Table 1.

Data and refinement statistics

	apo-MntRSeMet
	Anomalous data separated			Merged
	Se peak	Se edge	Se remote	Se peak	apo-MntRnat
Data collection
Space group		P212121			P3121
Unit cell dimensions (Å)		a=55.89, b =65.03, c=186.97			a=b=42.0, c=149.1
Beam line		ALS 8.2.1			ALS 8.3.1
Wavelength (Å)	0.9793	0.9796	0.9641	0.9793	1.1159
Resolution rangea (Å)	64.55–3.00 (3.16–3.00)	64.55–3.00 (3.16–3.00)	65.09–3.30 (3.48–3.30)	64.55–2.80 (2.95–2.80)	74.54–2.80 (2.95–2.80)
Observations	57,416	55,571	29,614	65,335	54,743
Unique reflections	24,633	25,373	9810	16,290	4162
Completeness (%)	92.8 (84.7)	90.2 (81.5)	78.4 (56.8)	93.6 (79.5)	99.9 (99.9)
I/σ(I)	7.8 (3.3)	7.1 (2.7)	7.7 (3.7)	6.3 (2.0)	8.8 (2.0)
Rmerge (%)b	8.2 (22.4)	9.0 (27.0)	8.9 (19.1)	10.6 (38.3)	4.1 (35.0)
Wilson B factor (Å2)				55	104
Phasing
Sites in asuc		5
Resolution range		15.0–3.8
Figure of merit		0.46
Refinement
Resolution (Å)				20.0–2.80	20.0–2.80
Rcryst/Rfree (%)d				21.8/27.6	27.4/34.2
Subunits per asue				4	1
Number of atoms
Protein				4423	1103
Solvent				22	1
Solute				60	0
RMSD bonds (Å)				0.008	0.009
RMSD angles (°)				1.20	1.09
Ramachandrand
Most favored (%)				88.8	88.5
Additionally allowed (%)				10.8	11.5
Generously allowed (%)				0.2	0
Disallowed (%)				0.2	0

Apo-MntR_nat was purified as described.¹⁷ Apo-MntR_SeMet was expressed in Escherichia coli cells grown in the presence of selenomethionine according to standard methods,²⁹ and purified in the same manner as the native protein. Crystals of apo-MntR_SeMet were obtained by the hanging-drop vapor diffusion method. Drops of protein solution (10 mg/ml) were mixed with an equal volume of reservoir solution (15% (w/v) PEG 4000, 0.2 M ammonium sulfate, 5 mM cobalt(II) chloride) and allowed to equilibrate. Crystals of apo-MntR_nat were obtained similarly, except that PEG 400 substituted for PEG 4000. Suitable crystals were transferred to a solution of the mother liquor with 10% (v/v) glycerol added as a cryo-protectant and flash-cooled in liquid nitrogen prior to data collection under a flowing stream of cold nitrogen (100 K). Data were processed using MOSFLM³⁰ and scaled using SCALA from the CCP4 program suite.³¹ The structure of apo-MntR_SeMet was determined using phases obtained by multiwavelength anomalous dispersion as implemented in Solve.³² Phases were extended to 2.8 Å using density modification as implemented in CNS.²³ Model building was performed in O³³ and subsequent refinement was performed using CNS software.²³ The structure of apo-MntR_nat was determined using molecular replacement, using a subunit of the apo-MntR_SeMet structure as a search model. Virtually identical rotation and translation functions were obtained using EPMR²⁴ and CNS. Following rigid body refinement and simulated annealing in CNS, further refinement in Refmac5 from the CCP4 program suite implementing TLS refinement²⁷ permitted model rebuilding using COOT software.³⁴ Despite the presence of Co²⁺ in the crystallization buffers, no evidence of bound cobalt was found in electron density maps or anomalous difference Fourier maps.

^aNumbers in parentheses reflect the highest resolution shell.

^bR_merge = ∑_h∑_j|I_h,j−<I_h>|/∑_j∑_h|I_h,j|, where I_h,j is the jth observation of reflection h.

^cAsymmetric unit.

^dR_cryst = ∑_h||F_o|−|F_c||/∑_h|F_o|, where F_o and F_c are the observed and calculated structure factors for reflection h. R_free is calculated similarly for 10% of the data not used in refinement.

^eDetermined using Procheck software.³⁵