Crystal structure of uronate isomerase (TM0064) from Thermotoga maritima at 2.85 Å resolution

Authors

  • Robert Schwarzenbacher,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Jaume M. Canaves,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Linda S. Brinen,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Xiaoping Dai,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
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  • Ashley M. Deacon,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Marc A. Elsliger,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
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  • Said Eshaghi,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Ross Floyd,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Adam Godzik,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Carina Grittini,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
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  • Slawomir K. Grzechnik,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Chittibabu Guda,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Lukasz Jaroszewski,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Cathy Karlak,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
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  • Heath E. Klock,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Eric Koesema,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • John S. Kovarik,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Andreas Kreusch,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Peter Kuhn,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Scott A. Lesley,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Daniel McMullan,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Timothy M. McPhillips,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Mark A. Miller,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Mitchell D. Miller,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Andrew Morse,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Kin Moy,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
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  • Jie Ouyang,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Alyssa Robb,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Kevin Rodrigues,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Thomas L. Selby,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
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  • Glen Spraggon,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Raymond C. Stevens,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
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  • Henry van den Bedem,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Jeff Velasquez,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
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  • Juli Vincent,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Genomics Institute of the Novartis Research Foundation, San Diego, California
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  • Xianhong Wang,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Bill West,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
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  • Guenter Wolf,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • Keith O. Hodgson,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California
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  • John Wooley,

    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. San Diego Supercomputer Center, La Jolla, California
    3. University of California, San Diego, La Jolla, California
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  • Ian A. Wilson

    Corresponding author
    1. Joint Center for Structural Genomics, Stanford University, Menlo Park, California
    2. Scripps Research Institute, La Jolla, California
    • Joint Center for Structural Genomics, Scripps Research Institute, BCC206, 10050 N. Torrey Pines Rd., La Jolla, CA 92037
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Introduction.

The TM0064 gene of Thermotoga maritima encodes a predicted uronate isomerase (EC 5.3.1.12) with a molecular weight of 52,174 Da and a calculated isoelectric point of 5.68. Uronate dehydrogenase catalyzes the conversion of D-glucuronate to D-fructuronate, or D-galacturonate to D-tagaturonate, which are the first steps in the pathway of glucuronic and galacturonic acid metabolism. This enzyme has no other paralogs, but a number of orthologs have been identified in other bacterial species. Here, we report the crystal structure of TM0064 determined with use of the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics.1 This is the first structure of a protein from this family to be determined.

We solved the structure of TM0064 to 2.85 Å resolution using the multiple-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I.The final model includes residues 1–450 for each of the three independent molecules in the asymmetric unit (the C-terminal Gly is not present in the final model), and 107 water molecules. The Matthews coefficient (Vm) for TM0064 is 2.73 Å3/Da, and the estimated solvent content is 58.9%. The Ramachandran plot produced by PROCHECK 3.42 shows that 90% of the residues are in the most favored regions, 9.8% in additional allowed regions, and 0.2% in generously allowed regions. No residues lie in disallowed regions.

Table I. Summary of Crystal Parameters, Data Collection and Refinement Statistics for TM0064 (PDB: 1J5S)
  1. Rsym = Σ|Ii 〈Ii〉| |/ΣIi|, where Ii is the scaled intensity of the ith measurement, and 〈Ii〉 is the mean intensity for that reflection.

  2. Rcryst = Σ| |Fobs| − |Fcalc| |/Σ|Fobs|, where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively.

  3. Rfree = as for Rcryst, but for 5% of the total reflections chosen at random and omitted from refinement.

Crystal characteristics and data statistics
 Space group P1
 Unit cell parameters a = 77.41 Å, b = 79.96 Å, c = 89.43 Å, α = 115.7°, β = 97.6°, γ = 110.4°
Data Collectionλ1MADSeλ2MADSeλ3MADSe
 Wavelength (Å)0.97940.91840.9792
 Resolution range()25.0–2.7525.0–2.7525.0–2.75
 Number of observations153,413153,787153,293
 Number of unique reflections42,71542,70242,728
 Completeness (%)96.496.496.4
 (In highest resolution shell, %)96.496.196.4
 Mean I/σ(I)5.85.24.2
 (In highest resolution shell, %)2.12.21.6
 Rsym on I0.0770.0750.175
 (In highest resolution shell, %)0.2440.2320.832
 Sigma Cutoff0.00.00.0
 Highest resolution shell (Å)2.90–2.752.90–2.752.90–2.75
Model and refinement statistics Data set used in refinementλ2MADSe
 Resolution range (Å)24.99–2.85Cutoff criteria|F|> 0
 No. of reflections (total)36,521Rcryst0.233
 No. of reflections (test)1938Rfree0.274
Stereochemical parameters   
 Restraints (RMS observed)   
  Bond length 0.020 Å 
  Bond angle 1.67° 
 Average isotropic B-value 31.3 Å2 
Luzzati Mean Coordinate error 0.36 Å 

The TM0064 monomer consists of a single polypeptide chain of 451 amino acids composed of 25 helices (21 α-helices, and four 310-helices), and 9 β-strands [Fig. 1(A, C, and E)].

Figure 1.

(A) Domain organization of Thermotoga maritima TM0064 uronate isomerase. Domain A contains two noncontiguous segments (yellow and blue). (B) Solvent accessible surface (1.4 Å probe radius) of TM0064. The red arrow indicates the channel leading to the active site. The black arrow indicates the position of the putative metal ion. (C) Ribbon representation showing the distribution of secondary structure elements in TM0064. Strands are shown in cyan and helices, in red. The arrow points at the active site. (D) Close-up view of the active site, showing residues interacting with the putative metal ion. (E) Diagram showing the secondary structure elements in TM0064 superimposed to its primary sequence. Residues interacting with the putative metal ion are indicated by green triangles. Helices are labeled H1–H25. Strands are labeled according to their respective β-sheets (i.e., A and B). The location of the β-hairpin formed by β-strands 6 and 7 (labeled C), as well as the location of β and γ turns, are also depicted in the diagram.

The total α-helix, 310-helix, and β-strand content is 58.8, 2.4, and 7.8%, respectively [Fig. 1(C)]. The 9 β-strands form two parallel β-sheets [strands labeled A and B in Fig. 1(E)] and a β-hairpin (β6–β7) [strands labeled C in Fig. 1(E)]. The first β-sheet is formed by strands β1–β3, with a topology 1X 1X. The second β-sheet (B) is formed by strands β4, β5, β8, and β9, with a 1X 1X 1X topology.

The predicted active site is defined by residues His30, His32, Trp366, and Asp397, which coordinate a putative metal ion that has been conservatively modeled as water 1 in the absence of any experimental data on the nature of the metal [Fig. 1(D)]. This finding is consistent with observations indicating that uronate isomerases are capable of binding metals, such as Zn2+ or Cu2+, which can act as inhibitors.3 The activity of this enzyme has been characterized in three different organisms: Escherichia coli,3Erwinia carotovora,4 and Flavobacterium heparinum.5

Twenty homologs belonging to this protein family have been identified in Bacteria, including Thermotogales, Cyanobacteria, Proteobacteria (Alpha and Gamma subdivisions), and Firmicutes (all in the Bacillus/Clostridium group). This enzyme family is absent in Archaea and Eukaryotes. Homology structural models of all these bacterial homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM0064.

The structure reported here represents the first structure of a member of the glucuronate isomerase family (Pfam 026014). The protein is organized in two domains. Domain A contains two noncontiguous segments (0–42 and 135–450). Domain B encompasses residues 43–134.

Predictions performed with use of the Protein Quaternary Structure Server at the European Bioinformatics Institute (http://www.pqs.ebi.ac.uk/pqs-bin/macmol. pl?filename=1j5s) strongly suggest that the asymmetric unit contains a biologically relevant oligomer. This notion is supported by the presence of 4 residues exposed in the isolated chain that are buried in the trimeric complex, 7 interchain salt bridges within the complex, a loss of 4223 Å2 of solvent-accessible surface area upon complex formation, and folding from isolated chains to form a complex that shows a gain in solvation free energy of −52.2 kcal/mol.

A structural similarity search, performed by the program DALI6 with the coordinates of TM0064, indicated that the closest structural homolog is a phosphotriesterase [Protein Data Bank (PDB): 1PSC chain A] from Brevundimonas diminuta,7 with a 3.4 Å root-mean-square deviation (RMSD) over 239 residues (52%), and a sequence identity of 10%. Subsequently, we aligned both structures, using combinatorial extension (CE).8 The CE Z score for the structural alignment was 5.2, with an RMSD of 3.3 Å. Although 233 residues were aligned by CE, there were 125 gap positions. The criteria used by the PDB to define a new fold are CE Z score 〈4, RMSD〉 = 3.0 Å and number of aligned positions <70% of the protein chain length. The similarity to the phosphotriesterase fold is limited to domain A. Therefore, we consider this region of the structure of this glucuronate isomerase to constitute a new fold, although structurally, it could be distantly related to the phosphotriesterase fold.

The approximately 90 residues (43–134) from the helical domain B, not covered by the previous structural alignment, were excised and examined independently with DALI. No structural homologs were found for this domain, indicating that it corresponds to a completely novel fold. In conclusion, the structure of TM0064 as a complete unit represents a novel fold, although domain A contains some structural features distantly related to the phosphotriesterase fold.

The functional relevance of the new structural domain B is unknown. The channel leading to the active site is located in Domain A [Fig. 1(B)]. Two of the residues interacting with the metal ion (His 30 and His 32) are located in the N-terminal segment of domain A [Fig. 1(C)] that tethers both domains, so motions in domain B could have profound effects on the active site. Taken together, this information suggests a possible role of domain B in the regulation of substrate access to the active site, substrate specificity, and/or modulation of catalysis.

Protein Production.

Uronate isomerase (TIGR: TM0064; SwissProt: Q9WXR9) was amplified by polymerase chain reaction (PCR) with the use of Pfu (Stratagene) from T. maritima strain MSB8 genomic DNA with primer pairs encoding the predicted 5′- and 3′-ends of TM0064. The PCR product was cloned into plasmid pMH2T7, which encodes a purification tag consisting of the amino acids MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. We performed protein expression in selenomethionine-containing medium using the E. coli methionine auxotrophic strain DL41. Bacteria were lysed by sonication after a freeze–thaw procedure in lysis buffer (50 mM Tris pH 7.9, 50 mM NaCl, 1 mM MgCl2, 0.25 mM Tri(2-carboxyethyl)phosphine hydrochloride (TCEP), 1 mg/mL lysozyme) and cell debris pelleted by centrifugation at 3600× g for 60 min. The soluble fraction was applied to a nickel chelate resin (Pharmacia) previously equilibrated with equilibration buffer (50 mM KH2PO4 pH 7.8, 0.25 mM TCEP, 10% v/v glycerol, 0.3 M NaCl) containing 20 mM imidazole. The resin was washed with equilibration buffer containing 40 mM imidazole, and protein was eluted with elution buffer (20 mM Tris pH 7.9, 10% glycerol, 0.25 mM TCEP, 300 mM imidazole). Buffer exchange was performed to remove imidazole from the protein eluate, and the protein in Buffer Q (20 mM Tris pH 7.9, 50 mM NaCl, 5% v/v glycerol, 0.25 mM TCEP) was then applied to a Resource Q column (Pharmacia). Protein was eluted, with a linear gradient of up to 500 mM NaCl. Appropriate fractions were buffer exchanged into size exclusion chromatography (SEC) buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 M TCEP). The protein was concentrated for crystallization assays by centrifugal ultrafiltration (Millipore).

Crystallization.

The protein was crystallized with the vapor diffusion method. Crystallization robotics with miniaturized volume assemblies were used to set up crystallization trials in specialized 96-well microtiter trays (Greiner) containing 50 nL of protein solution and 50 nL of crystallization solution in a sitting drop format.1 Each protein was set up with 480 standard crystallization conditions [Wizard I/II, Cryo I/II (Emerald BioStructures, Bainbridge Island, WA), Crystal Screen, Crystal Screen 2, Crystal Screen Cryo, PEG/Ion Screen, Grid Screen Ammonium Sulfate, Grid Screen PEG 6000, Grid Screen MPD, and Grid Screen PEG/LiCl (Hampton Research, Riverside, CA)] at 20°C. Images of each crystal trial were taken at 7 and 28 days after setup with an Optimag Veeco Oasis 1700 imager and evaluated manually. The crystallization buffer contained 50% (v/v) PEG-200 as the precipitant in 0.1 M Tris at pH 7.0. Crystals grew within 28 days at 20°C. The crystals were indexed in the triclinic space group P1 (Table I).

Data Collection.

We collected the MAD data at Stanford Synchrotron Radiation Laboratory (SSRL) (Stanford, CA) on beamline 9-2 using the BLU-ICE9 data collection environment (Table I). All data sets were collected at 100 K with a Quantum 4 charge-coupled device (CCD) detector. Data were integrated and reduced with Mosflm,10 then scaled with the program SCALA from the CCP4 suite.11 Data statistics are summarized in Table I.

Structure Solution and Refinement.

We determined the structure with SOLVE12 and RESOLVE.13 Structure refinement was performed with CNS14 and REFMAC5.11 The asymmetric unit contained three molecules. Tight positional and thermal non crystallographic symmetry (NCS) restraints were used during all stages of refinement for residues 5–440 of each monomer. Refinement statistics are summarized in Table I. The final model contains residues 1–450 for each monomer of the trimer. The C-terminal glycine, as well as the purification tag, was excluded from the model, because we found no electron density for these residues in the maps. The sidechains of 17 residues in each chain were disordered and not visible in the electron density maps (Arg12, Lys36, Lys112, Lys128, Val168, Arg178, Val185, Lys187, Trp190, Arg191, Val194, Glu195, Phe222, Lys223, Gly257, Glu258, and Ser398).

Validation and Deposition.

We analyzed the stereochemical quality of the models with the JCSG Validation Central suite, which integrates seven validation tools: Procheck 3.5.4, SFcheck 4.0, Prove 2.5.1, ERRAT, WASP, DDQ 2.0, and Whatcheck. The Validation Central suite is accessible at http://www.jcsg.org. Atomic coordinates of the final model and experimental structure factors of TM0064 have been deposited with the PDB and are accessible under the code 1J5S.

Acknowledgements

Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences).

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