Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme in Neisseria meningitidis

The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochrome c maturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogen Neisseria meningitidis. To help understand the structural and functional aspects of N. meningitidis DsbD, the two periplasmic domains which are required for electron transfer are being studied. Here, the expression, purification and biophysical properties of n-NmDsbD and c-NmDsbD are described. The crystallization and crystallo­graphic analysis of n-NmDsbD and c-NmDsbD are also described in both redox states, which differ only in the presence or absence of a disulfide bond but which crystallized in completely different conditions. Crystals of n-NmDsbD_Ox, n-NmDsbD_Red, c-NmDsbD_Ox and c-NmDsbD_Red diffracted to 2.3, 1.6, 2.3 and 1.7 Å resolution and belonged to space groups P2₁3, P321, P4₁ and P12₁1, respectively.

1. Introduction

In bacteria, disulfide-bond formation occurs in the periplasm and is mediated by the disulfide-bond (Dsb) family of proteins (Heras et al., 2007, 2009; Kadokura & Beckwith, 2010). In most Gammaproteobacteria, Dsb proteins form a two-pathway system. In the oxidation pathway, DsbA and its cognate oxidase DsbB introduce disulfide bonds into newly translocated proteins that are undergoing oxidative folding in the periplasm (Kamitani et al., 1992; Bardwell et al., 1993; Missiakas et al., 1993; Depuydt et al., 2011). The isomerase pathway is composed of the isomerase DsbC and the reductase DsbD, which are responsible for reshuffling non-native disulfide bonds formed by the DsbA/DsbB system (Cho & Collet, 2013; Missiakas et al., 1994, 1995; Rietsch et al., 1996).

DsbD is a 59 kDa integral membrane reductase that transfers electrons from the cytoplasm to periplasmic substrate proteins (Arts et al., 2015; Cho & Collet, 2013; Rietsch et al., 1997). Escherichia coli DsbD (EcDsbD) consists of a membrane-spanning domain, t-DsbD, flanked by two periplasmic domains, n-DsbD and c-DsbD (Katzen et al., 2002; Cho et al., 2007). The N- and C-terminal domains of DsbD adopt an immunoglobulin-like and a thioredoxin-like fold, respectively (Rozhkova et al., 2004). t-DsbD is membrane-embedded and consists of eight α-helices (Cho & Beckwith, 2009). Each DsbD module harbours two catalytic cysteines, which allow efficient electron flow through the three DsbD domains via a sequential cascade of thiol–disulfide exchange reactions (Stewart et al., 1999; Katzen & Beckwith, 2000). This process begins with oxidized t-DsbD, which accepts electrons from reduced cytoplasmic thioredoxin, leaving t-DsbD reduced and thioredoxin oxidized (Stewart et al., 1999). Reduced t-DsbD undergoes a conformational change which allows interaction with and reduction of c-DsbD (Cho & Collet, 2013). The latter domain reduces n-DsbD, which then reduces disulfides in its periplasmic substrates (Arts et al., 2015) such as DsbC, CcmG and DsbG (Stirnimann et al., 2005; Depuydt et al., 2009).

Dsb proteins promote multiple virulence phenotypes and regulate the redox balance in the cell envelope, but in general these proteins are not required for survival. However, this is not the case in Neisseria meningitidis, where DsbD (NmDsbD) was found to be essential for the viability of this obligate human pathogen (Kumar et al., 2011). We are interested in investigating the structural and functional characteristics of NmDsbD in order to understand the molecular basis of this uncommon phenotype. Furthermore, this information could form the basis for the future development of antineisserial agents targeting DsbD (Smith et al., 2016). Here, we report the expression, biophysical characterization, crystallization and preliminary X-ray diffraction data for the two periplasmic domains of NmDsbD in both redox states.

2. Materials and methods

2.1. Macromolecular production

For the expression of the N- and C-terminal domains of NmDsbD (residues 1–124 and 465–579, respectively, of the mature protein), the corresponding coding regions were PCR-amplified from N. meningitidis strain NMB genomic DNA. Ligation-independent cloning (LIC) was then used to insert the sequences into bacterial expression vector pMCSG7 (Eschenfeldt et al., 2009). The final constructs contained an N-terminal His₆ tag that preceded a Tobacco etch virus (TEV) protease cleavage site. This tag is used in the purification of the proteins and is cleaved during the process, leaving two additional residues (SN) on n-NmDsbD and three additional residues (SNA) on c-NmDsbD (Table 1).

Table 1
Macromolecule-production information

	n-NmDsbD	c-NmDsbD
Source organism	N. meningitidis strain NMB	N. meningitidis strain NMB
DNA source	N. meningitidis strain NMB	N. meningitidis strain NMB
Forward primer (5′–3′)	TACTTCCAATCCAATGCGAACGATCTGCTGCCGC	TACTTCCAATCCAATGCGATGTTTGCCGATACTGCCGCGC
Reverse primer (5′–3′)	TTATCCACTTCCAATGTCAGGTTTGCGGATGGTAAGTGC	TTATCCACTTCCAATGTCAGCGGTTTTGTTCATACCACTCG
Cloning vector	pMCSG7	pMCSG7
Expression vector	pMCSG7	pMCSG7
Expression host	E. coli BL21 (DE3) pLysS	E. coli BL21 (DE3) pLysS
Complete amino-acid sequence of the construct produced	MHHHHHHSSGVDLGTENLYFQSNANDLLPPEKAFVPELAVADDGVNVRFRIADGYYMYQAKIVGKTDPADLLGQPSFSKGEEKEDEFFGRQTVYHHEAQVAFPYAKAVGEPYKLVLTYQGCAEVGVCYPPVDTEFDISGNGTYHPQT	MHHHHHHSSGVDLGTENLYFQSNAMFADTAALKAAMDTALKEHPDKPVVLDFYADWCISCKEMAAYTLNQPEVHQAVDMERFFQIDVTANKPEHQALLKEYGLFGPPGVFVVRSDGSRSEPLLGFVKADKFIEWYEQN

Plasmid constructs pMCSG7::n-NmdsbD and pMCSG7::c-NmdsbD were transformed into E. coli BL21 (DE3) pLysS cells. n-NmDsbD and c-NmDsbD were expressed using the autoinduction method (Studier, 2005). Briefly, E. coli BL21 (DE3) pLysS cells harbouring n-NmDsbD- and c-NmDsbD-containing plasmids were grown at 303 K for 24 h with agitation at 180 rev min⁻¹ in rich (ZY) medium supplemented with 100 µg ml⁻¹ ampicillin and 34 µg ml⁻¹ chloramphenicol (typically to a final optical density at 600 nm in the range between 4 and 5). Bacterial cultures were harvested by centrifugation (7500g, 20 min, 277 K) and flash-frozen before storage at 183 K.

For purification, harvested pellets were resuspended in 25 mM Tris pH 8.0, 150 mM NaCl supplemented with EDTA-free protease-inhibitor cocktail tablets and 400 units of DNase I per litre of culture. Cells were lysed using a bench-top cell disruptor (one cycle at 241 MPa; TS series, Constant Systems Ltd). Cellular debris was removed from the lysate via centrifugation (30 000g, 20 min, 277 K) and the lysate was loaded onto a 5 ml HisTrap FF column (GE Healthcare). Proteins were eluted from the column in 25 mM Tris pH 8.0, 150 mM NaCl and a gradient of 0–500 mM imidazole using a Bio-Rad NGC system. Recombinant His-tagged TEV protease (Cabrita et al., 2007) was added to fractions containing His₆-n-NmDsbD and His₆-c-NmDsbD (0.2 and 0.4 mg TEV protease per 10 mg of protein, respectively) supplemented with 5 mM reduced dithiothreitol (DTT_Red) and samples were dialyzed overnight against 25 mM Tris pH 8.0, 150 mM NaCl. The samples were further purified using a 5 ml HisTrap FF column followed by size-exclusion chromatography (SEC) using a Superdex S-75 16/60 column (GE Healthcare) pre-equilibrated in 25 mM HEPES pH 6.7, 150 mM NaCl. The target proteins were oxidized using 1.7 mM copper phenanthro­lene or reduced using 40 molar equivalents of reduced DTT and incubated for 1 h at 277 K. Oxidized and reduced proteins were buffer-exchanged into 25 mM HEPES pH 6.7, 50 mM NaCl and the same buffer supplemented with 1 mM EDTA using a 10 ml PD10 column (GE Healthcare) prior to crystallization experiments. The redox state of the NmDsbD domains was confirmed by Ellman's assay (Evans & Ellman, 1959).

2.2. Small-angle X-ray scattering (SAXS) studies

SAXS data for n-NmDsbD and c-NmDsbD were collected on the SAXS-WAXS beamline at the Australian Synchrotron (Kirby et al., 2013; Table 2). Serial dilutions of an ∼5 mg ml⁻¹ stock were loaded into a 96-well plate. The estimated molecular mass was calculated using contrast and partial specific volumes determined from the protein sequences (Whitten et al., 2008). The pair-distance distribution function [p(r)] was generated from the experimental data using GNOM (Svergun, 1992), from which I(0), R_g and D_max were determined (Figs. 1a and 1b). The program DAMMIN (Svergun, 1999) was used to generate 16 molecular envelopes for each protein, which were averaged using the program DAMAVER (Volkov & Svergun, 2003), and the resolutions of the averaged structures were estimated using SASRES (Tuukkanen et al., 2016). The experimental scattering data were compared with scattering curves calculated from the crystal structures of the corresponding domains from E. coli [n-EcDsbD, PDB entry 1l6p (Goulding et al., 2002), and c-EcDsbD, PDB entry 1uc7 (Kim et al., 2003)] using CRYSOL (Svergun et al., 1995) (Fig. 1).

Table 2
SAXS data-collection and analysis details

	n-NmDsbD	c-NmDsbD
Data-collection parameters
Instrument	SAXS-WAXS (Australian Synchrotron)
Beam geometry	Point
Wavelength (Å)	1.127
Sample-to-detector distance (m)	1.480
q-range (Å^−1)	0.01–0.60
Exposure time (s)	60 (30 × 2 s exposures)
Protein concentration range† (mg ml^−1)	∼0.3–5.0
Temperature (K)	285
Standard	Water
Structural parameters
I(0) (cm^−1) (from Guinier)	0.00776 ± 0.00003	0.00997 ± 0.00002
Rg (Å) (from Guinier)	17.6 ± 0.2	15.3 ± 0.1
I(0) (cm^−1) [from p(r)]	0.007688 ± 0.000012	0.009905 ± 0.000011
Rg (Å) [from p(r)]	17.3 ± 0.1	14.8 ± 0.1
Dmax (Å)	57 ± 3	45 ± 3
Molecular-mass determination
Protein concentration (mg ml^−1)	0.74	1.12
Partial specific volume (cm^3 g^−1)	0.73	0.73
Contrast, Δρ (10^10 cm^−2)	2.91	2.97
Molecular mass Mr [from I(0)]	13200 ± 1000	11800 ± 1000
Molecular mass Mr (expected)	13911	13358
Software employed
Primary data reduction	scatterBrain (v.1.0)
Data processing	PRIMUS (v.3.2) and GNOM (v.4.6)
Ab initio modelling	DAMMIN (v.5.3)
Validation and averaging	DAMAVER (v.2.8.0)
Computation of model intensities	CRYSOL (v.2.8.3)

†The R_g for n-NmDsbD showed mild concentration dependence above a concentration of 1.0 mg ml⁻¹, decreasing from ∼17.5 Å at 0.37 mg ml⁻¹ to ∼16 Å at 5.88 mg ml⁻¹. The 0.74 mg ml⁻¹ data set was used for further analysis and modelling as it was deemed to be the highest concentration at which the effects of concentration were negligible. The change in R_g for c-NmDsbD showed no obvious systematic trend in the concentration range measured. A concentration of 1.12 mg ml⁻¹ was chosen for further analysis and modelling.

Figure 1 Small-angle X-ray scattering from n-NmDsbD and c-NmDsbD. (a) Measured scattering data for n-NmDsbD (red) and c-NmDsbD (blue; scaled by a factor of 0.1 for clarity). Overlaid are the calculated scattering curves for n-EcDsbD (PDB entry 1l6p; black curve, χ^2 = 2.38) and c-EcDsbD (PDB entry 1uc7; yellow curve, χ^2 = 1.65). Inset: Guinier plots of the scattering data. (b) The pair-distance distribution functions for n-NmDsbD (red) and c-­NmDsbD (blue). (c) Probable domain shapes for n-NmDsbD (SASBDB entry SASDCH7; red envelope, resolution = 19 ± 2 Å) and c-NmDsbD (SASBDB entry SASDCJ7; blue envelope, resolution = 18 ± 2 Å) obtained from ab intio modelling against the scattering data. The crystal structures of n-­EcDsbD (PDB entry 1l6p) and c-EcDsbD (PDB entry 1uc7) are shown as cartoons. The grey envelopes represent the total volume encompassed by the 16 aligned models for n-NmDsbD (χ^2 = 1.90 ± 0.01) and c-NmDsbD (χ^2 = 1.60 ± 0.01).

2.3. Crystallization

Initial high-throughput crystallization experiments were performed at 293 K either in-house using a Mosquito crystallization robot (TTP Labtech) or a Crystal Gryphon Liquid Handling System (Art Robbins Instruments) or at the CSIRO Collaborative Crystallisation Centre (https://www.csiro.au/C3), Melbourne, Australia. For n-NmDsbD_Ox, c-NmDsbD_Ox, n-NmDsbD_Red and c-NmDsbD_Red, 96-well hanging-drop plates were prepared using 71, 44, 60 and 20–40 mg ml⁻¹ protein solutions, respectively, and commercially available crystallization screens (Crystal Screen, Crystal Screen 2, PEG/Ion, PEG/Ion 2, Index and Salt RX from Hampton Research, PACT premier and JCSG-plus from Molecular Dimensions Ltd and Precipitant Synergy from Jena Bioscience) and an in-house malonate grid screen (pH 5–7 and 0.2–3.4 M malonate).

Initial crystallization experiments for the reduced proteins (n-NmDsbD_Red and c-NmDsbD_Red) were performed using a Crystal Gryphon Liquid Handling System (Art Robbins Instruments). Crystallization experiments for n-NmDsbD_Red were set up with 60 mg ml⁻¹ protein solution, whereas experiments for c-NmDsbD_Red were set up with both 20 and 40 mg ml⁻¹ protein solutions. 96-well sitting-drop plates (200 nl protein solution and 200 nl reservoir solution equilibrated against 70 µl reservoir solution) were prepared using commercially available crystallization screens (PEG/Ion 1 and 2, Salt RX, Index, Crystal Screen and Crystal Screen 2 from Hampton Research and PACT premier, JCSG-plus and Morpheus from Molecular Dimensions Ltd).

All crystal-optimization experiments were carried out at 293 K in 24-well hanging-drop VDXm plates using 18 mm siliconized cover slips (Hampton Research), a reservoir volume of 500 µl and a drop size of 2 µl (1 µl protein and 1 µl reservoir solution).

Crystals of n-NmDsbD_Ox only grew in 20%(w/v) PEG 6000, 0.01 M zinc chloride, 0.1 M MES pH 6 (PACT premier). This condition was optimized by varying different components and diffraction-quality single hexagonal crystals (Figs. 2a and 2c) grew overnight using protein at 66 mg ml⁻¹ in 18–20%(w/v) PEG 6000, 0.1 M MES pH 6.4, 0.02 M ZnCl₂.

Figure 2 Crystals and diffraction patterns for n-NmDsbD (this domain shares 30% sequence identity with n-EcDsbD; PDB entry 1l6p). (a) n-NmDsbDOx crystals with approximate dimensions of 0.30 × 0.25 × 0.40 mm. (b) n-NmDsbDRed crystals with approximate dimensions of 0.1 × 0.05 × 0.1 mm. Diffraction images are shown for (c) n-NmDsbDOx and (d) n-NmDsbDRed crystals.

Initial screening for n-NmDsbD_Red produced microcrystals in 2.5 M ammonium sulfate, 0.1 M Tris pH 8.5 (condition 1; from Salt RX), 0.1 M HEPES pH 7.5, 1.4 M sodium citrate tribasic dehydrate (condition 2; from Index), 5% Tacsimate pH 7.0, 0.1 M HEPES pH 7.0, 10%(w/v) PEG MME 5000 (condition 3; from Index), 0.1 M ammonium citrate tribasic pH 7.0, 12%(w/v) PEG 3350 (condition 4; from Index), 20%(w/v) PEG 3350, 0.2 M potassium nitrate (condition 5; from JCSG) and 0.1 M bis-tris propane pH 7.5, 20%(w/v) PEG 3350, 0.2 M sodium nitrate (condition 6; from PACT premier).

Optimization screens were prepared using protein at 30 and 60 mg ml⁻¹, and the best initial hits and small hexagonal diffraction-quality crystals (Figs. 2b and 2d) grew amongst salt crystals within 2 d in 2.5 M ammonium sulfate, 0.1 M Tris pH 9.1 with a protein concentration of 60 mg ml⁻¹.

Initial screening of c-NmDsbD_Ox yielded crystals in only one condition consisting of 5%(w/v) PEG 400, 2 M sodium citrate/citric acid pH 7.5 (Precipitant Synergy), and diffraction-quality crystals were obtained using protein at 42–38 mg ml⁻¹ in 5%(w/v) PEG 400, 1.7–2.2 M citrate/citric acid pH 7.0–7.8. These thin spear-shaped crystals grew under a skin after between 4 and 8 d (Figs. 3a and 3c).

Figure 3 Crystals and diffraction patterns for c-NmDsbD (this domain shares 38% sequence identity with c-EcDsbD; PDB entry 2fwe). (a) c-NmDsbDOx crystals with approximate dimensions of 0.10 × 0.05 × 0.50 mm. (b) c-NmDsbDRed crystal cluster. Diffraction images are shown for (c) c-NmDsbDOx and (d) c-­NmDsbDRed crystals.

Initial screening of c-NmDsbD_Red yielded crystals in the following conditions: 0.2 M zinc acetate dehydrate, 0.1 M sodium cacodylate trihydrate pH 6.5, 18%(w/v) PEG 8000 (condition 1), 2.4 M sodium malonate pH 7 (condition 2), 0.1 M Tris–HCl pH 8.5, 2 M ammonium sulfate (condition 3; from Crystal Screen), 2 M ammonium sulfate (condition 4; from Crystal Screen) and 2.1 M DL-malic acid (condition 5; from JCSG-plus). Diffraction-quality crystals of c-NmDsbD_Red were obtained using protein at 30 mg ml⁻¹ in 0.2 M zinc acetate dihydrate, 0.1 M sodium cacodylate tri­hydrate pH 7.5, 18%(w/v) PEG 8000. Clustered crystals grew in 3 d, or overnight with microseeding, and were subsequently manipulated to isolate single crystals for diffraction data collection (Figs. 3b and 3d).

All final crystallization conditions for the crystals used for data collection are described in Table 3.

Table 3
Crystallization

	n-NmDsbDOx	n-NmDsbDRed	c-NmDsbDOx	c-NmDsbDRed
Method	Hanging drop	Hanging drop	Hanging drop	Hanging drop
Plate type	VDXm plates	VDXm plates	VDXm plates	VDXm plates
Temperature (K)	291	291	291	291
Protein concentration (mg ml^−1)	66	61	40	30
Buffer composition of protein solution	0.025 M HEPES, 0.05 M NaCl pH 6.7	0.025 M HEPES, 0.05 M NaCl pH 6.7	0.025 M HEPES, 0.05 M NaCl pH 6.7	0.025 M HEPES, 0.05 M NaCl pH 6.7
Composition of reservoir solution	18–20%(w/v) PEG 6000, 0.1 M MES pH 6.4, 0.020 M ZnCl2	2.5 M ammonium sulfate, 0.1 M Tris pH 9.1	5%(w/v) PEG 400, 1.7–2.2 M citrate/citric acid pH 7.0–7.8	0.2 M zinc acetate dehydrate, 0.1 M sodium cacodylate trihydrate pH 7.5, 18%(w/v) PEG 8000
Volume and ratio of drop	2 µl, 1:1	2 µl, 1:1	2 µl, 1:1	2 µl, 1:1
Volume of reservoir (µl)	500	500	500	500

2.4. X-ray diffraction data measurements

For diffraction data measurements, n-NmDsbD_Ox, c-NmDsbD_Ox and n-NmDsbD_Red crystals were cryoprotected by soaking them for 2 min in mother liquor supplemented with 20% glycerol, PEG 400 and glycerol, respectively. c-NmDsbD_Red crystals were cryocooled in reservoir solution. X-ray diffraction data were collected at 100 K on the microfocus beamline (MX2) at the Australian Synchrotron using an ADSC Quantum 315r detector. Diffraction data were collected over a total angular rotation of 180° for both the n-NmDsbD_Ox and n-NmDsbD_Red crystals, with an oscillation angle of 1° and an exposure time of 1 s. A total of 180° of diffraction images were also collected for the c-NmDsbD_Ox crystal, with an oscillation angle of 0.5° and an exposure time of 1 s. For the c-NmDsbD_Red crystal the oscillation range per image was 1° over a total angular rotation of 200°, with an exposure time of 0.5 s. Diffraction data were indexed, integrated and scaled using HKL-2000 (Otwinowski & Minor, 1997) for the n-NmDsbD_Ox and c-NmDsbD_Ox data sets, and using iMosflm (Battye et al., 2011) and AIMLESS from the CCP4 suite (Winn et al., 2011) for the n-NmDsbD_Red and c-NmDsbD_Red data sets.

3. Results and discussion

The two periplasmic domains of the thiol-disulfide reductase DsbD from N. meningitidis, n-NmDsbD and c-NmDsbD, were recombinantly expressed in E. coli and purified to homogeneity using three chromatographic steps (affinity, reverse affinity and size-exclusion chromatography) for crystallization trials.

We first analyzed n-NmDsbD and c-NmDsbD using SAXS not only to determine the quality of the recombinant proteins in solution but also to obtain low-resolution information on the shape and conformation of these domains (Fig. 1). The SAXS data displayed a low experimental noise level, with a linear Guinier plot consistent with a homogeneous, monodisperse solution of both NmDsbD domains. The molecular weights of n-NmDsbD and c-NmDsbD estimated from extrapolation to I(0) were 13.2 and 11.9 kDa, respectively. Ab initio modelling was employed to determine the low-resolution structure of both domains. 16 low-resolution models were generated and subsequently aligned, averaged and filtered to produce the probable shape of the protein (Fig. 1c). The agreement between the crystal structures of the n-EcDsbD and c-EcDsbD domains and the red and blue envelopes provides convincing evidence that the n-NmDsbD and c-NmDsbD domains adopt similar three-dimensional structures in solution to their E. coli counterparts.

Despite the only difference between n-NmDsbD_Ox and n-NmDsbD_Red or c-NmDsbD_Ox and c-NmDsbD_Red being the presence or absence of a disulfide bond, the domains crystallized in different conditions in their two redox forms. Single crystals of n-NmDsbD_Ox grew in 18–20%(w/v) PEG 6000, 100 mM MES pH 6.4, 20 mM ZnCl₂. n-NmDsbD_Red crystals were obtained in 2.5 M ammonium sulfate, 0.1 M Tris pH 9.1. c-NmDsbD_Ox crystallized in 5%(w/v) PEG 400, 1.7–2.2 M citrate/citric acid pH 7.0–7.8 and crystals of c-NmDsbD_Red grew in 0.2 M zinc acetate dehydrate, 0.1 M sodium cacodylate trihydrate pH 7.5, 18%(w/v) PEG 8000 (Figs. 2 and 3). The n-NmDsbD_Ox, n-NmDsbD_red, c-NmDsbD_Ox and c-NmDsbD_Red crystals diffracted to resolutions of 2.3, 1.6, 2.3 and 1.7 Å and belonged to space groups P2₁3, P321, P4₁ and P12₁1, respectively. Assuming that the n-NmDsbD_Ox, n-NmDsbD_Red, c-NmDsbD_Ox and c-NmDsbD_Red crystals contain six, two, two and one molecules per asymmetric unit, respectively, their respective Matthews coefficients (V_M) are 3.19, 3.29, 2.29 and 2.09 ų Da⁻¹ and their corresponding solvent contents are 61.50, 62.65, 46.37 and 41.17%, respectively (Matthews, 1968). Diffraction data statistics are shown in Table 4. Further studies are in progress to elucidate the structure and mode of action of these catalytic domains in NmDsbD.

Table 4
Data collection and processing

Values in parentheses are for the highest resolution shell.

	n-NmDsbDOx	n-NmDsbDRed	c-NmDsbDOx	c-NmDsbDRed
Diffraction source	Australian Synchrotron	Australian Synchrotron	Australian Synchrotron	Australian Synchrotron
Wavelength (Å)	0.9537	0.9537	0.9537	0.9537
Temperature (K)	100	100	100	100
Detector	ADSC Quantum 315r	ADSC Quantum 315r	ADSC Quantum 315r	ADSC Quantum 315r
Rotation range per image (°)	1	1	0.5	1
Total rotation range (°)	180	180	180	200
Exposure time per image (s)	1	1	1	0.5
Space group	P213	P321	P41	P1211
a, b, c (Å)	147.64, 147.64, 147.64	116.98, 116.98, 45.79	35.80, 35.80, 188.74	43.25, 28.06, 45.80
α, β, γ (°)	90, 90, 90	90, 90, 120	90, 90, 90	90, 101, 90
Predicted solvent content (%)	61.50	62.65	46.37	41.17
Resolution range (Å)	50.00–2.60 (2.69–2.60)	45.79–1.60 (1.69–1.60)	50.00–2.30 (2.38–2.30)	42.46–1.70 (1.79–1.70)
Total No. of reflections	432319	504136	131158	46022
No. of unique reflections	32934	47439	10483	12150
Completeness (%)	99.9 (100)	100 (100)	99.8 (100)	99.8 (99.9)
Redundancy (multiplicity)	13.1 (12.6)	10.6 (10.5)	12.5 (12.7)	3.8 (3.9)
〈I/σ(I)〉	14.0 (4.6)	11.4 (1.8)	20.4 (6.3)	7.5 (2.2)
Rr.i.m.†	0.09 (0.365)	0.08 (1.06)	0.07 (0.281)	0.067 (0.357)
Rp.i.m.	0.051 (0.213)	0.046 (0.594)	0.039 (0.155)	0.059 (0.306)

†Calculated by multiplying R_merge = , where I(hkl) is the intensity of individual reflections, by the factor [N/(N − 1)]^1/2, where N is the data multiplicity.