1 | 7 | 0 | 2 | 0 | 0 | |`\beta` | | | | | | | |deviations| | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *RMSD* | 0.841 | 0.447 | 0.456 | 0.414 | 0.384 | 0.0 | +----------+----------+----------+--------+--------+--------+--------+ .. _`question10`: - | **Question 10**: :ref:`Validation CryoEM ` | ``NOTE:`` Actual values depend on previous fitting and they may differ from the ones shown in this appendix. .. container:: :name: table:refmac_question_10 .. table:: Validation statistics of human *metHgb* :math:`\beta`subunit *model*. *RSRAC* stands for *Real Space Refine* after *Coot*. *Rama* stands for Ramachandran. +----------+----------+----------+--------+--------+--------+--------+ | | | | | | | | | | | |*PHENIX*|*Refmac*|*Refmac*| | |Statistic |*ChimeraX*| *Coot* |*RSRAC* |after |after | *5NI1* | | | | | |*Coot* |*RSRAC* | | +----------+----------+----------+--------+--------+--------+--------+ |CC(mask) | 0.524 | 0.690 | 0.776 | 0.765 | 0.767 | 0.830 | +----------+----------+----------+--------+--------+--------+--------+ | :math:`E | 1.13 | 3.93 | 4.76 | 3.70 | 5.32 | 4.87 | | MRinger` | | | | | | | | *score* | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *RMS* | 0.0313 | 0.0169 | 0.0078 | 0.0191 | 0.0183 | 0.0117 | | (Bonds) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *RMS* | 2.17 | 1.97 | 1.33 | 1.95 | 1.87 | 1.40 | | (Angles) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Rama | 96.55 | 97.92 | 96.53 | 97.22 | 95.14 | 95.83 | | favored* | | | | | | | | (%) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Rama | 3.45 | 2.08 | 3.47 | 2.78 | 4.86 | 4.17 | | allowed* | | | | | | | | (%) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Rama | 0.0 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | | outliers*| | | | | | | | (%) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Rotamer | 1.68 | 29.66 | 0.85 | 27.97 | 5.93 | 0.00 | | outliers*| | | | | | | | (%) | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Clash | 75.93 | 34.24 | 3.89 | 25.57 | 2.16 | 4.32 | | score* | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *Overall | 2.75 | 3.16 | 1.40 | 3.14 | 1.92 | 1.50 | | score* | | | | | | | +----------+----------+----------+--------+--------+--------+--------+ |C | 0 | 8 | 0 | 1 | 0 | 0 | |`\beta` | | | | | | | |deviations| | | | | | | +----------+----------+----------+--------+--------+--------+--------+ | *RMSD* | 0.935 | 0.495 | 0.470 | 0.441 | 0.494 | 0.0 | +----------+----------+----------+--------+--------+--------+--------+ .. _`question11`: - | **Question 11**: :ref:`Building the asymmetric unit ` .. container:: :name: table:refmac_question_11 .. table:: Validation statistics of human *metHgb* unit cell *model*. *RSR* stands for *Real Space Refine*. *Rama* stands for Ramachandran. +----------+----------+--------+--------+--------+ | | | | | | | |*ChimeraX*|*PHENIX*|*Refmac*| | |Statistic |*rigid* |*RSRAC* |after | *5NI1* | | |*fit* | |*RSRAC* | | +----------+----------+--------+--------+--------+ |CC(mask) | 0.787 | 0.808 | 0.789 | 0.840 | +----------+----------+--------+--------+--------+ | :math:`E | 4.64 | 4.58 | 4.35 | 4.11 | | MRinger` | | | | | | *score* | | | | | +----------+----------+--------+--------+--------+ | *RMS* | 0.0187 | 0.0093 | 0.0182 | 0.0122 | | (Bonds) | | | | | +----------+----------+--------+--------+--------+ | *RMS* | 1.860 | 1.380 | 1.840 | 1.410 | | (Angles) | | | | | +----------+----------+--------+--------+--------+ | *Rama | 95.41 | 95.41 | 94.70 | 95.05 | | favored* | | | | | | (%) | | | | | +----------+----------+--------+--------+--------+ | *Rama | 4.59 | 4.59 | 5.30 | 4.95 | | allowed* | | | | | | (%) | | | | | +----------+----------+--------+--------+--------+ | *Rama | 0.00 | 0.00 | 0.00 | 0.00 | | outliers*| | | | | | (%) | | | | | +----------+----------+--------+--------+--------+ | *Rotamer | 3.90 | 0.00 | 3.90 | 0.43 | | outliers*| | | | | | (%) | | | | | +----------+----------+--------+--------+--------+ | *Clash | 5.31 | 3.54 | 3.32 | 3.53 | | score* | | | | | +----------+----------+--------+--------+--------+ | *Overall | 2.05 | 1.46 | 1.94 | 1.49 | | score* | | | | | +----------+----------+--------+--------+--------+ |C | 0 | 0 | 0 | 0 | |`\beta` | | | | | |deviations| | | | | +----------+----------+--------+--------+--------+ | *RMSD* | 0.494 | 0.509 | 0.537 | 0.00 | +----------+----------+--------+--------+--------+ .. _`question12`: - | **Question 12**: :ref:`The whole macromolecule ` .. container:: :name: table:refmac_question_12 .. table:: Validation statistics of whole human *metHgb* *model*. *RSR* stands for *Real Space Refine*. *Rama* stands for Ramachandran. +----------+----------+--------+--------+--------+ | | | | | | | |*ChimeraX*|*PHENIX*|*Refmac*| | |Statistic |*operate* |*RSR* |after | *5NI1* | | | | |*RSR* | | +----------+----------+--------+--------+--------+ |CC(mask) | 0.810 | 0.803 | 0.792 | 0.842 | +----------+----------+--------+--------+--------+ | :math:`E | 4.95 | 4.70 | 4.05 | 4.18 | | MRinger` | | | | | | *score* | | | | | +----------+----------+--------+--------+--------+ | *RMS* | 0.0093 | 0.0076 | 0.0181 | 0.0122 | | (Bonds) | | | | | +----------+----------+--------+--------+--------+ | *RMS* | 1.390 | 1.350 | 1.860 | 1.410 | | (Angles) | | | | | +----------+----------+--------+--------+--------+ | *Rama | 95.41 | 95.41 | 95.41 | 95.23 | | favored* | | | | | | (%) | | | | | +----------+----------+--------+--------+--------+ | *Rama | 4.59 | 4.59 | 4.59 | 4.77 | | allowed* | | | | | | (%) | | | | | +----------+----------+--------+--------+--------+ | *Rama | 0.00 | 0.00 | 0.00 | 0.00 | | outliers*| | | | | | (%) | | | | | +----------+----------+--------+--------+--------+ | *Rotamer | 0.00 | 0.00 | 5.41 | 0.43 | | outliers*| | | | | | (%) | | | | | +----------+----------+--------+--------+--------+ | *Clash | 4.97 | 3.21 | 2.54 | 3.53 | | score* | | | | | +----------+----------+--------+--------+--------+ | *Overall | 1.58 | 1.43 | 1.94 | 1.48 | | score* | | | | | +----------+----------+--------+--------+--------+ |C | 0 | 0 | 0 | 0 | |`\beta` | | | | | |deviations| | | | | +----------+----------+--------+--------+--------+ | *RMSD* | 0.579 | 0.642 | 0.454 | 0.00 | +----------+----------+--------+--------+--------+