Ford et al. (2010)

Supplementary information

Supplementary Table 1. Data Collection, Phasing and Refinement Statistics

Data Collection / PapC CTD F762M SeMet / PapC CTD WT
Peak / Inflection / Remote / Native
Wavelength (Å) / 0.9788 / 0.9791 / 0.9641 / 1.00
Space group / I4122 / P42212
Cell dimensions (Å) / a=b=133.35 c=285.52 / a=b=100.94 c=89.54
α, β, γ (°) / 90.0, 90.0, 90.0 / 90.0, 90.0, 90.0
Rmeas[a] / 5.1 (53.3) / 5.2 (46.7) / 6.6 (58.4) / 4.5 (26.3)
I/σI / 10.8 (2.3) / 9.7 (2.3) / 8.2 (2.1) / 12.8 (3.5)
Resolution (Å) / 39-2.85 / 39-2.85 / 39-2.85 / 45.1-2.1
Overall completeness (%) / 99.9 (100.0) / 100.0 (100.0) / 100.0 (100.0) / 100.0 (100.0)
Overall multiplicity / 12.6 (9.9) / 12.0 (9.2) / 12.2 (9.4) / 14.4 (14.2)
Anomalous completeness (%) / 99.9 (99.5) / 99.8 (99.2) / 99.8 (99.2)
Anomalous multiplicity / 6.5 (5.0) / 6.2 (4.7) / 6.3 (4.8)
Total reflections (unique) / 383032 (30454) / 366658 (30457) / 372549 (30457) / 396790 (27641)
MAD Phasing Statistics
SHELXD
Sites (occupancy > 0.1) / 24
CCall/CCweak / 57.91/43.23
SHELXE
Contrast / 1.09
Connectivity / 0.81
FOM / 0.63
Pseudo-free CC (%) / 67.0
Refinement Statistics (PapC CTD WT)
Resolution range (Å) / 45.1-2.1
Wilson B factor (Å2) / 24.9
Rwork/Rfree[b,c] / 19.8/22.9 (28.2/33.2)
Total atoms (non-H)[d] / 3343.00
RMSD bond lengths (Å) / 0.003
RMSD bond angles (Degrees) / 0.75
Ramachandran plot (%)[e] / 97.2/2.8/0.0

Values in parentheses are for the highest-resolution shell (2.95-2.85Å for SeMet PapC F732M CTD and 2.21-2.1Å for WT PapC CTD). Data were collected at beamline 4.2.2 at the Advanced Photon Source.

[a] Rmeas = åhkl {N / (N  1)}1/2 å i|Ii (hkl) - I(hkl) | / åhkl åi Ii(hkl), where N is the number of times a reflection has been observed (1).

[b] Rwork = åhklÌW || Fobs | - k | Fcalc || / åhklÌW | Fobs |, where W is the working set.

[c] Rfree = åhklÌT || Fobs | - k | Fcalc || / åhklÌT | Fobs |, where T is the test set obtained by randomly selecting 5% of the data.

[d] Includes 247 water molecules and 2 cobalt ions.

[e] Residues in favored/allowed/outlier regions of the Ramachandran plot, calculated with RAMPAGE (2).


Supplementary Table 2. Structural statistics for the NMR solution structure determination for PapC CTD.

Number of restraints
Interproton NOE-based distances
All / 1309
Intra-residue (i=j) / 596
Sequential (i-j=1) / 239
Short (1 < i-j 5) / 126
Long (i-j > 5) / 348
H-bonds / 76
Dihedral angles / 77
Disulfide bonds / 1
Conformer ensemble / Lowest energy conformer
RMSD to experimental restraints
Inter-proton distances (Å) / 0.064 / ± / 0.005 / 0.061
Dihedral angles (º) / 2.54 / ± / 0.69 / 2.36
Deviation from ideal geometry
Bonds (Å) / 0.0091 / ± / 0.0003 / 0.0090
Angles (º) / 1.08 / ± / 0.05 / 1.04
Dihedrals (º) / 3.45 / ± / 0.21 / 3.06
Experimental restraint violations
NOE > 0.5 Å / 2.6 / ± / 1.7 / 1
Dihedrals > 5º / 4.7 / ± / 1.2 / 3
Precision of the conformer ensemble
Average pairwise RMSD
Backbone atoms (residues 7-82) (Å) / 0.75 / ± / 0.28
Heavy atoms (Å) / 1.39 / ± / 0.21
Procheck Ramachandran statistics (%, by region)
Most favoured / 64.7 / ± / 4.4 / 66.2
Additional / 29.0 / ± / 4.4 / 29.4
Generous / 4.1 / ± / 1.2 / 2.9
Disallowed / 2.2 / ± / 1.4 / 1.5

References

1. Diederichs, K., and P. A. Karplus. 1997. Improved R-factors for diffraction data analysis in macromolecular crystallography. Nature Structural Biology 4:269-275.

2. Lovell, S. C., I. W. Davis, W. B. Arendall, P. I. W. de Bakker, J. M. Word, M. G. Prisant, J. S. Richardson, and D. C. Richardson. 2003. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50:437-450.


Supplementary figure legends

Supp Figure 1. Representative region of the experimental electron density, from SHELXE, around M732 of PapC CTD F732M. The gray mesh represents all experimental density contoured at 1 sigma, while the red mesh represents the anomalous difference Fourier map contoured at 6 sigma.

Supp Figure 2. 2D 15N,1H-heterononuclear single quantum coherence spectrum of PapC-CTD recorded at 600 MHz 1H frequency, 310 K, pH 6.5. Sequence-specific cross-peak assignments are labelled according to a numbering scheme wherein Val725 of the intact PapC polypeptide is denoted residue 1 for the NMR work.


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