7. Use P ¼ 0.5 for the analysis without regularization procedure.
Use P value of 0.68 or 0.95 (confidence level of one or two
standard deviations, respectively) for analysis with regularization. Increasing the P value will decrease the resolution of the
distribution but increase the accuracy of its information. Sedimenting species that will disappear in the c(s) distribution at
higher P-value should be interpreted with care.
8. In the presence of a contaminant species that does not participate in the interaction, it is possible with the program GUSSI
to define an s-value range that will be excluded from the calculation of the s w values [24].
9. A poor fit may indicate that the interaction model is incorrect.
Before drawing such conclusion, carefully verify there is no
error in the entered parameters (molar mass, molar extinction
coefficients, s A and s B values).
10. The refined values for s A , s B , and s AB should be in agreement
with values observed in the c(s) distributions of individual
components and titration series. If parameters are not well
restrained by experimental data, refined values may be unrealistic. In this case, s A and s B should be fixed in the
refinement [19].
11. It is noteworthy that GST-BTG2 (species A) is a dimer in
solution, and we can expect two independent binding sites
for PABP (species B). However, due to the low affinity of the
complex and the concentrations used, it is not possible to
determine the sedimentation coefficient and K d for the ABB
species.
Acknowledgments
The authors acknowledge the support and the use of resources of
the French Infrastructure for Integrated Structural Biology FRISBI
ANR-10-INBS-05 and of Instruct-ERIC: the platforms of the
Grenoble Instruct-ERIC center (ISBG: UMS 3518 CNRS-CEAUGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB) and the Strasbourg Instruct-ERIC center (Centre de
Biologie Inte ´grative, CBI) within IGBMC (CNRS UMR 7104Inserm U 1258-Universite ´ de Strasbourg). CE also acknowledges
the support of the COST action CA 15126 MOBIEU and GRAL
financed within the University Grenoble Alpes graduate school
(Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17EURE-0003).
Heterogeneity and Affinity Interaction Analysis by Sedimentation Velocity
169
Use P value of 0.68 or 0.95 (confidence level of one or two
standard deviations, respectively) for analysis with regularization. Increasing the P value will decrease the resolution of the
distribution but increase the accuracy of its information. Sedimenting species that will disappear in the c(s) distribution at
higher P-value should be interpreted with care.
8. In the presence of a contaminant species that does not participate in the interaction, it is possible with the program GUSSI
to define an s-value range that will be excluded from the calculation of the s w values [24].
9. A poor fit may indicate that the interaction model is incorrect.
Before drawing such conclusion, carefully verify there is no
error in the entered parameters (molar mass, molar extinction
coefficients, s A and s B values).
10. The refined values for s A , s B , and s AB should be in agreement
with values observed in the c(s) distributions of individual
components and titration series. If parameters are not well
restrained by experimental data, refined values may be unrealistic. In this case, s A and s B should be fixed in the
refinement [19].
11. It is noteworthy that GST-BTG2 (species A) is a dimer in
solution, and we can expect two independent binding sites
for PABP (species B). However, due to the low affinity of the
complex and the concentrations used, it is not possible to
determine the sedimentation coefficient and K d for the ABB
species.
Acknowledgments
The authors acknowledge the support and the use of resources of
the French Infrastructure for Integrated Structural Biology FRISBI
ANR-10-INBS-05 and of Instruct-ERIC: the platforms of the
Grenoble Instruct-ERIC center (ISBG: UMS 3518 CNRS-CEAUGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB) and the Strasbourg Instruct-ERIC center (Centre de
Biologie Inte ´grative, CBI) within IGBMC (CNRS UMR 7104Inserm U 1258-Universite ´ de Strasbourg). CE also acknowledges
the support of the COST action CA 15126 MOBIEU and GRAL
financed within the University Grenoble Alpes graduate school
(Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17EURE-0003).
Heterogeneity and Affinity Interaction Analysis by Sedimentation Velocity
169