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