CHAPTER 4 STRUCTURE OF αGlyBP……………………………………... 73-123
4.2 MATERIALS AND METHODS
4.2.4 Data collection, processing, structure solution, model building and
4.2.4 Data collection, processing, structure solution, model building and
checked and validated by programs PROCHECK (Laskowski et al., 1993) and MolProbity (Chen et al., 2010). The three-dimensional atomic coordinates and the structure factors of all the structures have been deposited in the RCSB Protein Data Bank (Berman et al., 2000) with accession codes 6J9W, 6J9Y, 6JAD, 6JAG, 6JAH, 6JAI, 6JAL, 6JAM, 6JAN, 6JAO, 6JAP, 6JAQ, 6JAR, 6JAZ, 6JB0, 6JB4, 6JBA, 6JBB and 6JBE for αGlyBP_WT•TRE, αGlyBP_WT•MAL, αGlyBP_WT•PAL, αGlyBP_WT•SUC, αGlyBP_WT•GLC, αGlyBP_D118A•MAL, αGlyBP_R356A,
αGlyBP_R356A•TRE, αGlyBP_R356A•MAL, αGlyBP_R356A•PAL,
αGlyBP_R356A•SUC, αGlyBP_R356A•GLC, αGlyBP_R49A•MAL,
αGlyBP_W287F•TRE, αGlyBP_W287A•TRE, αGlyBP_W287A•MAL,
αGlyBP_W287A•PAL, αGlyBP_W287A•SUC and αGlyBP_W287A•GLC, respectively. All the molecular graphics figures of protein structures were prepared using PyMOL (Molecular Graphics System, Version 1.7.2.2 Schrödinger, LLC).
Table 4.2. Data collection and refinement statistics of αGlyBP_WT protein (bound to trehalose, maltose and palatinose). The values in parenthesis are for the last resolution shell.
αGlyBP WT•TRE αGlyBP WT•MAL αGlyBP WT•PAL
Wavelength (Å) 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100
Space group P41212 P41212 P41212
Unit-cell parameters (Å, ᵒ)
a=b=84.91, c=145.80, α=β=γ=90
a=b=85.24, c=145.68, α=β=γ=90
a=b=84.77, c=145.83, α=β=γ=90 Resolution (Å) 48.60-1.80 (1.84-
1.80)
46.44-1.63 (1.66- 1.63)
59.94-1.90 (1.94- 1.90) No. of observed
reflections 611481 (35139) 611875 (26380) 279983 (18724) No. of unique
reflections 50048 (2860) 67774 (3302) 42666 (2695) Mn(I) CC(1/2) 0.999 (0.954) 0.998 (0.970) 0.997 (0.915) Completeness
(%) 99.8 (98.9) 100 (99.9) 99.9 (99.6)
VM (Å3 Da-1) 2.90 2.92 2.89
Solvent content
(%) 57.63 57.93 57.50
Mosaicity (°) 0.461 0.422 0.639
MeanI/σ(I) 18.8 (5.8) 18.5 (5.5) 13.2 (3.6)
Rmerge† (%) 0.088 (0.435) 0.067 (0.242) 0.083 (0.449)
Rpim (%) 0.037 (0.185) 0.033 (0.135) 0.050 (0.268) Rmeas (%) 0.095 (0.473) 0.075 (0.278) 0.097 (0.524)
Multiplicity 12.2 (12.3) 9.0 (8.0) 6.6 (6.9)
Rwork/Rfree (%) 13.25/16.41 13.69/16.27 13.94/18.16
Protein model No. of subunits
in ASU 1 1 1
Protein atoms 3242 3274 3250
Water molecules 533 575 517
Trehalose 1 - -
Maltose - 1 -
Palatinose - - 1
Others 2 3 2
Deviation from ideal geometry
Bond length (Å) 0.020 0.022 0.018
Bond angles (°) 1.848 2.057 1.804
Average B-factor (Å2)
Protein atoms 16.73 16.19 21.60
Water
molecules 30.60 31.05 34.18
Trehalose 11.56 - -
Maltose - 11.88 -
Palatinose - - 15.75
Ramachandran plot
Favored (%) 97.73 98.47 98.49
Allowed (%) 1.77 1.27 1.26
Remaining (%) 0.51 0.25 0.25
PDB id 6J9W 6J9Y 6JAD
† 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.
TRE: Trehalose; MAL: Maltose; PAL: Palatinose
Table 4.3. Data collection and refinement statistics of αGlyBP_WT (bound to sucrose and glucose) and αGlyBP_D118A mutant protein (bound to maltose). The values in parenthesis are for the last resolution shell.
αGlyBP WT•SUC αGlyBP WT•GLC αGlyBP D118A•M AL
Wavelength (Å) 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100
Space group P41212 P41212 P41212
Unit-cell parameters (Å, ᵒ)
a=b=84.93, c=145.95, α=β=γ=90
a=b= 84.23, c=145.90, α=β=γ=90
a=b= 84.72, c=146.02, α=β=γ=90 Resolution (Å) 60.05-1.85 (1.89-
1.85)
48.63-1.63 (1.66- 1.63)
59.91-2.10 (2.16- 2.10) No. of observed
reflections 823750 (47317) 1181510 (51925) 493303 (38585) No. of unique
reflections 46411 (2826) 66385 (3266) 31860 (2553) Mn(I) CC(1/2) 0.999 (0.944) 0.999 (0.929) 0.998 (0.957) Completeness
(%) 100 (100) 100 (100) 100 (100)
VM (Å3 Da-1) 2.91 2.86 2.89
Solvent content
(%) 57.70 56.98 57.51
Mosaicity (°) 0.573 0.521 0.730
Mean I/σ(I) 24.3 (5.8) 22.3 (4.3) 17.5 (6.0)
Rmerge† (%) 0.107 (0.573) 0.082 (0.647) 0.126 (0.481)
Rpim (%) 0.036 (0.203) 0.028 (0.239) 0.046 (0.180) Rmeas (%) 0.113 (0.608) 0.087 (0.690) 0.134 (0.514) Multiplicity 17.7 (16.7) 17.8 (15.9) 15.5 (15.1)
Rwork/Rfree (%) 12.93/16.68 13.48/16.05 14.17/18.99
Protein model No. of subunits
in ASU 1 1 1
Protein atoms 3255 3258 3246
Water
molecules 570 580 448
Sucrose 1 - -
Glucose - 1 -
Maltose - - 1
Calcium ion - - 1
Others 2 7 3
Deviation from ideal geometry
Bond length (Å) 0.019 0.021 0.017
Bond angles (°) 1.801 1.990 1.705
Average B-factor (Å2)
Protein atoms 14.93 17.28 20.22
Water
molecules 28.80 32.41 31.35
Sucrose 8.12 - -
Glucose - 10.12 -
Maltose - - 21.46
Calcium ion - - 18.07
Ramachandran plot
Favored (%) 97.73 97.73 97.78
Allowed (%) 1.77 1.77 1.51
Remaining (%) 0.51 0.51 0.50
PDB id 6JAG 6JAH 6JAI
† 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.
SUC: Sucrose; GLC: Glucose
Table 4.4. Data collection and refinement statistics of αGlyBP_R356A mutant protein in open and closed conformation (bound to trehalose and maltose). The values in parenthesis are for the last resolution shell.
αGlyBP R356A αGlyBP R356A•TRE αGlyBP R356A•M AL
Wavelength
(Å) 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100
Space group P21 P41212 P41212
Unit-cell parameters (Å, ᵒ)
a= 53.04, b=
85.13, c=55.34, α=γ=90, β=92.99
a=b=85.07, c=146.38, α=β=γ=90
a=b=84.41, c=145.55, α=β=γ=90 Resolution (Å) 44.97-1.56 (1.59-
1.56) 48.79-1.63 (1.66-1.63) 59.69-1.77 (1.81- 1.77) No. of
observed
reflections 318860 (14055) 1241773 (53313) 771334 (40657) No. of unique
reflections 69683 (3351) 67875 (3277) 49679 (2550) Mn(I) CC(1/2) 0.999 (0.994) 0.999 (0.953) 0.999 (0.967) Completeness
(%) 99.8 (96.4) 100 (100) 96.0 (87.8)
VM (Å3 Da-1) 2.76 2.92 2.86
Solvent content
(%) 55.39 57.96 57.06
Mosaicity (°) 0.357 0.542 0.559
Mean I/σ(I) 30.1 (15.0) 22.1 (4.6) 22.1 (6.2)
Rmerge† (%) 0.031 (0.061) 0.078 (0.532) 0.083 (0.389)
Rpim (%) 0.024 (0.048) 0.026 (0.194) 0.029 (0.137) Rmeas (%) 0.040 (0.077) 0.082 (0.567) 0.089 (0.413)
Multiplicity 4.6 (4.2) 18.3 (16.3) 15.5 (15.9)
Rwork/Rfree (%) 12.71/15.05 13.81/16.59 13.25/16.35
Protein model No. of subunits
in ASU 1 1 1
Protein atoms 3396 3279 3251
Water
molecules 666 587 528
Trehalose - 1 -
Maltose - - 1
Others 7 6 7
Deviation from ideal geometry Bond length
(Å) 0.022 0.021 0.019
Bond angles (°) 2.155 1.955 1.856
Average B-factor (Å2)
Protein atoms 10.39 18.23 16.50
Water
molecules 25.21 32.83 30.50
Trehalose - 12.59 -
Maltose - - 10.52
Ramachandran plot
Favored (%) 98.75 97.73 97.98
Allowed (%) 1.00 1.77 1.77
Remaining (%) 0.25 0.51 0.25
PDB id 6JAL 6JAM 6JAN
† 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.
TRE: Trehalose; MAL: Maltose
Table 4.5. Data collection and refinement statistics of αGlyBP_R356A mutant protein in closed conformation (bound to palatinose, sucrose and glucose). The values in parenthesis are for the last resolution shell.
αGlyBP R356A•PAL αGlyBP R356A•SU C
αGlyBP R356A•
GLC
Wavelength (Å) 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100
Space group P41212 P41212 P41212
Unit-cell parameters (Å, ᵒ)
a=b=85.42, c=146.15, α=β=γ=90
a=b= 85.41, c=146.29, α=β=γ=90
a=b=84.62, c=146.23, α=β=γ=90
Resolution (Å) 60.40-1.77 (1.81-1.77) 60.39-1.77 (1.81- 1.77)
59.84-1.95 (2.00- 1.95) No. of observed
reflections 940791 (48057) 911320 (46820) 580422 (40079) No. of unique
reflections 53234 (2987) 53038 (2950) 39565 (2725) Mn(I) CC(1/2) 0.999 (0.982) 0.999 (0.986) 0.999 (0.952) Completeness
(%) 100 (100) 99.7 (98.6) 100 (100)
VM (Å3 Da-1) 2.94 2.95 2.89
Solvent content
(%) 58.24 58.27 57.47
Mosaicity (°) 0.481 0.421 0.388
Mean I/σ(I) 24.0 (6.1) 31.2 (8.8) 22.2 (6.0)
Rmerge† (%) 0.080 (0.302) 0.063 (0.247) 0.108 (0.483)
Rpim (%) 0.027 (0.111) 0.022 (0.090) 0.041 (0.185) Rmeas (%) 0.085 (0.322) 0.067 (0.263) 0.116 (0.517)
Multiplicity 17.7 (16.1) 17.2 (15.9) 14.7 (14.7)
Rwork/Rfree (%) 15.49/19.94 15.04/19.53 13.14/16.71
Protein model No. of subunits
in ASU 1 1 1
Protein atoms 3273 3274 3247
Water molecules 557 556 531
Palatinose 1 - -
Sucrose - 1 -
Glucose - - 1
Others 4 3 5
Deviation from ideal geometry
Bond length (Å) 0.018 0.018 0.019
Bond angles (°) 1.827 1.799 1.783
Average B-factor (Å2)
Protein atoms 19.91 17.81 15.49
Water
molecules 34.33 32.16 28.71
Palatinose 12.68 - -
Sucrose - 10.13 -
Glucose - - 8.79
Ramachandran plot
Favored (%) 98.47 97.96 97.97
Allowed (%) 1.27 1.53 1.52
Remaining (%) 0.25 0.51 0.51
PDB id 6JAO 6JAP 6JAQ
† 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.
PAL: Palatinose; SUC: Sucrose; GLC: Glucose
Table 4.6. Data collection and refinement statistics of αGlyBP_R49A (bound to maltose), αGlyBP_W287F and αGlyBP_W287A mutant proteins (bound to trehalose).
The values in parenthesis are for the last resolution shell.
αGlyBP R49A•MA L
αGlyBP W287F•TR E
αGlyBP W287A•TR E
Wavelength
(Å) 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100
Space group P41212 P41212 P41212
Unit-cell parameters (Å, ᵒ)
a=b=84.98, c=145.46, α=β=γ=90
a=b=85.27,
c=145.65, α=β=γ=90 a=b=84.76, c=146.21, α=β=γ=90 Resolution
(Å)
48.49-1.63 (1.66- 1.63)
48.55-1.85 (1.89- 1.85)
48.74-1.63 (1.66- 1.63) No. of
observed reflections
1006044 (44383) 690786 (41102) 804773 (35559) No. of
unique
reflections 61325 (2721) 46653 (2839) 65184 (2980) Mn(I)
CC(1/2) 0.999 (0.954) 0.999 (0.949) 0.999 (0.942) Completenes
s (%) 91.3 (83.4) 100 (99.9) 96.9 (91.5)
VM (Å3 Da-1) 2.90 2.92 2.90
Solvent
content (%) 57.61 57.95 57.61
Mosaicity
(°) 0.558 0.370 0.332
Mean I/σ(I) 24.3 (5.1) 21.6 (5.8) 24.4 (5.7)
Rmerge† (%) 0.064 (0.391) 0.092 (0.511) 0.061 (0.381)
Rpim (%) 0.022 (0.139) 0.035 (0.196) 0.025 (0.160) Rmeas (%) 0.068 (0.416) 0.098 (0.547) 0.066 (0.414) Multiplicity 16.4 (16.3) 14.8 (14.5) 12.3 (11.9) Rwork/Rfree
(%) 13.99/16.62 13.26/16.83 13.42/16.02
Protein model
No. of subunits in
ASU 1 1 1
Protein
atoms 3262 3232 3249
Water
molecules 601 539 620
Maltose 1 - -
Trehalose - 1 1
Others 3 2 4
Deviation from ideal geometry Bond length
(Å) 0.020 0.019 0.020
Bond angles
(°) 1.932 1.835 1.947
Average B-factor (Å2) Protein
atoms 17.85 17.06 14.81
Water
molecules 33.00 30.34 30.05
Maltose 17.93 - -
Trehalose - 18.36 9.46
Ramachandran plot
Favored (%) 97.96 97.97 97.73
Allowed (%) 1.53 1.52 1.77
Remaining
(%) 0.51 0.51 0.51
PDB id 6JAR 6JAZ 6JB0
† 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.
TRE: Trehalose
Table 4.7. Data collection and refinement statistics of αGlyBP_W287A mutant protein (bound to maltose, palatinose and sucrose). The values in parenthesis are for the last resolution shell.
αGlyBP W287A•MA L
αGlyBP W287A•P AL
αGlyBP W287A•
SUC Wavelength
(Å) 1.5418 1.5418 1.5418
Temperature
(K) 100 100 100
Space group P41212 P41212 P41212
Unit-cell parameters (Å, ᵒ)
a=b=85.13, c=145.54, α=β=γ=90
a=b=84.10, c=146.02, α=β=γ=90
a=b=84.48, c=145.93, α=β=γ=90 Resolution
(Å) 48.51-1.63 (1.66-1.63) 59.47-2.00 (2.05- 2.00)
59.74-1.95 (2.00- 1.95) No. of
observed reflections
628881 (27856) 308917 (22315) 311670 (20698) No. of unique
reflections 67595 (3297) 36237 (2647) 38891 (2614) Mn(I)
CC(1/2) 0.999 (0.891) 0.990 (0.917) 0.997 (0.869) Completeness
(%) 100 (100) 100 (100) 99.0 (96.9)
VM (Å3 Da-1) 2.91 2.85 2.87
Solvent
content (%) 57.78 56.88 57.24
Mosaicity (°) 0.400 0.60 0.590
Mean I/σ(I) 17.1 (3.9) 10.4 (3.4) 13.4 (3.2)
Rmerge† (%) 0.074 (0.457) 0.140 (0.574) 0.118 (0.557)
Rpim (%) 0.037 (0.246) 0.073 (0.308) 0.061 (0.285) Rmeas (%) 0.082 (0.520) 0.158 (0.653) 0.133 (0.629)
Multiplicity 9.3 (8.4) 8.5 (8.4) 8.0 (7.9)
Rwork/Rfree (%) 13.68/16.17 15.06/18.63 14.27/17.69
Protein model No. of
subunits in ASU
1 1 1
Protein atoms 3246 3254 3241
Water
molecules 593 448 473
Maltose 1 - -
Palatinose - 1 -
Sucrose - - 1
Others 5 4 5
Deviation from ideal geometry Bond length
(Å) 0.020 0.017 0.017
Bond angles
(°) 1.876 1.787 1.796
Average B-factor (Å2)
Protein atoms 16.13 22.37 18.07
Water
molecules 30.57 33.84 30.21
Maltose 11.31 - -
Palatinose - 20.84 -
Sucrose - - 10.82
Ramachandran plot
Favored (%) 97.73 97.72 97.48
Allowed (%) 1.77 1.77 2.02
Remaining
(%) 0.51 0.51 0.50
PDB id 6JB4 6JBA 6JBB
† 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.
MAL: Maltose; PAL: Palatinose; SUC: Sucrose
Table 4.8. Data collection and refinement statistics of αGlyBP_W287A mutant protein (bound to glucose). The values in parenthesis are for the last resolution shell.
αGlyBP_W287A•GLC
Wavelength (Å) 1.5418
Temperature (K) 100
Space group P41212
Unit-cell parameters (Å, ᵒ) a=b=84.99, c=145.83, α=β=γ=90
Resolution (Å) 55.56-1.75 (1.78-1.75)
No. of observed reflections 645449 (32906)
No. of unique reflections 52985 (2590)
Mn(I) CC(1/2) 0.999 (0.918)
Completeness (%) 97.0 (88.3)
VM (Å3 Da-1) 2.91
Solvent content (%) 57.72
Mosaicity (°) 0.448
Mean I/σ(I) 17.4 (3.9)
Rmerge† (%) 0.092 (0.606)
Rpim (%) 0.037 (0.238)
Rmeas (%) 0.099 (0.652)
Multiplicity 12.2 (12.7)
Rwork/Rfree (%) 13.89/17.57
Protein model
No. of subunits in ASU 1
Protein atoms 3246
Water molecules 573
Glucose 1
Others 6
Deviation from ideal geometry
Bond length (Å) 0.018
Bond angles (°) 1.770 Average B-factor (Å2)
Protein atoms 18.64
Water molecules 32.31
Glucose 10.92
Ramachandran plot
Favored (%) 97.73
Allowed (%) 1.77
Remaining (%) 0.51
PDB id 6JBE
† 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.
GLC: Glucose