Table 1.

Crystallographic data and refinement statistics

A. Data collection statistics
Crystal form	I	III
Temperature (K)	100	100
X-ray sourcea	Photon Factory	In-house
Space group	C2221	P31
Cell dimensions (Å)
a (Å)	47.0	130.4
b (Å)	178.0	130.4
c (Å)	174.2	129.9
Resolution (Å)	2.2	2.9
No. observations	108,275	148,733
No. unique reflns	32,396	59,700
Multiplicity	3.3	2.5
Data completeness (%)	85.7 (88.0)	97.8 (97.4)
No. data >2σI	73.4 (50.4)	73.4 (38.5)
I/σI	20.7 (4.9)	16.8 (1.8)
Rmerge (%)	5.5 (26.1)	6.1 (57.1)
B. Refinement statistics
Non-hydrogen atoms
Protein	3705	14,628
Water	330	306
Resolution (Å)	2.2	2.9
Rfactor (%)	21.1	23.3
Rfree (%)	26.8	29.9
rms deviations from ideality
Bond lengths (Å)	0.007	0.006
Bond angles (deg.)	1.5	1.1
Impropers (deg.)	0.7	0.6
Dihedrals (deg.)	28.0	26.5
Residues in most favored region of Ramachandran plot (%)	99.8	99.6
Average B-factors (Å2)
Main chain	44.9	68.0
Side-chain	46.4	68.7
Water	47.4	47.7
r.m.s deviation bonded B-factors	2.7	4.3

Notes PFO was over-expressed and purified as described.²⁰ Both crystal forms were grown using the hanging-drop, vapor-diffusion technique at room temperature. The procedure for growing crystal form I was as described.²⁰ Crystal form III grow as long hexagonal rods in 100 mM Mes (pH 5.4) and using 8% (w/v) PEG 8000 as the precipitant and a protein concentration of 10 mg/ml in 10 mM Hepes (pH 7.5), 1 mM DTT. The crystals grow at room temperature and take about a week to reach final size. All crystals were flash-frozen. The diffraction data were processed using DENZO and SCALEPACK.³⁷ The structures were refined using similar protocols, with either X-PLOR³⁸ or CNS.³⁹ Tightly-restrained individual B-factor refinement was used and bulk solvent corrections were applied to all data sets. In cases where there was more than one molecule in the asymmetric unit, non-crystallographic symmetry restraints were applied. The structure of PFO was determined originally from crystal form I using 2.7 Å resolution X-ray data collected at room temperature. A high-resolution data set was collected on the 6A2 beamline of the Photon Factory (Tsukuba, Japan) from a flash-frozen crystal. To avoid unacceptably high mosaicity, the crystals had to be transferred from 5% to 20% (v/v) MPD, with a soak for 30 minutes for each 5% increment. The crystals were indexed in space group C222₁ with unit cell dimensions a=47.0 Å, b=178.0 Å and c=174.2 Å. Notably, the b cell edge had shrunk by 4 Å compared to the previous room temperature data set.²⁰ The starting model for the refinement was the medium-resolution structure after omitting water molecules. Rigid body refinement was used initially, followed by a number of iterative rounds of simulated annealing refinement and model building. The final model includes all residues (30 to 500; the first 20 residues constitute the signal peptide that was cleaved from the construct) and 330 water molecules. Although the first nine residues have very high B-factors (>80 Ų), there was clear electron density for them and their inclusion lowered the R_free value. All residues lie within the allowed regions of the Ramachandran plot with only one residue in the generously allowed region.⁴⁰ This residue, Ile31, is located at the mobile N terminus and hence its placement is uncertain. The high-resolution structure superposes closely with the published structure with an overall r.m.s. deviation of 0.4 Å for all C^α atoms. The major outliers (>1.5 Å) fall between residues 56 and 61. This is a region of relatively poor electron density that reflects local disorder within the structure. The model includes seven residues at the N terminus that were not observed in the 2.7 Å resolution published structure.²

Crystal form III was initially assigned to space group P6₂ with unit cell dimensions a=b=130.4 Å, c=129.9 Å. The crystals diffracted very strongly to the water ring, but did not diffract much beyond this limit, suggesting a degree of disorder within the crystals. Subsequently, it was realized that the crystals were heavily twinned merohedrally with a twinning fraction greater than 0.4.⁴¹ The data were subsequently reprocessed in space group P3₁ and detwinned using CCP4 programs.⁴² The new space group was consistent with up to four molecules in the asymmetric unit. Molecular replacement attempts using the high-resolution model from crystal form I failed. However, a search model comprising only domains 1, 2 and 3 yielded two convincing solutions in the translation function, although superposing the intact molecule resulted in clashes involving domain 4. Domain 4 was then used as a search probe, after fixing the positions of the other domains based on the initial molecular replacement solution. Given the low signal expected from using such a search probe, normalized structure factors were used. Two rotation function peaks, listed 27th and 28th in the peak list, gave peaks in the translation function that were significantly above the noise level (correlation coefficients of 0.28 and 0.23, respectively, against a noise level of 0.19). Rigid body fitting of the four solutions resulted in a correlation coefficient of 0.39, and an R_factor of 41%. Surprisingly, the two domain 4 solutions did not relate to the two identified domain 1–3 solutions, and hence represented two independent PFO molecules within the asymmetric unit. Thus, it seemed there were four partial models within the asymmetric unit, two encompassing domains 1–3 only (molecules A and B) and two comprising domain 4 only (molecules C and D). Map calculations using the rigid-body refined partial model revealed unbiased density for domain 4 for molecules A and B. Because of the high twin fraction of the data set being used, further data sets were collected from a number of crystals in an effort to obtain one where there was less of a problem. From about 30 crystals, a complete data set from one crystal was collected where the twin fraction was negligible (0.02). The structure was solved and refined using this data set. The unbiased density for domain 4 from molecules A and B was clearly visible in this data set also. Subsequent maps revealed density that enabled fitting of domains 1–3 of molecules C and D. During energy minimization, the four independent molecules were refined with NCS restraints over individual domains, (excluding domain 2). However, no NCS restraint was used during temperature factor refinement, as the electron density suggested the same domain in each molecule exhibited very different temperature factors.

The values in parentheses are for the highest resolution bin (approximate interval of 0.1 Å). R_merge =∑hkl∑i∣Ii–<I>∣∣<I>∣, where Ii is the intensity for the ith measurement of an equivalent reflection with indices h,k,l.

^aPhoton Factory is the Weissenberg camera at beamline 6A2, Photon Factory, Tsukuba, Japan. In-house is an MARResearch detector (Hamburg, Germany) on an in-house Rikagu RU-200 rotating anode X-ray generator.