Solving phase problem using a Se-Met derivative of the flavoenzyme NAD(P)H:acceptor oxidoreductase (FerB)
T. Klumpler1, J. Marek1, V. Sedláček2 , I. Kučera2
1">Laboratory of Molecular Plant Physiology, Department of Functional Genomics and Proteomics, Institute of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5/A2, CZ 625 00 BRNO, <cny>Czech Republic</cny></aff>
2Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5/A5, CZ 625 00 BRNO, <cny>Czech Republic</cny></aff></aug>.
Keywords: ferric reductase B, FerB, phasing, MRSAD, MAD
The flavin adenine dinucleotide-dependent enzyme FerB from Paracoccus denitrificans reduces a range of substrates, including chromate, ferric complexes, benzoquinones and naphtoquinones. The reduced form of nicotinamide adenine dinucleotide serves as a source of electrons. Recombinant unmodified and selenometionine-substituted (Se-Met) FerB derivatives were crystallized, diffraction data for both forms were collected and the phase problem for Se-Met FerB dimer was solved by three-wavelength multiple anomalous dispersion, followed by the combination of molecular replacement and single-wavelength anomalous diffraction phasing. A molecular-replacement solution of unmodified FerB tetramer was obtained using Se-Met structure as a search model.
The easiest way to crystal structure determination is the molecular replacement (MR). Only one native dataset, the coordinates of homologous structure and software that find the right number, orientation and translation of initial search model are needed. Two-third of more than 60 000 protein structures deposited in January 2010 in the Protein Data Bank  has been determined using MR techniques. Easy-to-use MR software is available and over 95% of deposited X-ray structures solved by MR has been determined using MR protocols of CNS , or one of specialized MR packages AMoRe , MOLREP  and PHASER . More recently, software for automatic choice of phasing models from databases has been released, such as MrBUMP  and BALBES . The solution of phase problem by MR could be very fast in principle, but often first model requires multiple rounds of manual refinement.
The second most important phasing techniques are based on intensity differences arising from the presence of heavy atoms implemented in single or multiple isomorphous replacement with or without anomalous scattering (SIR/MIR or SIRAS/MIRAS) or in single or multiple wavelength anomalous diffraction methods (SAD or MAD, ). MIR methods were used to determine the first X-ray structures of macromolecules and still have potential to determine unknown structure directly from experimental data. Heavy atoms are introduced to the protein crystal by soaking the crystal in the ionic solution of heavy atom. On the contrary, SAD and MAD methods incorporate heavy (typically selenium) atoms into protein crystals using protein molecules containing selenomethionine instead of methionine . Se-Met methods becomes more and more popular recently, because they eliminate problems associated with heavy-metal screening, the lack of isomorphism between native and heavy-atoms structures and mainly the only one single crystal is needed to perform complete diffraction experiment. Heavy-atom (or selenium) substructure could be determined using the Patterson or direct-methods programs such as SnB , SHELXD , CNS or SOLVE . Determination of heavy-atom substructure via SAD can be initialized using preliminary positions of heavy-atom from an MR solution. This combined technique is called molecular replacement with single-wavelength anomalous diffraction (MRSAD) .
A broad scale of available programs indicates that crystallographer chooses the most appropriate individual programs for the specific sub-tasks executed during effective pass through the complete process of the protein crystal structure determination. At least partial automation of this multi-step decision process has become recent initiative. Different automated pipeline have been built up, e.g. ACrS , Auto-Rickshaw , autoSHARP , CRANK , ELVES , HKL-3000 , PHENIX , SGXPro .
The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors, NADH or NADPH [22, 23]. Enzymes utilizing flavin cofactors, (flavin mononucleotide, FMN, or flavin adenine dinucleotide, FAD), are unique in their ability to catalyze a wide variety of mechanistically different reactions, such as dehydrogenation, oxygen activation, halogenation, non-redox conversions, light sensing and emission, and DNA repair . The function of all of these enzymes in cell metabolism has not yet been fully elucidated. Finding the molecular basis of the catalysis by FerB would be greatly aided by knowledge of the three-dimensional structure of the enzyme. Here we report the solution of the phase problem of the Se-Met derivative of flavin dependent enzyme FerB from Paracoccus denitrificans using the advanced 3W-MAD and MRSAD protocols of Auto-Rickshaw: the EMBL-Hamburg automated crystal structure determination platform after MR solution of the native protein FerB had failed.
Materials and methods
1. Production, purification and crystallization of Se-Met FerB
Se-Met FerB was prepared using the methionine biosynthesis inhibition method . Purification in a single chromatography step using a HisPrep FF 16/10 column (GE Healthcare)  resulted in almost homogenous protein preparations (> 95% homogeneity). Purity and monodisperzity of a sample were controlled by SDS-PAGE electrophoresis and dynamic light scattering. For protein crystallization micro-seeding technique was exploited and crystals of native and Se-Met FerB were obtained (Fig. 1.). For details see .
2. MALDI-TOF mass spectrometry
Native and Se-Met FerB were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry on a ULTRAFLEX III mass spectrometer (Bruker Daltonics, Germany). Samples were co-crystallized with 2,5-dihydroxybenzoic acid and analyzed in linear mode using an accelerating voltage of 25 kV. The instrument was calibrated with [MH]+ and [MH]2+ peaks using a mixture of peptide standards (Bruker Daltonics). Peaks at molecular masses of 21289 and 21616 detected for intact and Se-Met FerB correspond well to the predicted mass difference of 328 Da (seven methionine residues per chain).
3. Auto-Ricksaw structure determination
Se-Met diffraction data were collected at tunable beamline X12 of the DORIS-III storage ring at EMBL/DESY (Hamburg, Germany), processed and merged using the XDS system  (for details see ). The structure of Se-Met FerB was solved using the advanced 3W-MAD and MRSAD protocols of Auto-Rickshaw: the EMBL-Hamburg automated crystal structure determination platform. The output diffraction data from XDS were converted for use in Auto-Rickshaw using programs of the CCP4 suite (CCP4,1994), and FA values were calculated using the program SHELXC . Based on an initial analysis of the data, the maximum resolution for FerB substructure determination and initial phase calculation was set to 1.8 Ĺ. 14 selenium atoms were found using the program SHELXD. The correct hand for the substructure was determined using the programs ABS  and SHELXE  and initial phases were calculated after density modification using the program SHELXE. The initial phases were improved by density modification and phase extension using the program DM . Then the twofold non-crystallographic symmetry (NCS) operator was found and then density modification with solvent flattening and NCS averaging was applied, both using the program RESOLVE . Resulting phases from RESOLVE were used as input for model building mode of program ARP/wARP.
The model building and refinement protocol implemented into MRSAD pipeline of Auto-Rickshaw started with rigid-body refinement of individual protein chains at 4 Ĺ resolution followed by positional, B-factor and once more positional refinement at 3.0 Ĺ using CNS. The CNS result was used for refinement and phase extension to 1.75 Ĺ resolution using REFMAC5 . In the next step of the protocol, quality of electron density map was improved using density modification and NCS-averaging by RESOLVE (Fig. 2.). New, more complete model of FerB was prepared consequently building of polyalanine model by beta version of SHELXE, side-chain docking with RESOLVE, REFMAC5 refinement, and finally by run of ARP/wARP [33, 34] in the model building regime.
We had tried to solve FerB structure by the molecular replacement (MR) methods using an NAD(P)H dependent FMN reductase flavoprotein from Pseudomonas aeruginosa PA01 (PDB code1RTT)  identified by a FASTA search  as a model. Unfortunately, all MR trials were unsuccessful. We therefore collected data with selenomethionine derivative of FerB. An excellent data quality and their maximum resolution allow us to try to solve phase problem of FerB using the advanced 3W-MAD protocol of Auto-Rickshaw: the EMBL-Hamburg automated crystal structure determination platform. The twofold NCS operator connecting expected two FerB monomers close to x, -y, ?-z was found by RESOLVE. The model building mode of program ARP/wARP employed at the end of the advanced 3W-MAD protocol of Auto-Rickshaw was able find 309 (from expected 364) residues divided to 11 chains, 100% of them have been docked in FerB sequence, and this intermediate model was completed by the MRSAD pipeline of Auto-Rickshaw to almost complete model of Se-Met FerB homodimer containing 349 residues divided into 9 chains (340 of them correctly docked) with final R/Rfree= 0.2144/0.2682.
The structure of native FerB tetramer was successfully solved by molecular replacement technique implemented in PHASER (the final translation function Z-score TFZ= 66.6) with manually unrefined structure of one of Se-Met FerB monomers as the search model. Refinement of both FerB structures is now in progress.
Figure 1. Rod-like crystals of Se-Met FerB.
Figure 2. 2FO-FC map of Se-Met FerB contoured at 1σ.
We wish thank to the EMBL/DESY Hamburg for providing us with synchrotron facilities and D. Tucker for his assistance with data collection on beamlines X12 and X13 of the DORIS-III storage ring at DESY Hamburg. The authors are grateful to Ondrej Šedo for measuring the MALDI-TOF MS spectra and the Meta Center for computer time. This research was supported by grants from the Czech Science Foundation (grant Nos. 204/08/H054 and 525/07/1069 and P503/10/217) and the Ministry of Education, Youth and Sports (grant Nos. MSM0021622413, MSM0021622415 and LC06034).
1 H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, Nucleic Acids Res., 28 (2000), 235.
2 A.T. Brünger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. Grosse-Kunstleve, J.S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Acta Cryst., D54, (1998), 905.
3 J. Navaza, Acta Cryst., A50, (1994), 157.
4 A. Vagin, A. Teplyakov, J. Appl. Cryst., 30, (1997), 1022.
5 A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni, R.J. Read, J. Appl. Cryst., 40, (2007), 658.
6 R.M. Keegan, M.D. Winn, Acta Cryst., D63, (2007), 447.
7 F. Long, A.A. Vagin, P. Young, G.N. Murshudov, Acta Cryst., D64, (2008), 125.
8 W.A. Hendrickson, Science, 254, (1991), 51.
9 W.A. Hendrickson, J.R. Horton, D.M. LeMaster, EMBO J., 9, (1990), 1665.
10 R. Miller, N. Shah, M.L. Green, W. Furey, C.M. Weeks, J. Appl. Cryst., 40, (2007), 938.
11 G.M. Sheldrick, Acta Cryst., A64, (2008), 112.
12 T.C. Terwilliger, J. Berendzen, Acta Cryst., D55, (1999), 849.
13 J.P. Schuermann, J.J. Tanner, Acta Cryst., D59, (2003), 1731.
14 J.S. Brunzelle, P. Shafaee, X. Yang, S. Weigand, Z. Ren, W.F. Anderson, Acta Cryst., D59, (2003), 1138.
15 S. Panjikar, V. Parthasarathy, V.S. Lamzin, M.S. Weiss, P.A. Tucker, Acta Cryst., D65, (2009), 1089.
16 C. Vonrhein, E. Blanc, P. Roversi, G. Bricogne, Methods Mol. Biol., 364, (2006), 215.
17 S.R. Ness, R.A. de Graaff, J.P. Abrahams, N.S. Pannu, Structure, 12 (2004), 1753.
18 J. Holton, T. Alber, Proc. Natl Acad. Sci. USA, 101, (2004), 1537.
19 W. Minor, M. Cymborowski, Z. Otwinowski, M. Chruszcz, Acta Cryst., D62, (2006), 859.
20 P.D. Adams, R.W. Grosse-Kunstleve,L.W. Hung, T.R. Ioerger, A.J. McCoy, N.W. Moriarty, R.J. Read, J.C. Sacchettini, N.K. Sauter, & T.C. Terwilliger, Acta Cryst., D58, (2002), 1948.
21 Z.Q. Fu, J. Rose, B.C. Wang, Acta Cryst., D61, (2005), 951.
22 J. Mazoch, R. Tesařík, V. Sedláček, I. Kučera, J. Turánek, Eur. J. Biochem., 271, (2004), 553.
23 V. Sedláček, R.J.M. van Spanning, I. Kučera, Arch. Biochem. Biophys., 483, (2009), 29.
24 A. Mattevi, Trends Biochem. Sci., 31, (2006), 276.
25 G.D. Van Duyne, R. Standaert, P.A. Karplus, S.L. Schreiber, J. Clardy, J. Mol. Biol., 229, (1993), 105.
26 R. Tesařík, V. Sedláček, J. Plocková, M. Wimmerová, J. Turánek, I. Kučera, Protein Expres. Purif., 68,(2009), 233.
27 T. Klumpler, V. Sedláček, J. Marek, M. Wimmerová, I. Kučera, Acta Cryst., F66, (2010), (in press).
28 W. Kabsch, J. Appl. Cryst., 26, (1993), 795.
29 Q. Hao, J. Appl. Cryst., 37, (2004), 498.
30 K. Cowtan, Joint CCP4 and ESF-EACBM Newsletter on protein crystallography, 31, (1994), 34.
31 T.C. Terwilliger, Acta Cryst., D59, (2003), 38.
32 G.N. Murshudov, A.A. Vagin, E.J. Dodson, Acta Cryst., D53, (1997), 240.
33 A. Perrakis, R.J. Morris, V.S. Lamzin, Nature Struct. Biol., 6, (1999), 458.
34 S.X. Cohen, M. Ben Jelloul, F. Long, A. Vagin, P. Knipscheer, J. Lebbink, T.K. Sixma, V.S. Lamzin, G. N. Murshudov, A. Perrakis, Acta Cryst., D64, (2008), 49.
35 R. Agarwal, J.B. Bonanno, S.K. Burley, S. Swaminathan, Acta Cryst., D62, (2006), 383.
36 W.R. Pearson, D.J. Lipman, Proc. Natl. Acad. Sci. USA, 85, (1998), 2444.