Crystal structure of a novel manganese-containing cupin (TM1459) from Thermotoga maritima at 1.65 Å resolution

Authors

  • Lukasz Jaroszewski,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The San Diego Supercomputer Center, 9500 Gilman Drive, La Jolla, California
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  • Robert Schwarzenbacher,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The San Diego Supercomputer Center, 9500 Gilman Drive, La Jolla, California
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  • Frank von Delft,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
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  • Daniel McMullan,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Linda S. Brinen,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Jaume M. Canaves,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The San Diego Supercomputer Center, 9500 Gilman Drive, La Jolla, California
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  • Xiaoping Dai,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
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  • Ashley M. Deacon,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Mike DiDonato,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Marc-André Elsliger,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
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  • Said Eshagi,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Ross Floyd,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Adam Godzik,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The San Diego Supercomputer Center, 9500 Gilman Drive, La Jolla, California
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  • Carina Grittini,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
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  • Slawomir K. Grzechnik,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The San Diego Supercomputer Center, 9500 Gilman Drive, La Jolla, California
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  • Eric Hampton,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Inna Levin,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Cathy Karlak,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Heath E. Klock,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Eric Koesema,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • John S. Kovarik,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Andreas Kreusch,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Peter Kuhn,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
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  • Scott A. Lesley,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Timothy M. McPhillips,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Mitchell D. Miller,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Andrew Morse,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Kin Moy,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
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  • Jie Ouyang,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Rebecca Page,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
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  • Kevin Quijano,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Ron Reyes,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Fred Rezezadeh,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Alyssa Robb,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Eric Sims,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Glen Spraggon,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Raymond C. Stevens,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Henry van den Bedem,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Jeff Velasquez,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
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  • Juli Vincent,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, California
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  • Xianhong Wang,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Bill West,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Guenter Wolf,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. Stanford Synchrotron Radiation Laboratory, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Qingping Xu,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • Keith O. Hodgson,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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  • John Wooley,

    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
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    3. The University of California, San Diego, 9500 Gilman Drive, La Jolla, California
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  • Ian A. Wilson

    Corresponding author
    1. The Joint Center for Structural Genomics, Stanford University, 2575 Sand Hill Rd, Menlo Park, California
    2. The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California
    • JCSG, The Scripps Research Institute, BCP3206, 10550 North Torrey Pines Road, La Jolla, CA 92037
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The TM1459 gene of Thermotoga maritima encodes a conserved hypothetical protein with a molecular weight of 12,977 Da (residues 1–114) and a calculated isoelectric point of 5.6. Currently, no functional annotation has been made for this protein, made fold recognition methods such as Fold and Function Assignment System (FFAS)1 have recognized significant sequence similarity to the family of cupins.2 Here, we report the crystal structure of TM1459 with an endogenous manganese ligand that was determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).3

The structure of TM1459 [Fig. 1(a)] was determined to 1.65 Å resolution using the molecular replacement (MR) method using a search model constructed from oxalate oxidase (PDB: 1FI2),4 despite a very low sequence identity (18%). Data collection, modeling and refinement statistics are summarized in Table I. The final model includes two protein molecules (residues 1–114), 2 manganese ions, and 252 water molecules. The Matthews' coefficient (Vm) for TM1459 is 2.71 Å3/Da, and the estimated solvent content is 54.2%. The Ramachandran plot, produced by Procheck 3.4,5 shows that 92.7% of the residues are in the most favored regions, and 7.3% are in additional allowed regions.

Figure 1.

Crystal structure of TM1459 shown as (a) ribbon diagram of Thermotoga maritima TM1459 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization and the location of the manganese ion (purple sphere). The 310-helices (H1, H2), β-strands (β1–β11 and β-sheets A and B are indicated; and (b) ribbon diagram of TM1459 dimer. β-strands β1 and β9 of the crossover domain-swap interaction are indicated and are labeled β1′ and β9′ in the opposing subunit.

Table I. Summary of Crystal Parameters, Data Collection, and Refinement Statistics for TM1459 (PDB: 1o5n)
  • a

    highest resolution shell

  • bESU = Estimated overall coordinate error13, 17

  • cRmeas=[Σhi|〈Ih〉−Ih,i|]/ΣhΣiIh,i where w=[nh/(nh−1)]1/2 and 〈Ih〉=[ΣinIh,i]/nh. This is the multiplicity-weighted Rsym18

  • d

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

  • e

    Rfree = as for Rcryst, but for 4.9% of the total reflections chosen at random and omitted from refinement.

Space groupP32  
Unit cell parametersa = b = 52.55 Å, c = 96.27 Å, α = β = 90 °, γ = 120 °
Data collectionλ0  
Wavelength (Å)0.9724  
Resolution range (Å)48.13–1.65  
Number of observations124,395  
Number of unique reflections35,029  
Completeness (%)97.8 (87.6)a  
Mean I/σ(I)15.6 (1.9)a  
Rmeas on I0.067 (0.577)a  
Sigma cutoff0.0  
Highest resolution shell (Å)1.74–1.65  
Model and refinement statistics   
Resolution range (Å)33.07–1.65Data set used in refinementλ0
No. of reflections (total)34,991Cutoff criteria|F| > 0
No. of reflections (test)1,729Rcrystd0.208
Completeness (% total)97.7Rfreee0.249
Stereochemical parameters   
Restraints (RMS observed)   
 Bond length0.018 Å  
 Bond angle1.65°  
Average isotropic B-value24.6 Å2  
ESU-based on free R value0.10 Å  
Protein residues/atoms288/1854  
Solvent molecules252  

The final model of the TM1459 monomer is composed of 11 β-strands (β1–β11) and two short 310-helices (H1, H2). The total β-strand content is 43.6%. The TM1459 structure is characterized by two antiparallel β-sheets (A and B) that form a jellyroll β-sandwich with a topology that is characteristic of the cupin-barrel fold3, 4 [Fig. 1(a)]. The seven-stranded β-sheet A (β1′, β2–β4, β6, β9, β11) has a 2347561 topology, where β-strand β1′ is contributed from the neighboring subunit of the dimer by domain-swapping (Fig. 1). The four-stranded β-sheet B (β5, β7, β8, β10) has 1423 topology [Fig. 1(a)]. Each of the 11 β-strands runs approximately perpendicular to the barrel axis. TM1459 forms a dimer linked by a pair of crossovers between the adjacent edge β-strands β1 and β9 from different subunits in the dimer [Fig. 1(b)]. The dimer interface corresponds to interactions between the two A β-sheets with a buried surface area of 863 Å2 per monomer.6

Each TM1459 domain has a metal-binding site in the mouth of the β-barrel [Fig. 2(b)]. The metal ion has octahedral coordination in which four ligands are contributed by conserved histidine side chains that define a new sub-family of cupins. The metal coordination is different from the His, His, Glu, His metal-coordination typically found in cupins and is the second example of a metal coordinated by four histidine residues similar to that observed for manganese in the photosynthetic reaction center of Rhodobacter sphaeroides (PDB: 1YST).7 The metal-binding residues are His52, His54, His58, and His92 with metal-to-atom distances of 2.19, 2.19, 2.25 and 2.13 Å, respectively. The remaining coordination sites are occupied by two water molecules (W1, W2) at distances of 2.06 and 2.24 Å [Fig. 2(b)]. The metal has been assigned as manganese because it gave the best refined B-factor agreement with surrounding atoms (tested for a series of metals) and also based on the identity (Mn) and similar octahedral coordination of the metal in the structural homologue oxalate oxidase.4 Sigma-A-weighted OMIT maps show density continuous with the sulfhydryl group of Cys106, which in subunit A extends as a continuous tube past the Mn-coordinated water molecules. Although the density could not be identified and was modeled as an unknown ligand (UNL) H-bonded to Cys106 [Fig. 2(b)], it does suggest a catalytic role for Cys106.

Figure 2.

(a) Diagram showing the secondary structure elements in TM1459 superimposed on its primary sequence. β-Hairpins are depicted as red loops. Residues coordinating the metal ion are marked with blue dots. (b) The active site of TM1459 showing the proposed manganese ion (Mn), its coordinating residues (His52, His54, His58, His92, and two waters) and Cys106, adjacent to the unidentified density modeled as UNL, is depicted in ball and stick. The atoms are indicated as follows: carbon (grey), oxygen (red), nitrogen (blue), sulfur (orange) and manganese (purple). Metal-ligand bonds are represented as dashed yellow lines.

A structural similarity search, performed with the coordinates of TM1459 using the DALI server,8 indicated that the closest structural homologue is oxalate oxidase (germin) from Hordeum vulgare (PDB: 1FI2), which was used here as a MR search model.4 The RMSD between TM1459 and oxalate oxidase is 1.4 Å over 74 aligned residues with 20% sequence identity. Another structural homologue is TM1287, a putative oxalate decarboxylase whose structure was determined recently (PDB: 1O4T).9 The RMSD between TM1459 and TM1287 is 1.7 Å over 80 aligned residues with 18% sequence identity.

According to FFAS,1 TM1459 has at least five distant homologues in the Thermotoga proteome: TM1287 (18% sequence identity), TM1010 (16%), TM0656 (13%), TM1112 (10%), and TM0736 (8%). Sequence similarity searches with the TM1459 sequence against the non-redundant protein sequence database revealed more than 1000 homologues in the three kingdoms of life, with about 100 homologues from this new sub-family that contain metals coordinated by four histidines. This subfamily comprises single-domain proteins like TM1459, as well as multi-domain proteins like the family of mannose phosphorylases. Models for TM1459 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1459.

The crystal structure reported here is the first representation of a novel subfamily of cupins that contains a metal site coordinated by four histidines. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional determinants of this protein family and the thermostability of these organisms.

Materials and Methods.

Protein production and crystallization.

TM1459 (TIGR: TM1459; Swissprot:Q9X1H0) was amplified by PCR from Thermotoga maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM1459. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth [24 g/L yeast extract, 12 g/L tryptone, 1% (v/v) glycerol, 50 mM 3-(N-Morpholino) propanesulfonic acid (MOPS) pH 7.6] using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 1 mg/mL. 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 Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)] and the cell debris pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a metal chelate affinity resin (Amersham Biosciences) and equilibrated with Equilibration Buffer [50 mM potassium phosphate pH 7.8, 0.25 mM TCEP, 10% (v/v) glycerol, 300 mM NaCl] containing 20 mM imidazole. The Ni-resin was washed with Equilibration Buffer containing 40 mM imidazole, and the protein was eluted with Elution crystallization buffer [20 mM Tris pH 7.9, 10% (v/v) glycerol, 0.25 mM TCEP, 300 mM imidazole]. The eluate was buffer exchanged into crystallization buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated to ≈10 mg/mL for crystallization trials by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method10 with standard JCSG crystallization protocols.3 The crystallization solution contained 50% PEG 200, 0.2M NaCl, and 0.1M Na-phosphate-citrate (pH 4.2) (final pH 5.2). The crystals were indexed in the trigonal space group P32 (Table I).

Data collection.

Native diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, USA) on beamline 11-1 using the BLU-ICE11 data collection environment (Table I). The data set was collected at 100K using a Quantum 315 charge-coupled device (CCD) detector. Data were integrated and reduced using Mosflm12 and then scaled with the program SCALA from the CCP4 suite.13 The crystal suffered from partial twinning (twinning fraction 0.35) and data were detwinned with program XPREP. Data statistics are summarized in Table I.

Structure solution and refinement.

The structure was determined by molecular replacement using program MOLREP from the CCP4 suite.13 A homology model based on the FFAS1 alignment between TM1459 and oxalate oxidase (PDB: 1FI2), with a sequence identity of only 18%, was constructed with the modeling program Whatif14 and used as a search model. Structure refinement was performed using REFMAC5,13 O15 and Xfit.16 Refinement statistics are summarized in Table I. The final model includes a protein dimer (residues 1–114), two manganese ions, and 252 water molecules in the asymmetric unit. No electron density was observed for the expression or purification tag.

Validation and deposition.

Analysis of the stereochemical quality of the models was accomplished using Procheck 3.4 and SFcheck 4.0.5, 13 Protein quarternary structure analyis and buried surface area were taken from the PQS server (http://pqs.ebi.ac.uk/). Atomic coordinates of the final model and experimental structure factors of TM1459 have been deposited with the PDB and are accessible under the code 1o5n.

Acknowledgements

This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). 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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