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Logo of actaf2this articlesearchopen accesssubscribesubmitActa Crystallographica. Section F, Structural Biology CommunicationsActa Crystallographica. Section F, Structural Biology Communications
Acta Crystallogr F Struct Biol Commun. 2014 March 1; 70(Pt 3): 347–349.
Published online 2014 February 19. doi:  10.1107/S2053230X14002143
PMCID: PMC3944699

Tetartohedral twinning in IDI-2 from Thermus thermophilus: crystallization under anaerobic conditions


Type-2 isopentenyl diphosphate isomerase (IDI-2) is a key flavoprotein involved in the biosynthesis of isoprenoids. Since fully reduced flavin mononucleotide (FMNH2) is needed for activity, it was decided to crystallize the enzyme under anaerobic conditions in order to understand how this reduced cofactor binds within the active site and interacts with the substrate isopentenyl diphosphate (IPP). In this study, the protein was expressed and purified under aerobic conditions and then reduced and crystallized under anaerobic conditions. Crystals grown by the sitting-drop vapour-diffusion method and then soaked with IPP diffracted to 2.1 Å resolution and belonged to the hexagonal space group P6322, with unit-cell parameters a = b = 133.3, c = 172.9 Å.

Keywords: isopentenyl diphosphate isomerase, IDI-2, flavoprotein, anaerobic, isoprenoid, twinning

1. Introduction  

Type-2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) is a key enzyme involved in the biosynthesis of isoprenoids, which are essential compounds for all organisms. IDI-2 is the common isoform in some pathogenic bacteria and is not found in humans. Thus, IDI-2 is a logical target for antibiotics. Discovered by Kaneda et al. (2001 [triangle]), IDI-2 requires a divalent cation and reduced flavin (FMNH2) for activity. Crystal structures with the oxidized cofactor reveal that isopentenyl diphosphate substrate is parallel and adjacent to the flavin in the active site (Berthelot et al., 2012 [triangle]; de Ruyck et al., 2011 [triangle]). The structural information that we seek will facilitate the rational design of covalent and noncovalent inhibitors for the enzyme.

2. Materials and methods  

2.1. Cloning and expression  

Steven C. Rothman generously provided a plasmid encoding His-tagged IDI-2 from Thermus thermophilus (tt-IDI-2). Escherichia coli M15 cells (Qiagen) were cultivated on MDG and (LB + 1% glucose) media containing ampicillin and kanamycin at 100 and 25 µg ml−1, respectively. Overexpression and purification were performed as described previously (Rothman et al., 2007 [triangle]).

2.2. Crystallization  

Recombinant tt-IDI-2 was concentrated to 17 mg ml−1 (Tris pH 8.0, 10% glycerol) as calculated by a BCA assay. The His tag was not removed prior to the crystallization trials. After setting up the anaerobic chamber (COY Laboratories) to ensure an atmosphere with <5 p.p.m. O2, the crystallization solutions (100 mM HEPES pH 6.5–8.0, 25–50% PEG 400) were gas-exchanged for 1 h by vacuum pumping for 2 min, then filled for 30 s with argon before they were placed in the anaerobic chamber. The solution containing the enzyme was also gas-exchanged, placed in the chamber and reduced by the addition of 100 mM Na2S2O4. Anaerobic crystallization was performed using the sitting-drop vapour-diffusion method in 24-well crystallization plates at 298 K. Drops were prepared by mixing 4 µl protein solution with 4 µl reservoir solution. After one month, colourless hexagonal crystals appeared in 100 mM HEPES pH 7.5, 40% PEG 400 (Fig. 1 [triangle]). A portion of the crystals were soaked in modified assay buffer (2 mM IPP, 10 mM MgCl2, 100 mM HEPES pH 7.5, 100 mM Na2S2O4).

Figure 1
Crystals of colourless tt-IDI-2.

2.3. Data collection and processing  

One soaked and one native crystal were separately mounted in a loop and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K on beamline BL7-1 at the Stanford Synchrotron Radiation Lightsource (SSRL) and data sets were processed with XDS (Kabsch, 2010 [triangle]) (Table 1 [triangle]).

Table 1
Data-collection and processing statistics for T. thermophilus IDI-2

3. Results and discussion  

The novelty of this experiment was to crystallize reduced flavin-bound IDI-2 to investigate the IDI-2–IPP–FMNH2 complex.

Crystallization conditions were adapted from those for the recently solved IDI-2 structure from Sulfolobus shibatae (Nagai et al., 2011 [triangle]; Nakatani et al., 2012 [triangle]). The initial crystals diffracted to ~4 Å resolution (Fig. 2 [triangle] a) in the absence of a ligand. When the crystal was soaked with IPP, the resolution increased to 2.1 Å (Fig. 2 [triangle] b).

Figure 2
Diffraction patterns of (a) native and (b) soaked crystals. These patterns were taken on an in-house diffractometer prior to synchrotron data collection.

We collected a complete data set. The crystals belonged to space group P6322, according to XDS, with unit-cell parameters a = b = 133.3, c = 172.9 Å. However, the L and H twinning tests output by phenix.xtriage indicated that the crystal was perfectly twinned (twin fraction = 42.6%). We obtained a starting structure by molecular replacement (AutoMR) using the previously solved structure of oxidized tt-IDI-2 (PDB entry 3dh7; de Ruyck et al., 2008 [triangle]). Unfortunately, refinement using phenix.refine was not successful (R cryst [similar, equals] 50%), even applying the −k, −h, −l twin law (Adams et al., 2010 [triangle]).

4. Conclusions  

The reduced flavin structure should provide the location and orientation of the reduced cofactor and active-site amino acids of the catalytically active state of IDI-2. Of particular interest is the increase in resolution when IPP is bound owing to conformational changes in the enzyme. This is in agreement with evidence that the flexible N-terminal segment becomes a structured part of the active site upon binding IPP (Nakatani et al., 2012 [triangle]).


This work was supported by NIH grant GM 25521. JDR thanks the FNRS for the position of postdoctoral researcher. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a Directorate of SLAC National Accelerator Laboratory and an Office of Science User Facility operated for the US Department of Energy Office of Science by Stanford University. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS, NCRR or NIH. The authors want to thank Professor J. Wouters for his help with data processing.


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