Supplemental Data Insights into the Catalytic Mechanism of PPM Ser ...

Structure 15

Supplemental Data Insights into the Catalytic Mechanism of PPM Ser/Thr Phosphatases from the Atomic Resolution Structures of a Mycobacterial Enzyme Marco Bellinzoni, Annemarie Wehenkel, William Shepard, and Pedro M. Alzari

Determination of the nature of ligands in the active site To ascertain the presence of manganese in the active site of MspP, two complete datasets were collected for each crystal form (MspP-phosphate complex, space group P21212; and MspP-cacodylate complex, space group P212121) at the ESRF beamline ID23-1 using Xray wavelengths of 1.9074 Å and 1.8785 Å, corresponding to the Mn K-edge low-remote energy (6500 eV) and high remote energy (6600 eV), respectively. Similarly, to confirm the identification of the bound cacodylate ion, two complete datasets were collected at energies corresponding to the As K-edge peak (E = 11875 eV, λ = 1.0440 Å), determined with a fluorescence scan, and to the nearest low-remote region (E = 11840 eV, λ = 1.0472 Å). In all cases, data processing and reduction were carried out with the programs XDS and XSCALE1 and the statistics are reported in Table S1. Control datasets were also collected from the same crystals at the X-ray wavelength of 0.9537 Å (E = 13000 eV; data not shown) and used to calculate the phases from both models by 20 cycles of refinement with either REFMAC52 (for the cacodylate complex) or SHELXL3 (for the phosphate complex). Model phases were then used to produce double difference anomalous maps (Dano(hr/pk) – Dano(lr)) with the programs SFTOOLS and FFT from the CCP4 suite4 (Fig. S1). The presence of a manganese ion and a bound sulfate in the MspP-sulfate complex was also confirmed with an anomalous map

calculated from the anomalous differences measured in the dataset used for refinement, which was collected at λ=0.939 Å (see Table 2 in the text; Fig. S1, C).

1. Kabsch, W. (1993). Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795-800. 2. Murshudov, G.N., Vagin, A.A., Lebedev, A., Wilson, K.S., and Dodson, E.J. (1999). Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D Biol. Crystallogr. 55, 247-255. 3. Sheldrick, G.M., and Schneider, T.R. (1997). SHELXL: high resolution refinement. Meth. Enzymol. 277, 319-343. 4. CCP4 (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760-763.

Table S1. Anomalous Diffraction Statistics MspP - cacodylate Complex MspP - phosphate (space group P212121) (space group P21212) Mn K-edge Mn K-edge Mn K-edge Mn K-edge As K-edge As K-edge Edge low remote high remote low remote high remote low remote peak Energy (eV) 6500.0 6600.0 6500.0 6600.0 11840.0 11875.4 Wavelength (Å) 1.907 1.879 1.907 1.879 1.047 1.044 Resolution range (Å) a,b 42.0-2.1 (2.2-2.1) 42.0-2.1 (2.2-2.1) 42.0-2.7 (2.8-2.7) 42.0-2.7 (2.8-2.7) 66.2-1.8 (1.9-1.8) 66.1-1.7 (1.8-1.7) Unique reflections 22841 22892 14578 14090 25741 30314 a 27.1 (16.4) 29.1 (19.6) 10.9 (3.7) 11.4 (3.6) 11.7 (2.1) 13.4 (2.7) I/σ(I) Multiplicity a 3.8 (3.7) 3.8 (3.8) 1.5 (1.2) 1.6 (1.4) 3.5 (3.5) 3.5 (3.5) Completeness (%) a 94.0 (90.2) 94.2 (90.9) 98.2 (93.6) 98.4 (98.3) 99.7 (99.8) 99.8 (99.9) Rmerge c 0.034 (0.067) 0.031 (0.055) 0.052 (0.192) 0.051 (0.218) 0.075 (0.568) 0.073 (0.413) All data were collected at the ESRF beamline ID23-1. a Numbers in parentheses correspond to the highest resolution shell. b Collection of diffraction data beyond 2.1 Å resolution was impaired at long wavelengths (Mn K-edge) by the beamline configuration. c Rmerge = ∑j |Îh – Ih,j|}/∑h,j I h,j where Îh = (∑jIh,j)/nh and nh is the multiplicity of reflection h.

Figure S1. Identification of the Ligands Bound to MspP by Analysis of the Anomalous Scatterers in the Catalytic Center (a) MspP-cacodylate complex. The double difference anomalous map calculated with diffraction data above (6600 eV) and below (6500 eV) the Mn K- edge is shown in orange and contoured at 8σ. Similarly, the double difference anomalous map calculated from data at the As K-edge (11875 eV) and below the edge (11840 eV) is shown in gray-cyan and contoured at 8σ. (b) MspP-phosphate complex. The double difference anomalous map for Mn, calculated as above and countoured at 8σ, is shown in orange. (c) MspP-sulfate complex. Anomalous density calculated at 13200 eV (from data used in refinement) is shown in green and countured at 4σ.