Crystal structure and domain characterization of ScpB from Mycobacterium tuberculosis
2008; Wiley; Volume: 71; Issue: 3 Linguagem: Inglês
10.1002/prot.21981
ISSN1097-0134
AutoresJeong‐Sun Kim, Sujin Lee, Beom Sik Kang, Myung Hee Kim, Heung‐Soo Lee, Kyung‐Jin Kim,
Tópico(s)RNA and protein synthesis mechanisms
ResumoProteins: Structure, Function, and BioinformaticsVolume 71, Issue 3 p. 1553-1556 Structure NoteFree Access Crystal structure and domain characterization of ScpB from Mycobacterium tuberculosis Jeong-Sun Kim, Jeong-Sun Kim Department of Chemistry, Chonnam National University, Gwangju, 500-757, KoreaSearch for more papers by this authorSujin Lee, Sujin Lee Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784, KoreaSearch for more papers by this authorBeom Sik Kang, Beom Sik Kang School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, KoreaSearch for more papers by this authorMyung Hee Kim, Myung Hee Kim Systems Microbiology Research Center, Korea Research Institute of Biosciences and Biotechnology, Yusung, Daejon 305-806, KoreaSearch for more papers by this authorHeung-Soo Lee, Heung-Soo Lee Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784, KoreaSearch for more papers by this authorKyung-Jin Kim, Corresponding Author Kyung-Jin Kim kkj@postech.ac.kr Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784, KoreaBeamline Division, Pohang Accelerator Laboratory, San31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, Korea===Search for more papers by this author Jeong-Sun Kim, Jeong-Sun Kim Department of Chemistry, Chonnam National University, Gwangju, 500-757, KoreaSearch for more papers by this authorSujin Lee, Sujin Lee Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784, KoreaSearch for more papers by this authorBeom Sik Kang, Beom Sik Kang School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, KoreaSearch for more papers by this authorMyung Hee Kim, Myung Hee Kim Systems Microbiology Research Center, Korea Research Institute of Biosciences and Biotechnology, Yusung, Daejon 305-806, KoreaSearch for more papers by this authorHeung-Soo Lee, Heung-Soo Lee Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784, KoreaSearch for more papers by this authorKyung-Jin Kim, Corresponding Author Kyung-Jin Kim kkj@postech.ac.kr Beamline Division, Pohang Accelerator Laboratory, Pohang, Kyungbuk 790-784, KoreaBeamline Division, Pohang Accelerator Laboratory, San31, Hyoja-dong, Nam-gu, Pohang, Kyungbuk 790-784, Korea===Search for more papers by this author First published: 25 February 2008 https://doi.org/10.1002/prot.21981Citations: 8AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat INTRODUCTION Structural maintenance of chromosomes (SMC) proteins, which are found in prokaryotes and eukaryotes, participate in various DNA processes such as sister chromatid cohesion, chromosome condensation and segregation, recombination, and DNA repairing.1, 2 They are proteins of a large molecular weight and construct five structural domains; an N-terminal globular domain, a long helix, a hinge domain, another long helix, and a C-terminal globular domain. They form a peculiar circular structure upon dimerization. The N- and C-terminal domains of SMC proteins assemble to form a globular head domain, which shows a weak ATPase activity.3, 4 The dimeric circular structure can be opened and closed through the head domain when it interacts with ATP4 or through the hinge domain when it binds to DNA.5, 6 SMC proteins require non-SMC proteins for their diverse functions and form intermolecular multi-subunit protein complexes. In eukaryotes, non-SMC protein, Scc1, binds to the SMC head domain using its C-terminal winged-helix domain (cWHD) and form a tertiary complex with an SMC protein.7, 8 Escherichia coli also has a functional SMC analog MukB, whose loss leads to a critical defect in chromosome segregation. MukB also requires two non-SMC proteins, MukE and MukF.9, 10 The N-terminal fragment of MukB comprises two-domain structure (a WHD and a four-helix bundle) and forms a dimeric structure.11 The prokaryotic SMC protein in Bacillus subtilis also has two non-SMC proteins, segregation and condensation proteins (ScpA_Bs and ScpB_Bs). The deletion of non-SMC proteins has shown the same phenotypes as that of SMC proteins. ScpA that interacts directly with SMC proteins mediates the ternary complex formation between SMC proteins and ScpB.12-14 M. tuberculosis has a gene Rv1710, which codes of a polypeptide of 231 amino acids. Amino acid sequence analysis (http://pfam.sanger.ac.uk/) shows that it belongs to an ScpB protein with a common structural motif, DUF387 domain at the N-terminus (residues 30–188), hereafter called as ScpB_Mt. Another closely related gene, Rv1709, is located at the upstream of ScpB_Mt coding gene in the genome loci of M. tuberculosis with a high sequence similarity to the ScpA family, hereafter called as ScpA_Mt. Recent structural studies of ScpB from Chlorobium Tepidum (ScpB_Ct) revealed that the monomer is composed of two WHDs and a dimeric structure was formed through the interaction of the two sequence-conserved cWHDs.15 However, the biochemical function and biological implication of an ScpB protein, for example domain characterization upon binding with ScpA protein, could not be deduced with the reported ScpB_Ct structure alone. Therefore, we determined the crystal structure of ScpB_Mt. The ScpB_Mt structure closely resembles that of the ScpB_Ct except for the domain–domain interaction in forming a dimer. We also show that ScpB_Mt uses both the nWHD and the cWHD for forming an ScpA/ScpB complex. MATERIALS AND METHODS Cloning, expression, purification, and crystallization of ScpB_Mt will be described elsewhere (Kwon et al., in preparation). Briefly, the recombinant ScpB_Mt protein was expressed using the bacterial expression system and purified through sequential chromatographic steps. Suitable crystals for diffraction experiments were obtained at 22°C within 5 days from the precipitant of 40 mM Tris-HCl at pH 7.5, 2M NaCl, and 10% (w/v) polyethyleneglycol 6000. For data collection, 30% (w/v) glycerol was added to the crystallizing precipitant as a cryoprotectant and the crystals were immediately placed in a −173°C nitrogen-gas stream. X-ray diffraction data of native crystals were collected at a resolution of 2.3 Å at the 4A beamline of the Pohang Accelerator Laboratory (PAL, Korea) using a QUANTUM 210 CCD detector (San Diego, CA). The data were then indexed, integrated, and scaled using the HKL2000 suite.16 The crystals belonged to the space group R32, with the unit cell parameters a = b = 136.69 Å, c = 78.55 Å, γ = 120°. With one ScpB_Mt molecule in the asymmetric unit, the crystal volume per unit of protein weight was 2.95 Å3 Da−1, corresponding to a solvent content of 58%. The Seleno-L-methionine (SeMet) substituted protein was expressed in a minimal medium supplemented with SeMet and purified using the same sequential chromatographic steps as those used of the native protein. The SeMet-substituted protein crystals were obtained from the same crystallizing condition as that of the native protein crystal. The single wavelength anomalous dispersion (SAD) data with the SeMet crystal were collected at the 6C1 beamline of the PAL at a wavelength of 0.97950 Å. Three Se atoms out of the expected four in the asymmetric unit were identified at 2.9 Å resolution using the program SOLVE.17 The electron density was improved by density modification using the program RESOLVE,18 resulting in 58% of the cloned residues being automatically built. Further model building was performed manually using the program WinCoot19 and the refinement was performed with CCP4 refmac520 and CNS.21 The data statistics are summarized in Table I. The refined model was deposited in the Protein Data Bank (PDB code 2Z99). Table I. Data Collection and Refinement Statistics Parameters Native Se-Peak Synchrotron 4A(MXW), PAL 6C1(MXII), PAL Wavelength (Å) 1.0000 0.97950 Space group R32 R32 Cell parameters a = b = 136.69 Å a = b = 136.93 Å c = 78.55 Å c = 78.39 Å γ = 120° γ = 120° Resolution (Å) 50.0–2.3 (2.38–2.3) 50.0–2.5 (2.59–2.5) Completeness (%) 98.4 (97.0) 99.7 (98.8) Rsymaa Rsym = Σhkl Σj|Ij−〈I〉|/ΣhklΣjIj, where 〈I〉 is the mean intensity of reflection hkl. (%) 5.5 (21.4) 4.6 (23.1) Reflections (total/unique) 263,491/12,625 171,875/9,902 I/σ 33.9 (4.7) 52.9 (4.5) FOMbb Figure of merit = |∑ P(α)eiα/∑ P(α)|, where P(α) is the phase probability distribution and α is the phase (50.0–2.9 Å). (SOLVE/RESOLVE) 0.35/0.76 Rfactorcc Rfactor = Σhkl||Fobs|−|Fcalc||/Σhkl|Fobs|, where Fobs and Fcalc are, respectively, the observed and calculated structure factor amplitude for reflections hkl included in the refinement. (%) 24.4 Rfreedd Rfree is the same as Rfactor but calculated over a randomly selected fraction (5%) of reflection data not included in the refinement. (%) 28.5 Average B-factor (Å2) 57.34 No. of atoms (protein/water) 1,220/117 RMSD [Bonds (Å)/Angles (°)] 0.007/1.25 Geometry (%) Most favored 92.4 Additionally allowed 7.6 Values in parentheses are for the highest-resolution shell. FOM, figure of merit; RMSD, root-mean-square-deviation. a Rsym = Σhkl Σj|Ij−〈I〉|/ΣhklΣjIj, where 〈I〉 is the mean intensity of reflection hkl. b Figure of merit = |∑ P(α)eiα/∑ P(α)|, where P(α) is the phase probability distribution and α is the phase (50.0–2.9 Å). c Rfactor = Σhkl||Fobs|−|Fcalc||/Σhkl|Fobs|, where Fobs and Fcalc are, respectively, the observed and calculated structure factor amplitude for reflections hkl included in the refinement. d Rfree is the same as Rfactor but calculated over a randomly selected fraction (5%) of reflection data not included in the refinement. The M. tuberculosis genes coding for ScpA, ScpB(N) (Asp16∼Ala106), ScpB(C-cVR (cVR, C-terminal variable region)) (Arg107∼Asp231), and ScpB(N-C) (Asp16∼ Leu185) were amplified from M. tuberculosis chromosomal DNA by a polymerase chain reaction (PCR). The PCR products were then cloned into the bacterial expression vector pPROEX HTa (Novagen). The expression constructs were transformed into the E. coli B834 strain and were grown in a Luria-Bertani medium containing 100 μg/mL ampicillin at 37°C. After induction with 1.0 mM IPTG for a further 20 h at 22°C, the culture was harvested by centrifugation at 5000g for 20 min at 4°C. The cell pellet was resuspended in ice-cold buffer A (50 mM Tris-HCl at pH 8.0 and 5 mM of β-mercaptoethanol) and disrupted by ultrasonication. The cell debris was removed by centrifugation at 11,000g for 1 h, and the clear lysate was bound to Ni-NTA agarose (QIAGEN). After washing with buffer A containing 10 mM imidazole, the bound proteins were eluted with buffer A containing 300 mM imidazole. The eluted sample was cleaved with rTEV protease (GIBCO) to remove the 6-His tag. The cleaved proteins were further purified by applying the sequential chromatographic steps of HiTrap Q anion exchange and Superdex75 size exclusion. Sedimentation equilibrium experiments were performed using an analytical ultracentrifuge Beckman Optima XL-I (Fullerton, CA) at 20,000 rpm for 20 h at 15°C. The used protein concentrations were 50 μM for ScpB and 100 μM for ScpB(N) in 40 mM Tris-HCl at pH 8.0. RESULTS AND DISCUSSION The structure ScpB_Mt (Asp16-Asp231) was solved by SAD analysis with the SeMet substituted protein crystal (Table I). The asymmetric unit contains one ScpB_Mt molecule. Total 171 amino acid residues out of the 216 cloned residues were built into the experimental electron density map and the polypeptide region of Pro186-Asp231(cVR) was disordered in the present crystal. The monomeric structure is composed of two continuous WHDs; nWHD (Pro20-Arg91) and cWHD (Thr108-Thr170). A linker region (Ala92-Arg107) connects two WHDs. Both WHD motifs consist of three α-helices (H1, H2, and H3), which are walled by two β-strands (S1 and S2) [Fig. 1(A)]. A series of conserved nonpolar residues constitute the hydrophobic cores of nWHD and cWHD structures. An additional two-turn helix (H4′, Glu171-Gly178) and an extended structure (Leu179-Ser183) are attached at the cWHD. The partially open hydrophobic pocket in cWHD is occupied with nonpolar residues of Phe173, Leu174, and Leu177. Figure 1Open in figure viewerPowerPoint The architecture of ScpB_Mt. (A) Overall shape of a ScpB_Mt monomer. Two similarly folded domains are connected by a linker. nWHD and cWHD are indicated as red and blue colors, respectively, and the secondary structure elements are appropriately labeled. (B) The dimeric structure of ScpB_Mt. Each monomer is differentiated by colors and labeled. The right figure was produced by rotating the left figure by 90°. (C) Stereo-presentation of an ScpB_Mt dimer. Both nWHDs are symmetrically oriented. Residues (E27, R30, E33, R75, and R93) for ionic interactions (cyan) and residues for hydrophobic interaction (gray) are shown as stick models. (D) Sedimentation equilibrium of ScpB and ScpB(N). The residuals shown above the panel represent the difference between the calculated fit and the experimental data. The molecular weights of ScpB and ScpB(N) were calculated as 47,000 and 20,000 Da, respectively. The ScpB_Ct has shown a dimeric structure.15 The ScpB_Mt exists as a dimer in solution, which will be described later. Even though only a monomeric protein was present in the asymmetric unit of our present ScpB_Mt structure, the dimeric structure of ScpB_Mt could easily be generated by applying the crystallographic R32 symmetry. In the generated dimeric structure of ScpB_Mt, three α-helices and two β-strands of the nWHDs are symmetrically oriented [Fig. 1(B)]. Two H1 helices of nWHDs occupy the central position of this newly generated globular domain and are enclosed by two helices (H2 and H3), two strands (S1 and S2), and by two linker helices (H4) [Fig. 1(B)]. Both ionic and hydrophobic interactions are formed at the dimeric interface [Fig. 1(C)]. Glu33 from the H1 helix interacts with Arg73 of the H3 helix of a neighboring monomer. The nonpolar residues of Leu37, Leu70, Ile77, and Met88 from both nWHDs are intertwined and form a new hydrophobic pocket. The Arg93 in the linker helices (H4) additionally contribute to the ScpB_Mt dimerization by forming an ionic interaction with Glu27 on the H1 helix. The H4 helices form a local hydrophobic core using the residues of Phe94, Val98, Leu101, and Leu102, together with the solvent-exposed hydrophobic residues of Ala31, Ala34, Ala35, Leu37, Val38, Ala47, and Ala51 of the H1 and H2 in nWHD [Fig. 1(C)]. The dimeric structure of ScpB_Ct is formed through the interaction of two cWHDs by providing several hydrophobic and hydrophilic interactions at the interface.15 As aforementioned, the generated dimeric structure of ScpB_Mt is constructed by exchanging its nWHDs, not cWHDs. To confirm the involvement of nWHD in the ScpB_Mt dimer formation, we constructed two deletion mutants of ScpB_Mt, ScpB(N) and ScpB(C-cVR). On a size exclusion chromatographic column, the ScpB(N) domain migrated as a dimer, while the ScpB(C-cVR) was eluted as a monomer (data not shown). The involvement of nWHD in the ScpB_Mt dimer formation was further confirmed by an analytical ultracentrifugation experiment, which showed that the calculated molecular weights for ScpB and ScpB(N) were 47 and 20 kDa, respectively. This result obviously indicated the dimeric sizes of the respective proteins [Fig. 1(D)]. On the basis of the biochemical data as well as the observed dimeric interaction in the crystal structure, we suggest that the ScpB_Mt protein forms a dimer through the interaction of nWHD and H4 linker regions, not through cWHDs. To constitute the functional SMC machinery for DNA assembly, ScpA mediates the interaction between SMC and ScpB by forming an ScpA/ScpB complex.12-14 To identify the responsible region of ScpB_Mt for forming a complex with ScpA_Mt, we performed the Ni-affinity pull-down assays using the ScpA_Mt protein with 6-His tag as a bait and ScpB_Mt proteins without 6-His tag as a prey. Neither the ScpB(N) nor ScpB(C-cVR) alone was enough to form the complex with ScpA_Mt. Instead, the ScpA/ScpB complex formation was only detected when ScpB or ScpB (N-C) lacking the cVR was mixed with ScpA_Mt (see Fig. 2). This result clearly show that both WHDs of ScpB_Mt are simultaneously involved in the complex formation with ScpA_Mt and the cVR region is not necessary. Interestingly enough, the analysis of amino acid composition of ScpBs among various microorganisms shows the presence of several ScpBs lacking cVR region. Figure 2Open in figure viewerPowerPoint Domain characterization of ScpB_Mt for forming a ScpA/ScpB complex. To identify the characteristic domain of ScpB_Mt for a complex formation with ScpA_Mt, 6-His tagged ScpA_Mt was mixed with various constructs of ScpB_Mt without 6-His tag, and incubated overnight at 4°C. The mixtures were then applied to Ni-NTA chromatography, and the flow-through (FT) and elution (E) fractions were loaded onto an SDS gel. Complex formations were detected from the mixtures of ScpA/ScpB(N-C) and ScpA/ScpB, but not from those of ScpA/ScpB(N) or ScpA/ScpB(C-cVR), indicating both of N- and C- terminal domains of ScpB are required for the complex formation with ScpA. N, N-C, and C-cVR represent N-terminal, N- and C-terminal without cVR, and C-terminal and cVR of ScpB_Mt, respectively. In summary, we determined the crystal structure of ScpB_Mt. Two continuous WHDs with a C-terminal extended region constitute a monomeric structure of ScpB_Mt and the dimer is constructed through the interactions of nWHDs. Deletion mutants and pull-down data show that both WHDs of ScpB_Mt are required for the complex formation with ScpA_Mt. REFERENCES 1 Hirano T. SMC-mediated chromosome mechanics: a conserved scheme from bacteria to vertebrates? Genes Dev 1999; 13: 11– 19. CrossrefCASPubMedWeb of Science®Google Scholar 2 Strunnikov AV,Jessberger R. Structural maintenance of chromosomes (SMC) proteins: conserved molecular properties for multiple biological functions. Eur J Biochem 1999; 263: 6– 13. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 3 Hopfner KP,Karcher A,Shin DS,Craig L,Arthur LM,Carney JP,Tainer JA. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell 2000; 101: 789– 800. CrossrefCASPubMedWeb of Science®Google Scholar 4 Lowe J,Cordell SC,Van Den Ent F. Crystal structure of the Smc head domain: an ABC ATPase with 900 residues antiparallel coiled-coil inserted. J Mol Biol 2001; 306: 25– 35. CrossrefCASPubMedWeb of Science®Google Scholar 5 Gruber S,Arumugam P,Katou Y,Kuglitsch D,Helmhart W,Shirahige K,Nasmyth K. Evidence that loading of cohesin onto chromosomes involves opening of its SMC hinge. Cell 2006; 127: 523– 537. CrossrefCASPubMedWeb of Science®Google Scholar 6 Hirano M,Hirano T. Opening closed arms: long-distance activation of SMC ATPase by hinge-DNA interactions. Mol Cell 2006; 21: 175– 186. CrossrefCASPubMedWeb of Science®Google Scholar 7 Hirano T. At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol 2006; 7: 311– 322. CrossrefCASPubMedWeb of Science®Google Scholar 8 Haering C,Schoffnegger D,Nishino T,Helmhart W,Nasmyth K,Lowe J. Structure and stability of Cohesin's Smc1-Kleisin interaction. Mol Cell 2004; 15: 951– 964. CrossrefCASPubMedWeb of Science®Google Scholar 9 Niki H,Jaffe A,Imamura R,Ogura T,Hiraga S. The new gene MukB codes for a 177kD protein with coiled-coil domains involved in chromosome partitioning of Escherichia coli. EMBO J 1991; 10: 183– 193. Wiley Online LibraryGoogle Scholar 10 Van Den Ent F,Lockhart A,Kendrick-Jones J,Lowe J. Crystal structure of the N-Terminal domain of Mukb: a protein involved in chromosome partitioning. Struct Folds Des 1999; 7: 1181– 1187. CrossrefCASPubMedWeb of Science®Google Scholar 11 Fennell-Fezzie R,Gradia SD,Akey D,Berger JM. The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins. EMBO J 2005; 24: 1921– 1930. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 12 Soppa J,Kobayashi K,Noirot-Gros MF,Oesterhelt D,Ehrlich SD,Dervyn E,Ogasawara N,Moriya S. Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB. Mol Microbiol 2002; 45: 59– 71. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 13 Lindow JC,Kuwano M,Moriya S,Grossman AD. Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein. Mol Microbiol 2002; 46: 997– 1009. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 14 Volkov A,Mascarenhas J,Andrei-Selmer C,Ulrich HD,Graumann PL. A prokaryotic condensin/cohesin-like complex can actively compact chromosomes from a single position on the nucleoid and binds to DNA as a ring-like structure. Mol Cell Biol 2003; 23: 5638– 5650. CrossrefCASPubMedWeb of Science®Google Scholar 15 Kim JS,Shin DH,Pufan R,Huang C,Yokota H,Kim R,Kim SH. Crystal structure of ScpB from Chlorobium tepidum, a protein involved in chromosome partitioning. Proteins 2006; 62: 322– 328. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 16 Otwinowski Z,Minor W. Procession of X-ray diffraction data collected in oscillation mode. Methods Enzymol 1997; 276: 307– 326. CrossrefCASPubMedWeb of Science®Google Scholar 17 Terwilliger TC,Berendzen J. Automated MAD and MIR structure solution. Acta Crystallogr D Biol Crystallogr 1999; 55: 849– 861. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 18 Terwilliger TC. Maximum-likelihood density modification. Acta Crystallogr D Biol Crystallogr 2000; 56: 965– 972. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 19 Emsley P,Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004; 60: 2126– 2132. Wiley Online LibraryCASPubMedGoogle Scholar 20 Murshudov GN,Vagin AA,Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 1997; 53: 240– 255. Wiley Online LibraryCASPubMedGoogle Scholar 21 Brunger AT,Adams PD,Clore GM,DeLano WL,Gros P,Grosse-Kunstleve RW,Jiang JS,Kuszewski J,Nilges M,Pannu NS,Read RJ,Rice LM,Simonson T,Warren GL. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 1998; 54: 905– 921. Wiley Online LibraryCASPubMedGoogle Scholar Citing Literature Volume71, Issue315 May 2008Pages 1553-1556 FiguresReferencesRelatedInformation
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