CHAPTER 5 STRUCTURE OF βGlyBP……………………………………… 124-183
5.2 MATERIALS AND METHODS
5.2.6 Data collection, processing and structure determination
As all the crystals of the wild type and mutant protein βGlyBP (apo and holo) were grown in high concertation of PEG 8000, hence no further cryo-protectant was used during flash cooling. X-ray diffraction data for all the crystals were collected at -173°C using Cu_Kα radiation (wavelength 1.5418 Å) generated by Rigaku MicroMax-007 HF diffractometer (operated at 40 kV and 30 mA) and R-Axis IV++ image-plate detector available at Central Instruments Facility (CIF) of Indian Institute of Technology Guwahati (IITG), India. The data for all the crystals were collected with 1° oscillation and 300s exposure time. All the X-ray data sets were processed and scaled using the programs iMosflm (Battye et al., 2011) and Aimless (Evans and Murshudov, 2013) embedded in the package CCP4 (Winn, 2011).
The X-ray intensities were converted to structure factors using program module ctruncate of CCP4. The structure solution of βGlyBP_WT was obtained by molecular replacement method using the program Phaser (McCoy et al., 2007) using the crystal structure of sugar- binding protein MalE from Xanthomonas citri (PDB ID: 3UOR) as the search model, which shares a sequence identity (query coverage) of 27(97)% with the protein βGlyBP. The Matthews coefficient (Matthews, 1968) indicated the presence of one molecule in the asymmetric unit. The structure solution of holo βGlyBP_WT and apo & holo forms of mutant proteins βGlyBP_W41A, βGlyBP_W67A, βGlyBP_E117A and βGlyBP_W177X were obtained by the direct molecular replacement method using the program Phaser and the three-dimensional atomic coordinates of the apo βGlyBP_WT as the search model.
A total of 5% of the reflections were kept aside from the data set for the calculation of Rfree
(Brunger, 1992). All the structural refinement of the model was carried out by using program Refmac5 (Vagin, 2004) with a default set of parameters. All the model building was carried out manually using the program Coot (Emsley et al., 2010). Although, in case of all the complexed structures, a clear Fo−Fc electron density maps could be observed for the bound ligand(s) after the first cycle of the refinement, the protein atoms were firstly fitted into the electron density contoured at 3.0σ and 1.0σ for the Fo-Fc and 2Fo-Fc maps,
respectively. Subsequently, electron density observed for water molecules and other molecules present in the protein buffer and the crystallization conditions were modeled.
After each cycle of the model building, structure refinement was performed using the same set of parameters. Finally, the ligands bound to the protein molecule were fitted in the electron density using the difference map contoured at 3.0σ. The geometric parameters of all the refined structures were checked and validated by the programs Procheck (Laskowski et al., 1993) and MolProbity (Chen et al., 2010). All the refinement and validation statistics of the refined models are provided in the Table 5.2-5.7. The atomic coordinates and structure factors of all the refined models have been deposited in the RCSB Protein Data Bank (Berman et al., 2000). All the molecular graphic figures presented in this study were generated using the program PyMOL (Molecular Graphics System, Version 2.1.1 Schrödinger, LLC).
Table 5.2. X-ray crystallographic data collection and refinement statistics for the protein βGlyBP_WT (apo and complexed with CEL2 and CEL3). The values in parenthesis represent the statistics for the last resolution shell.
βGlyBP_WT_F ormI
βGlyBP_WT_
FormII
βGlyBP_WT•
CEL2
βGlyBP_WT•
CEL3 Wavelength
(Å) 1.5418 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100 100
Space group P212121 P21 P212121 P212121
Unit-cell parameters (Å, ᵒ)
a=58.52, b=62.93, c=104.02, α=β=γ=90
a=57.46, b=100.71,
c=66.26, α=γ=90, β=104.19
a=64.24, b=110.82, c=110.91, α=
β=γ=90
a=62.67, b=109.88, c=110.89, α=
β=γ=90 Resolution
(Å)
51.00-1.63 (1.66-1.63)
50.35-1.63
(1.66-1.63) 64.24-2.30
(2.38-2.30) 62.67-2.00 (2.05-2.00) No. of
observed reflections
440139 (19653) 415172 (18698) 252397 (22922)
368548 (25412) No. of
unique reflections
48845 (2397) 91079 (4434) 35932 (3469) 52166 (3744) Mn(I)
CC(1/2) 0.999 (0.961) 0.998 (0.963) 0.984 (0.911) 0.996 (0.882)
Completenes
s (%) 100.0 (99.8) 99.9 (98.0) 99.9 (99.9) 99.4 (98.3)
VM (Å3 Da-1) 2.09 2.03 2.15 2.08
Solvent
content (%) 41.16 39.36 42.90 40.96
Mosaicity (°) 0.40 0.70 0.70 0.80
Mean I/σ(I) 24.7 (4.9) 19.3 (6.5) 8.2 (3.4) 9.6 (3.1)
Rmerge† (%) 4.7 (28.8) 4.5 (16.8) 17.8 (47.3) 10.1 (48.8)
Rpim (%) 2.4 (15.8) 3.6 (13.2) 10.2 (28.7) 5.8 (29.0) Rmeas (%) 5.3 (33.0) 5.8 (21.4) 20.4 (55.6) 11.7 (56.9) Multiplicity 9.0 (8.2) 4.6 (4.2) 7.0 (6.6) 7.1 (6.8) Rwork/Rfree
(%) 16.40/20.80 13.26/16.58 19.22/24.84 16.98/23.06 Protein model
No. of subunits in ASU
1 2 2 2
Protein
atoms 3237 6436 6407 6421
Water
molecules 464 934 423 419
Carbohydrate
s (number) - - CEL2 (2) CEL3 (2)
Other
molecules 7 23 10 14
Deviation from ideal geometry Bond length
(Å) 0.017 0.021 0.013 0.013
Bond angles
(°) 2.163 2.380 1.748 1.766
Average B-factor (Å2) Protein
atoms 16.51 10.99 22.27 23.13
Water
molecules 32.24 25.06 27.45 34.33
Carbohydrate - - CEL2 (17.15) CEL3 (26.65)
Ramachandran plot (%)
Favored 98.02 97.28 97.72 97.57
Allowed 1.98 2.72 2.28 2.43
Remaining 0.00 0.00 0.00 0.00
PDB ID 7C63 7C64 7C66 7C67
† Rmerge = ∑hkl ∑i|Ii(hkl) - 〈I(hkl)〉|/ ∑hkl∑iIi(hkl), where I(hkl) is the intensity of reflection hkl, ∑hkl is the sum overall reflections and ∑i is the sum over i measurements of reflection hkl.
Abbreviations: CEL2, cellobiose; CEL3, cellotriose.
Table 5.3. X-ray crystallographic data collection and refinement statistics of the protein βGlyBP_WT (complexed with CEL4 and SOP2) and mutant βGlyBP_W177X (apo and complexed with GEN2). The values in parenthesis represent the statistics for the last resolution shell.
βGlyBP_WT•C EL4
βGlyBP_WT•
SOP2
βGlyBP_W17 7X
βGlyBP_W177 X•GEN2 Wavelength
(Å) 1.5418 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100 100
Space group P212121 P212121 P212121 P21
Unit-cell parameters (Å, ᵒ)
a=62.63, b=109.74, c=111.57, α=β=γ=90
a=62.79, b=108.93, c=109.99, α=β=γ=90
a=58.21, b=63.10, c=103.79, α=β=γ=90
a=62.34, b=91.54, c=69.39, α=γ=90, β=112.51 Resolution
(Å)
55.78-2.05 (2.11-2.05)
62.79-2.35 (2.43-2.35)
53.92-1.70 (1.73-1.70)
48.75-1.90 (1.94-1.90) No. of
observed reflections
352325 (27050) 279970 (25078)
278893
(12758) 252521 (14977) No. of
unique reflections
48888 (3760) 32179 (3110) 41913 (2061) 53435 (3258) Mn(I)
CC(1/2) 0.993 (0.920) 0.984 (0.901) 0.995 (0.922) 0.995 (0.891) Completene
ss (%) 99.7 (99.4) 99.9 (99.7) 98.1 (94.0) 94.5 (89.3)
VM (Å3 Da-1) 2.09 2.05 2.08 1.99
Solvent
content (%) 41.21 40.08 40.87 38.38
Mosaicity
(°) 0.90 0.60 0.60 0.80
Mean I/σ(I) 9.6 (3.2) 9.6 (3.8) 14.5 (4.2) 10.6 (2.8)
Rmerge† (%) 12.3 (46.4) 19.0 (47.9) 8.1 (34.2) 7.6 (38.7)
Rpim (%) 7.2 (27.8) 9.8 (26.1) 4.8 (20.9) 6.0 (29.4) Rmeas (%) 14.3 (54.3) 21.4 (54.7) 9.4 (40.2) 9.7 (48.9) Multiplicity 7.2 (7.2) 8.7 (8.1) 6.7 (6.2) 4.7 (4.6) Rwork/Rfree
(%) 18.18/22.41 19.96/26.23 17.38/21.58 17.35/22.73 Protein model
No. of subunits in ASU
2 2 1 2
Protein
atoms 6373 6388 3211 6439
Water
molecules 463 264 401 470
Carbohydrat
e (number) CEL4 (2) SOP2 (2) - GEN2 (2)
Other
molecules 15 6 10 10
Deviation from ideal geometry Bond length
(Å) 0.013 0.011 0.014 0.014
Bond angles
(°) 1.828 1.764 1.909 1.920
Average B-factor (Å2) Protein
atoms 19.89 19.10 19.30 21.69
Water
molecules 30.78 26.04 31.44 32.03
Carbohydrat
e CEL4 (21.42) SOP2 (17.98) - GEN2 (35.70)
Ramachandran plot (%)
Favored 97.09 97.23 97.56 96.96
Allowed 2.91 2.65 2.44 3.04
Remaining 0.00 0.12 0.00 0.00
PDB ID 7C68 7C69 7C6F 7C6G
† Rmerge = ∑hkl ∑i|Ii(hkl) - 〈I(hkl)〉|/ ∑hkl∑iIi(hkl), where I(hkl) is the intensity of reflection hkl, ∑hkl is the sum overall reflections and ∑i is the sum over i measurements of reflection hkl.
Abbreviations: CEL4, cellotetraose; SOP2, sophorose; GEN2, gentiobiose.
Table 5.4. X-ray crystallographic data collection and refinement statistics of the mutant βGlyBP_W177X (complexed with LAM2, SOP2, CEL2 and CEL3). The values in parenthesis represent the statistics for the last resolution shell.
βGlyBP_W177 X•LAM2
βGlyBP_W177 X•SOP2
βGlyBP_W177 X•CEL2
βGlyBP_W177 X•CEL3_Form
I Wavelength
(Å) 1.5418 1.5418 1.5418 1.5418
Temperatur
e (K) 100 100 100 100
Space
group P21 P21 P212121 P212121
Unit-cell parameters (Å, ᵒ)
a=62.51, b=92.25, c=69.78, α=γ=90, β=112.42
a=62.64, b=92.06, c=70.01, α=γ=90, β=112.85
a=60.90, b=100.25, c=134.05, α=β=γ=90
a=58.58, b=62.96, c=109.89, α=β=γ=90 Resolution
(Å)
54.62-1.85 (1.89-1.85)
54.90-1.70 (1.73-1.70)
55.72-2.10 (2.16-2.10)
58.58-1.77 (1.81-1.77) No. of
observed reflections
271996 (16488) 377894 (17665) 364377 (26595) 355851 (18113) No. of
unique reflections
62461 (3890) 80275 (4085) 48058 (3677) 39933 (2178) Mn(I)
CC(1/2) 0.995 (0.814) 0.993 (0.883) 0.994 (0.928) 0.999 (0.910) Completene
ss (%) 100.0 (100.0) 99.8 (96.5) 98.7 (93.9) 99.4 (96.9) VM (Å3 Da-
1) 2.03 2.03 2.33 2.21
Solvent
content (%) 39.40 39.41 44.92 40.39
Mosaicity
(°) 0.50 0.60 0.60 0.25
Mean I/σ(I) 10.5 (2.6) 11.1 (2.9) 12.8 (4.0) 21.2 (4.0)
Rmerge† (%) 8.3 (50.3) 8.3 (39.1) 10.8 (45.4) 6.1 (46.5)
Rpim (%) 6.9 (41.0) 6.4 (30.2) 5.9 (24.5) 3.0 (24.7) Rmeas (%) 10.9 (65.2) 10.5 (49.6) 12.3 (51.7) 6.8 (52.9) Multiplicity 4.4 (4.2) 4.7 (4.3) 7.6 (7.2) 8.9 (8.3) Rwork/Rfree
(%) 16.82/21.67 16.57/20.73 17.84/23.66 16.58/21.74 Protein model
No. of subunits in
ASU 2 2 2 1
Protein
atoms 6466 6450 6383 3182
Water
molecules 630 817 502 378
Carbohydra
te (number) LAM2 (2) SOP2 (2) CEL2 (2) CEL3 (1)
Other
molecules 11 10 6 6
Deviation from ideal geometry Bond
length (Å) 0.013 0.014 0.014 0.013
Bond
angles (°) 1.849 1.984 1.866 1.949
Average B-factor (Å2) Protein
atoms 18.19 17.47 18.86 18.82
Water
molecules 30.25 30.29 29.60 32.20
Carbohydra
te LAM2 (19.04) SOP2 (23.52) CEL2 (18.00) CEL3 (19.18) Ramachandran plot (%)
Favored 97.33 97.94 97.32 98.04
Allowed 2.67 2.06 2.55 1.96
Remaining 0.00 0.00 0.12 0.00
PDB ID 7C6H 7C6I 7C6J 7C6K
† Rmerge = ∑hkl ∑i|Ii(hkl) - 〈I(hkl)〉|/ ∑hkl∑iIi(hkl), where I(hkl) is the intensity of reflection hkl, ∑hkl is the sum overall reflections and ∑i is the sum over i measurements of reflection hkl.
Abbreviations: LAM2, laminaribiose; SOP2, sophorose; CEL2, cellobiose; CEL3, cellotriose.
Table 5.5. X-ray crystallographic data collection and refinement statistics of the mutant βGlyBP_W177X (complexed with CEL3, CEL4 and CEL5). The values in parenthesis represent the statistics for the last resolution shell.
βGlyBP_W177 X•CEL3_FormI
I
βGlyBP_W177 X•CEL4_FormI
βGlyBP_W177 X•CEL4_FormI
I
βGlyBP_W 177X•CEL5 Wavelength
(Å) 1.5418 1.5418 1.5418 1.5418
Temperatur
e (K) 100 100 100 100
Space group P212121 P212121 P212121 P212121
Unit-cell parameters (Å, ᵒ)
a=61.00, b=100.34, c=133.23, α=β=γ=90
a=58.32, b=62.93, c=110.26, α=β=γ=90
a=60.85, b=100.17, c=133.06, α=β=γ=90
a=58.23, b=63.23, c=110.08, α=β=γ=90 Resolution
(Å)
66.61-2.40 (2.49- 2.40)
58.32-1.90 (1.94- 1.90)
66.53-2.05 (2.11- 2.05)
58.23-2.10 (2.16-2.10) No. of
observed reflections
217551 (20686) 279959 (17278) 450232 (31718) 192624 (15215) No. of
unique reflections
32723 (3337) 32723 (2063) 50982 (3827) 22403 (1863)
Mn(I)
CC(1/2) 0.994 (0.905) 0.998 (0.920) 0.998 (0.924) 0.992 (0.912) Completene
ss (%) 99.9 (99.5) 99.9 (99.6) 98.5 (96.6) 92.0 (94.9) VM (Å3 Da-
1) 2.22 2.21 2.21 2.21
Solvent
content (%) 44.72 44.30 44.42 44.39
Mosaicity
(°) 0.60 0.50 0.40 0.70
Mean I/σ(I) 11.4 (3.6) 15.8 (3.5) 17.1 (4.4) 10.3 (3.6)
Rmerge† (%) 12.1 (47.7) 7.7 (46.9) 8.8 (44.1) 12.7 (44.6)
Rpim (%) 7.4 (29.6) 4.0 (24.3) 4.5 (23.2) 6.7 (23.2) Rmeas (%) 14.2 (56.3) 8.7 (53.0) 9.8 (49.9) 14.4 (50.4) Multiplicity 6.6 (6.2) 8.6 (8.4) 8.8 (8.3) 8.6 (8.2) Rwork/Rfree
(%) 19.11/26.33 17.85/22.90 16.12/21.98 23.01/29.16 Protein model
No. of subunits in
ASU 2 1 2 1
Protein
atoms 6361 3173 6371 3174
Water
molecules 327 240 648 94
Carbohydrat
e (number) CEL3 (2) CEL4 (1) CEL4 (2) CEL5 (1)
Other
molecules 8 4 8 3
Deviation from ideal geometry Bond length
(Å) 0.013 0.014 0.014 0.011
Bond angles
(°) 1.846 1.928 1.829 1.803
Average B-factor (Å2) Protein
atoms 22.05 23.32 20.52 19.93
Water
molecules 26.31 34.17 31.30 29.15
Carbohydrat
e CEL3 (25.34) CEL4 (31.20) CEL4 (25.33) CEL5
(31.15) Ramachandran plot (%)
Favored 96.74 98.54 98.05 95.85
Allowed 3.26 1.46 1.95 4.15
Remaining 0.00 0.00 0.00 0.00
PDB ID 7C6L 7C6M 7C6N 7C6R
† Rmerge = ∑hkl ∑i|Ii(hkl) - 〈I(hkl)〉|/ ∑hkl∑iIi(hkl), where I(hkl) is the intensity of reflection hkl, ∑hkl is the sum overall reflections and ∑i is the sum over i measurements of reflection hkl.
Abbreviations: CEL3, cellotriose; CEL4, cellotetraose; CEL5, cellopentaose.
Table 5.6. X-ray crystallographic data collection and refinement statistics of the mutants βGlyBP_W177X (complexed with LAM3 and LAM4) and βGlyBP_W41A (apo). The values in parenthesis represent the statistics for the last resolution shell.
βGlyBP_W177 X•LAM3_For
mI
βGlyBP_W177 X•LAM3_For
mII
βGlyBP_W177 X•LAM4
βGlyBP_W41 A_FormI Wavelength
(Å) 1.5418 1.5418 1.5418 1.5418
Temperatur
e (K) 100 100 100 100
Space
group P212121 P3121 P212121 P1
Unit-cell parameters (Å, ᵒ)
a=57.93, b=63.55, c=110.23, α=β=γ=90
a=b=185.41, c=74.18, α=β=90, γ=120
a=58.11, b=62.60, c=109.2, α=β=γ=90
a=80.99, b=116.40, c=132.14, α=83.39, β=88.73, γ=89.95 Resolution
(Å)
57.93-2.30 (2.38-2.30)
80.28-2.80 (2.92-2.80)
58.11-1.88 (1.92-1.88)
82.19-2.65 (2.70-2.65) No. of
observed reflections
165548 (15036) 354628 (40473) 240945 (14248) 463080 (20071) No. of
unique reflections
18713 (1771) 36336 (4404) 32582 (1978) 130991 (6096) Mn(I)
CC(1/2) 0.996 (0.961) 0.999 (0.933) 0.998 (0.920) 0.793 (0.806) Completene
ss (%) 99.8 (99.3) 100.0 (100.0) 98.5 (95.6) 94.1 (88.3) VM (Å3 Da-
1) 2.21 2.68 2.16 2.25
Solvent
content (%) 44.46 54.07 43.16 45.34
Mosaicity
(°) 0.60 0.44 0.75 0.80
Mean I/σ(I) 16.1 (5.6) 13.8 (4.5) 15.2 (3.5) 3.5 (2.0)
Rmerge† (%) 9.9 (33.8) 9.8 (50.1) 7.7 (43.3) 23.6 (37.2)
Rpim (%) 4.9 (16.7) 4.8 (25.8) 4.2 (24.1) 23.6 (37.2) Rmeas (%) 11.1 (37.8) 10.9 (56.5) 8.8 (49.6) 33.4 (52.7) Multiplicity 8.8 (8.5) 9.8 (9.2) 7.4 (7.2) 3.5 (3.3) Rwork/Rfree
(%) 20.26/26.49 18.66/23.56 18.71/24.23 24.30/26.80 Protein model
No. of subunits in ASU
1 3 1 12
Protein
atoms 3162 9469 3183 37819
Water
molecules 139 52 319 484
Carbohydra
te (number) LAM3 (1) LAM3 (3) LAM4 (1) -
Other
molecules 6 13 5 22
Deviation from ideal geometry Bond length
(Å) 0.014 0.013 0.013 0.012
Bond
angles (°) 1.931 1.841 1.880 1.622
Average B-factor (Å2) Protein
atoms 17.96 32.80 16.98 7.70
Water
molecules 27.50 36.99 33.74 4.60
Carbohydra
te LAM3 (26.91) LAM3 (45.92) LAM4 (29.94) -
Ramachandran plot (%)
Favored 97.07 95.20 96.80 96.80
Allowed 2.93 4.80 3.20 3.20
Remaining 0.00 0.00 0.00 0.00
PDB ID 7C6T 7C6V 7C6W 7C6X
† Rmerge = ∑hkl ∑i|Ii(hkl) - 〈I(hkl)〉|/ ∑hkl∑iIi(hkl), where I(hkl) is the intensity of reflection hkl, ∑hkl is the sum overall reflections and ∑i is the sum over i measurements of reflection hkl.
Abbreviations: LAM3, laminaritriose; LAM4, laminaritetraose.
Table 5.7. X-ray crystallographic data collection and refinement statistics of the mutants βGlyBP_W41A (apo), βGlyBP_W67A (apo and complexed form with GEN2) and βGlyBP_E117A (apo). The values in parenthesis represent the statistics for the last resolution shell.
βGlyBP_W41A _FormII
βGlyBP_W67 A
βGlyBP_W67
A•GEN2 βGlyBP_E117 A Wavelength
(Å) 1.5418 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100 100
Space group P212121 P21 P21 P21
Unit-cell parameters (Å, ᵒ)
a=58.35, b=63.25, c=103.95, α=β=γ=90
a=55.26, b=97.79, c=66.61, α=γ=90, β=103.42
a=55.24, b=98.96, c=66.56, α=γ=90, β=103.25
a=57.40, b=100.96,
c=66.52, α=γ=90, β=104.60 Resolution
(Å)
54.03-1.70 (1.73-1.70)
48.90-1.63 (1.66-1.63)
49.48-1.63 (1.66-1.63)
55.55-2.10 (2.16-2.10) No. of
observed reflections
373282 (17715) 349654
(16083) 618337 (27904) 173623 (13800) No. of
unique reflections
41739 (2100) 78922 (3823) 85916 (4142) 42869 (3480) Mn(I)
CC(1/2) 0.997 (0.939) 0.996 (0.838) 0.995 (0.918) 0.981 (0.812) Completenes
s (%) 97.2 (94.3) 92.4 (90.8) 99.0 (97.0) 100.0 (100.0)
VM (Å3 Da-1) 2.09 1.91 1.93 2.03
Solvent
content (%) 41.25 35.62 36.36 39.58
Mosaicity
(°) 0.60 1.07 0.60 0.80
Mean I/σ(I) 21.3 (5.7) 9.4 (2.6) 15.9 (4.0) 5.9 (2.6)
Rmerge† (%) 7.1 (32.9) 7.4 (40.1) 7.6 (38.9) 14.7 (39.7)
Rpim (%) 3.6 (17.4) 6.0 (32.0) 4.6 (24.7) 13.4 (36.2) Rmeas (%) 7.9 (37.3) 9.6 (51.4) 8.8 (46.3) 20.0 (54.0) Multiplicity 8.9 (8.4) 4.4 (4.2) 7.2 (6.7) 4.1 (4.0) Rwork/Rfree
(%) 17.23/21.08 15.75/19.59 13.65/17.81 17.90/24.39 Protein model
No. of subunits in ASU
1 2 2 2
Protein
atoms 3241 6392 6495 6362
Water
molecules 396 782 832 611
Carbohydrat
e (number) - - GEN2 (2) -
Other
molecules 7 17 17
12 Deviation from ideal geometry
Bond length
(Å) 0.017 0.015 0.017 0.011
Bond angles
(°) 2.128 1.963 2.147
1.646 Average B-factor (Å2)
Protein
atoms 16.28 13.37 12.74
14.99 Water
molecules 29.09 26.29 26.17
22.50 Carbohydrat
e - - GEN2 (30.79)
- Ramachandran plot (%)
Favored 98.27 97.79 98.00 96.47
Allowed 1.73 2.21 2.00 3.53
Remaining 0.00 0.00 0.00 0.00
PDB ID 7C6Y 7C6Z 7C70 7C71
† Rmerge = ∑hkl ∑i|Ii(hkl) - 〈I(hkl)〉|/ ∑hkl∑iIi(hkl), where I(hkl) is the intensity of reflection hkl, ∑hkl is the sum overall reflections and ∑i is the sum over i measurements of reflection hkl.
Abbreviation: GEN2, gentiobiose.
5.2.7 Measurement of ligand binding affinity using isothermal titration calorimetry