• Method: X_Ray diffraction.
Resolution: 2.5 Å
Chains: 2; A (\alpha chain) and B (\beta chain)
• NOTE: Actual values depend on previous fitting and they may differ from the ones shown in this appendix.
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)).
• 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).
• 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.
• Question 5: CCP4 Refmac
NOTE: Actual values depend on previous fitting and they may differ from the ones shown in this appendix.
Tab. 1.2 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.

• Question 6: CCP4 Refmac
Tab. 1.3 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.

• Question 7: CCP4 Refmac
NOTE: Actual values depend on previous fitting and they may differ from the ones shown in this appendix.
Tab. 1.4 :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).
• Question 8: 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.
• Question 9: Validation CryoEM
NOTE: Actual values depend on previous fitting and they may differ from the ones shown in this appendix.
 Statistic ChimeraX Coot PHENIX RSRAC Refmac after Coot Refmac after RSRAC 5NI1 CC(mask) 0.569 0.725 0.787 0.803 0.801 0.843 EMRinger score 3.86 2.37 5.38 2.87 5.34 3.98 RMS (Bonds) 0.0188 0.0183 0.0090 0.020 0.0191 0.0126 RMS (Angles) 2.41 2.02 1.30 1.94 1.84 1.43 Rama favored (%) 97.14 97.12 95.68 97.12 95.68 94.24 Rama allowed (%) 2.15 2.88 4.32 2.88 4.32 5.76 Rama outliers (%) 0.71 0.00 0.00 0.00 0.00 0.00 Rotamer outliers (%) 1.75 24.78 0.00 23.01 1.77 0.88 Clash score 70.34 26.24 1.81 21.73 1.36 2.26 Overall score 2.66 3.12 1.24 3.02 1.35 1.39 C beta deviations 1 7 0 2 0 0 RMSD 0.841 0.447 0.456 0.414 0.384 0.0
• Question 10: Validation CryoEM
NOTE: Actual values depend on previous fitting and they may differ from the ones shown in this appendix.
 Statistic ChimeraX Coot PHENIX RSRAC Refmac after Coot Refmac after RSRAC 5NI1 CC(mask) 0.524 0.690 0.776 0.765 0.767 0.830 E MRinger score 1.13 3.93 4.76 3.70 5.32 4.87 RMS (Bonds) 0.0313 0.0169 0.0078 0.0191 0.0183 0.0117 RMS (Angles) 2.17 1.97 1.33 1.95 1.87 1.40 Rama favored (%) 96.55 97.92 96.53 97.22 95.14 95.83 Rama allowed (%) 3.45 2.08 3.47 2.78 4.86 4.17 Rama outliers (%) 0.0 0.00 0.00 0.00 0.00 0.00 Rotamer outliers (%) 1.68 29.66 0.85 27.97 5.93 0.00 Clash score 75.93 34.24 3.89 25.57 2.16 4.32 Overall score 2.75 3.16 1.40 3.14 1.92 1.50 C beta deviations 0 8 0 1 0 0 RMSD 0.935 0.495 0.470 0.441 0.494 0.0
•  Statistic ChimeraX rigid fit PHENIX RSRAC Refmac after RSRAC 5NI1 CC(mask) 0.787 0.808 0.789 0.840 E MRinger score 4.64 4.58 4.35 4.11 RMS (Bonds) 0.0187 0.0093 0.0182 0.0122 RMS (Angles) 1.860 1.380 1.840 1.410 Rama favored (%) 95.41 95.41 94.70 95.05 Rama allowed (%) 4.59 4.59 5.30 4.95 Rama outliers (%) 0.00 0.00 0.00 0.00 Rotamer outliers (%) 3.90 0.00 3.90 0.43 Clash score 5.31 3.54 3.32 3.53 Overall score 2.05 1.46 1.94 1.49 C beta deviations 0 0 0 0 RMSD 0.494 0.509 0.537 0.00
•  Statistic ChimeraX operate PHENIX RSR Refmac after RSR 5NI1 CC(mask) 0.810 0.803 0.792 0.842 E MRinger score 4.95 4.70 4.05 4.18 RMS (Bonds) 0.0093 0.0076 0.0181 0.0122 RMS (Angles) 1.390 1.350 1.860 1.410 Rama favored (%) 95.41 95.41 95.41 95.23 Rama allowed (%) 4.59 4.59 4.59 4.77 Rama outliers (%) 0.00 0.00 0.00 0.00 Rotamer outliers (%) 0.00 0.00 5.41 0.43 Clash score 4.97 3.21 2.54 3.53 Overall score 1.58 1.43 1.94 1.48 C beta deviations 0 0 0 0 RMSD 0.579 0.642 0.454 0.00