Table 1.

Statistics for MAD data and phase determination from a selenomethionyl crystal. The substrate-binding unit of DnaK (389 to 607) was expressed in E. coli (29). A hexahistidine tag followed by a factor Xa cleavage site was introduced at the NH₂-terminus of the protein for the purpose of purification. The protein was chromatographed on a Ni-NTA-agarose column and digested with factor Xa. The cleaved protein was then chromatographed on Superdex-200. Crystals were grown from 2.4 M ammonium sulfate and phosphate buffer at pH 7.0 by either the hanging drop or the dialysis method. Most of the crystals are body-centered orthorhombic Se K edge (see Table 2). All data collection was done at the X4A beamline at Brookhaven National Laboratory, using image plates. The crystals were frozen at 100 K with xylitol as a cryoprotectant. The MAD data were collected at four different wavelengths (which were before the Se K edge, at the edge, at the peak, and after the peak, respectively) with 1^° oscillation range without overlap. The selenomethionyl crystal was oriented such that the a axis of the crystal was parallel to the oscillation axis. The data were processed with DENZO (53) and SCALEPACK (53). The MADSYS program package (27) was used to evaluate phase differences and amplitudes for the selenium scattering component of the structure factors. Five out of the six selenium atoms were located with the program HASSP (53), and the last site was found by difference Fourier analysis.

Wavelength (λ) (Å)			Reflections		Complete (%)	(l/σ)		Rsym* (%)
			Total	Unique
			Data collection (20 to 2.0 Å)
0.9873 (remote 1)			59,575	24,415	94.9	19.5		5.2
0.9793 (edge)			59,962	24,662	95.6	18.6		5.2
0.9792 (peak)			58,583	24,560	93.9	18.6		5.1
0.9641 (remote 2)			60,800	25,014	96.7	17.0		5.3
			Anomalous diffraction ratios†
	20.0 < d <3.0 Å				3.0 < d <2.3 Å
	X1	λ2	X3	λ4	λ1	λ2	λ3	λ4
X1	.033 (.033)	.051	.041	.029	.059 (.051)	.067	.058	.050
X2		.063 (.033)	.026	.057		.090 (.057)	.048	.074
X3			.084 (.037)	.045			.109 (.056)	.063
X4				.058 (.031)				.082 (.053)
			MAD phasing statistics‡
	R(°|Ft|) =0.051		R(°|FA|) = 0.356		<Δ(Δϕ)> = 36.5°		<σ(Δϕ)> = 17.2°

^*R_sym = 100 × Σ_hkl Σ_i |l_i − <l>|/Σ_hkl Σ_il_i, where I_i is the ith measurement and <l> is the weighted mean of all measurements of l. Unique reflections distinguish Bijvöet mates.

^†Anomalous diffraction ratios = <Δ|F|²>^1/2/<|F]²>^1/2. where Δ/F| is the absolute value of the Bijvöet (diagonal elements) or dispersive difference (off-diagonal elements), respectively. Values in parentheses are for centric data.

^‡R = Σ_hkl Σ_i ||F_i| − <F> |/Σ|F|.

^°F_T is the structure factor due to normal scattering from all the atoms; °F_A is the structure factor due to normal scattering from the anomalous scatterers only, and Δϕ is the phase difference between °F_T and °F_A. Δ(Δϕ) is the difference between two independent determinations of Δϕ.