vikram babu kasaragod and hermann schindelin babu kasaragod and hermann schindelin supplemental...
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Structure, Volume 24
Supplemental Information
Structural Framework for Metal Incorporation
during Molybdenum Cofactor Biosynthesis
Vikram Babu Kasaragod and Hermann Schindelin
SUPPLEMENTAL FIGURES
Figure S1, related to Figure 1. Structural Features of GephE Complexes: (A) Crystal
packing of GephE and its various complexes in spacegroup I222. The single molecule present
in the asymmetric unit is colored in blue and its symmetry related molecules in gray. (B)
Enlarged view of the nucleotide recognition loop (red) clearly shows that its conformation is
not influenced by crystal contacts. (C) 2Fo-Fc map, contoured at an rmsd of 1.5, of the
nucleotide binding pocket for the GephE-Mn2+
-ADP bound structure.
Figure S2, related to Figure 2. Comparison of Structures: (A) Superposition of GephE-
apo, and its binary and ternary complexes. (B) Enlarged view of the nucleotide recognition
loop illustrating its conformational heterogeneity and flexibility. (C) Superposition of the
ADP moieties in the binary and ternary complexes indicating the limited flexibility in the
phosphates and associated metal ions.
Figure S3, related to Figure 3. Mo and W Cluster: (A) Anomalous density (green, rmsd of
6) and 2Fo-Fc (blue, rmsd of 1.5) for the tungsten cluster in the GephE-ADP-tungsten ternary
complex. (B) Superposition of the GephE-ADP-Mo complex (yellow) with the W-cluster in
the GephE-ADP-W structure. The apical molybdenum (blue) and tungsten (black) atoms are
shown with their bound oxygen atoms (red). The Arabic numbers 1-7 indicate the conserved
positions in the metal clusters. (C) Electrostatic surface potential of the nucleotide and metal
binding pocket in the GephE-ADP-Mo complex (electropositive in blue and electronegative
in red, contoured at ±5 kT). ADP is represented with sticks and metals as spheres, with the
apical Mo in blue and all other Mo atoms in purple. Energy scans at the Mo-K (D) and W-LIII
(E) edge. (F) ITC titration curves of the molybdate and tungstate titrations to the GephE-ADP
complex. (G)-(J) Two different views of the molybdenum and the tungsten clusters with
inter-metallic distances given in Å. All sites in the molybdenum cluster (G and H) are colored
in purple except for one (blue), which is in close proximity of Ser630. Mo3, Mo4, Mo6 and
Mo7 as well as the apical Mo1 constitute the first plane and the second plane of metals is
defined by Mo2, Mo5, Mo8 and Mo9. All sites in the tungsten cluster (I and J) are colored in
brown except for one (black), which is in close proximity of Ser630. The first plane of metals
is constituted by W2, W5, W6, W7, W9 and W10, while W3, W4 and W8 define the second
plane in the metal cluster. In this case, the apical W is located in between both planes.
Figure S4, related to Figure 4. Comparison of GephE with MoeA: (A) Structure of the
GephE-ADP complex viewed along the twofold axis of symmetry. (B) Enlarged view of the
nucleotide binding pocket illustrating the importance of Gly414 from subdomain II of the
second monomer in nucleotide binding. (C) Superposition of GephE and MoeA based on
subdomain III. (D and E) Enlarged views of the nucleotide and metal binding regions in
GephE and MoeA colored as in (C). Critical residues are represented as sticks.
SUPPLEMENTAL TABLES
Table S1, related to Table 1. Data Collection and Refinement Parameters.
Parameters Structure
GephE-Mn2+-ADP
Beamline ESRF ID 30A-3
) 0.9640
Space group I 222
Unit cell Parameters (a, b, c in ) 87.93, 99.61, 112.32
) 43.96 – 2.0
Rsyma 0.195 ( 1.589 )
Rpimb 0.081 ( 0.67)
CC1/2 0.991 (0.532)
Redundancy 12.9 (12.8 )
Unique reflections 33,205
Completeness 1.0 (1.0)
<I/σI>c 11.6 ( 1.8)
Rd /Rfreee 0.162/ 0.201
Deviation from ideal values in
) 0.009
Bond angles (°) 0.990
Ramachandran statistics (%)f
(Preferred/Allowed/Outliers)
97.6/ 2.4/ 0.0
2) 34.54
Coordinate error (Å)g 0.20
aRsym= ΣhklΣi | Ii - <I> | / ΣhklΣiIi where Ii is the ith measurement and <I> is the weighted mean of all
measurements of I.
bRpim = Σhkl1/ (N-1)
½ Σi| Ii(hkl) – I (hkl) | / ΣhklΣiI( hkl) , where N is the redundancy of the data and
I (hkl ) the average intensity.
c<I/σI> indicates the average of the intensity divided by its standard deviation.
dR = Σhkl ||Fo| - |Fc|| / Σhkl|Fo| where Fo and Fc are the observed and calculated structure factor
amplitudes.
eRfree same as R for 5% of the data randomly omitted from the refinement. The number of reflections
includes the Rfree subset.
fRamachandran statistics were calculated with MolProbity in PHENIX.
gThe coordinate errors were calculated based on maximum likelihood.
Numbers in parentheses refer to the respective highest resolution data shell in each dataset.
Table S2, related to the experimental procedures. Summary of Crystallization Conditions.
Protein Crystallization condition
GephE-apo 0.1 M Sodium acetate pH 4.5,
25% 2-Methyl-2,4-pentanediol
GephE-ADP-MgCl2 0.1 M Sodium acetate pH 4.5,
20% 2-Methyl-2,4-pentanediol
GephE-AMP-MgCl2 0.1 M Sodium acetate pH 4.5,
34% 2-Methyl-2,4-pentanediol
GephE-ADP-Na2WO4 0.1 M Sodium acetate pH 4.5, 0.02 M Calcium
chloride, 30% 2-Methyl-2,4-pentanediol
GephE-ADP-Na2MoO4 0.1 M Sodium acetate pH 4.5, 0.02 M Calcium
chloride, 24% 2-Methyl-2,4-pentanediol
GephE-ADP-MnCl2 0.1 M Sodium acetate pH 4.5,
26% 2-Methyl-2,4-pentanediol