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The structure of Ski8p, a protein regulating mRNA degradation: Implications for WD protein structure

Section of Molecular and Cellular Biology, University of California, Davis, California 95616, USA

Reprint requests to: David K. Wilson, Section of Molecular and Cellular Biology, 1 Shields Ave., University of California, Davis, CA 95616, USA; e-mail: dave{at}alanine.ucdavis.edu; fax: (530) 752-3085.

(RECEIVED February 20, 2004; FINAL REVISION March 19, 2004; ACCEPTED March 22, 2004)

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Ski8p is a 44-kD protein that primarily functions in the regulation of exosome-mediated, 3' 5' degradation of damaged mRNA. It does so by forming a complex with two partner proteins, Ski2p and Ski3p, which complete a complex that is capable of recruiting and activating the exosome/Ski7p complex that functions in RNA degradation. Ski8p also functions in meiotic recombination in complex with Spo11 in yeast. It is one of the many hundreds of primarily eukaryotic proteins containing tandem copies of WD repeats (also known as WD40 or -transducin repeats), which are short ~40 amino acid motifs, often terminating in a Trp–Asp dipeptide. Genomic analyses have demonstrated that WD repeats are found in 1%–2% of proteins in a typical eukaryote, but are extremely rare in prokaryotes. Almost all structurally characterized WD-repeat proteins are composed of seven such repeats and fold into seven-bladed propellers. Ski8p was thought to contain five WD repeats on the basis of primary sequence analysis implying a five-bladed propeller. The 1.9 Å crystal structure unexpectedly exhibits a seven-bladed propeller fold with seven structurally authentic WD repeats. Structure-based sequence alignments show additional sequence diversity in the two undetected repeats. This demonstrates that many WD repeats have not yet been identified in sequences and also raises the possibility that the seven-bladed propeller may be the predominant fold for this family of proteins.

Keywords: Ski8; YDR267c; Rec103

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04704704.

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Proteins containing multiple tandem copies of WD repeats are involved in a diverse range of physiological roles including signal transduction, RNA splicing and export, protein degradation, cell cycle regulation, transcriptional regulation, apoptosis, and many other critical eukaryotic cellular functions (Neer et al. 1994; Yu et al. 2000; van Nocker and Ludwig 2003). The common function between the majority of these is that they appear to be involved in protein–protein interactions. Sequences contained within the repeat are divergent and can also contain insertions that complicate their identification on the basis of sequence information alone. Structural characterizations of these proteins are beginning to reveal the diversity possible within these repeats, and will provide a basis for making more accurate predictions in the future.

Ski8p is a WD-repeat protein that was originally identified as one of a number of genes functioning in the suppression of the production of a killer toxin encoded by double-stranded satellite RNA from the virus L-A (Toh-e et al. 1978; Ridley et al. 1984). This activity gave them the name superkiller (SKI) in yeast. More recently, several of these Ski proteins, including Ski8p, have been found to function in a mechanism that degrades mRNA lacking a stop codon (Frischmeyer et al. 2002; van Hoof et al. 2002; Parker and Song 2004). This pathway is specific for mRNAs lacking a stop codon, and does not follow the normal sequence of 3'-deadenylation, 5'-decapping, and 5' 3' degradation. Rather, stalled ribosomes at the end of mRNA are used to identify nonstop message and are used to recruit a large complex of proteins that degrade the RNA in a 3'5' manner. This process is executed by the exosome, a core complex of at least 10 proteins that are all believed to possess RNAse activity. The exosome acts in conjunction with the G protein Ski7p (Araki et al. 2001) and the Ski complex consisting of the RNA helicase Ski2p, the tetratricopeptide repeat protein Ski3p and Ski8p (van Hoof and Parker 1999). In addition to its major role in surveillance of RNA integrity, it also appears to function in 5.8S rRNA maturation (Mitchell et al. 1996) in a manner independent of the Ski complex (Anderson and Parker 1998).

The exosome appears to be generally present in eukaryotes as a large, 300–400-kD complex of 10 or more subunits. It has been identified in Trypanosoma brucei and Caenorhabditis elegans (Estevez et al. 2001), and it appears to exist in Arabidopsis thaliana (Chekanova et al. 2000). It is also present in human and is composed of homologous proteins (Allmang et al. 1999; Brouwer et al. 2001).

Ski8p contains tandem WD repeats (also known as WD40 and -transducin repeats), a poorly conserved sequence that can contain 40 or more amino acids (Anderson and Parker 1998). On the basis of the roles assigned to hundreds of other WD-repeat proteins, it is likely that Ski8p functions as a scaffolding or protein-binding protein. In general, these proteins either regulate the function of other proteins by modulating binding, or act to colocalize two or more other proteins. Coimmunoprecipitation experiments have demonstrated that Ski8p associates with at least two other proteins, Ski2p and Ski3p, forming a 1:1:1 heterotrimeric complex (Brown et al. 2000). The identical phenotypes of Ski2p, Ski3p, and Ski8p mutants support the idea that these proteins interact with one another in accomplishing a common task. Furthermore, the binding of Ski2p has been shown to be dependent on the initial formation of the Ski8p–Ski3p (Brown et al. 2000).

Ski8p homologs functioning in exosome regulation have yet to be found in organisms outside of Saccharomyces cerevisiae. Clues about a true homolog in other organisms have been obtained from yet another function in S. cerevisiae as REC103, one of a family of genes required for meiotic recombination (Gardiner et al. 1997). The frequency of crossing over for Rec103 as measured by both the frequency of heterozygous drug resistance markers and by spore dissection revealed that Rec103 mutants exhibited a reduction of over 100-fold, similar to spontaneous recombination frequencies (Gardiner et al. 1997). Very recently, Ski8p has been identified as an essential partner for Spo11p in the formation of double-stranded breaks necessary for meiotic recombination (Arora et al. 2004).

The S. pombe protein, Rec14, is significantly smaller (33 kD), but has 24% sequence identity (40% homology) and performs a similar role in recombination. Rather than the five WD repeats found in the Ski8p sequence (Brown et al. 2000), Rec14 was found to contain six WD repeats in its sequence (Evans et al. 1997). The lack of clear Ski8p homologs in other organisms could stem from the fact that Ski8p is a WD repeat-containing protein and a member of a very large family of such proteins with significant sequence homology obscuring the identity of the exact functional homolog.

Our goal was to structurally characterize Ski8p so that we could begin to understand its role in exosome regulation and its other apparent functions. We also want to explore the structure of WD-repeat proteins to determine whether a common seven-bladed propeller folding motif exists for these proteins. Here, we report the X-ray structure of one of these proteins, Ski8p, in hopes that it will give us insight into a more general structural motif formed by WD-repeat proteins. The Ski8p structure, as well as those of the subunit of the G-protein (Lambright et al. 1996; Sondek et al. 1996), the carboxy-terminal domain of Tup1p (Sprague et al. 2000), Aip1p (Voegtli et al. 2003), and several others, all show evidence of a common seven-bladed propeller motif. In addition to these three proteins, many other WDs are also involved in protein–protein binding. Further structural and biochemical studies may enable a greater understanding as to the relationship between a common WD structural motif and protein–protein binding.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Overall structure
We have determined the crystal structure of Ski8p to 1.9 Å using multiwavelength anomalous dispersion from crystals of selenomethionine-substituted protein (Table 1). The final structure contains 379 of 397 total expected residues. The model starts at amino acid 3 and continues through residue 395. Gaps in the model occur in segments from residues 134–137, 228–230, and 278–285, which occur in loop regions. The structure was refined using CNS (Brünger et al. 1998), which yielded a final R_cryst of 19.1% (R_free = 24.6%) and r.m.s. deviations from ideal bond distances and angles of 0.012 Å and 1.7°, respectively. Using PROCHECK, we found that 88.2% of the residues fell within the most favored region of a Ramachandran plot, whereas no residues fell into the disallowed regions (Laskowski et al. 1993).

View larger version (49K): [in this window] [in a new window]   Figure 1. Stereo views of the C trace of Ski8p oriented (A) down the propeller axis (colored by temperature factor), and (B) normal to the propeller axis ( strands are colored red; loop regions are shown in blue). Molecular graphics in Figs. 1, 2, 3, and 5 were created using BOBSCRIPT (Esnouf 1997).

The number of super secondary-blade structures and WD repeats are equal; however, each blade is not produced by a single repeat. As seen in other propeller proteins, the repeat yields the D strand of one blade and the A, B, and C strands of the next one. The D strand of the first repeat interlocks with the C strand of the last repeat, a feature that is conserved among all propeller proteins and believed to be important for protein stability (Jawad and Paoli 2002). Thus, although there are an equivalent number of WD repeats and blades, there is not a one-to-one correspondence. The A strands of each repeat line the inner circumference of the propeller structure. The top of the molecule contains the loops that connect the D strand of one blade to the A strand of another. The circular structure is closed by a D strand in the last blade, which contains the first 15 residues of Ski8p.

Several polar residues (Lys 128, Lys 240, Lys 361, and Ser 298) project into the pore from the inner A strand of different blades. These and numerous water molecules form hydrogen bonding and charged contacts with a well-ordered sulfate. Another sulfate is located on the exterior of the protein within 3.2 Å of Thr 328, Lys 314, and Arg 325. Neither of these sulfates is involved in intermolecular contacts with adjacent Ski8p molecules.

Blade structure
Overlaying the backbones of the individual blades of Ski8p reveals that they are structurally very similar in the core regions, but show considerable divergence in the loops connecting the strands. With the exception of the amino-terminal helical region, G can be considered a minimal WD-repeat protein, with each repeat consisting of ~40 canonical amino acids and almost no excursions in the loops connecting individual strands. In comparison, the repeats in Ski8p range in size from 40 residues in the second WD to 70 in the seventh as a result of sequence insertions in the loop regions. Similar diversity is also seen in some of the other WD-repeat structures that have been determined. In Ski8p, the additional residues can be generally assigned to larger DA and CD loops rather than to a change in the core parts of the blade. The seventh WD repeat, for instance, contains an extended 20-residue insertion between its D and A strands, whereas the fifth and sixth WD repeats contain ~50 residues, with two 10-residue insertions composed of residues 226 through 235 and 277 through 288. All of these loops project into solvent, whereas the last two contained no identifiable |F_o - F_c| density, probably owing to their flexibility.

Structural neighbors
As expected, this fold is similar to other WD-repeat proteins, such as the subunit of the cell-signaling G protein transducin (Wall et al. 1995; Lambright et al. 1996; Sondek et al. 1996) and later in a fragment of the Tup1p corepressor protein (Sprague et al. 2000), the p40 subunit of the Arp2/3 complex (Robinson et al. 2001), the actin-binding protein Aip1p (Voegtli et al. 2003) and a domain of the Groucho/TLE1 corepressor protein (Pickles et al. 2002). Lower, but detectable structural homology is seen with non-WD repeat containing -propeller proteins such as RCC1 and kelch-repeat proteins.

An overlay of Ski8p with the representative Tup1p and G structures reveals that the core of these three proteins are strikingly similar, giving r.m.s. deviations of 1.38 Å and 1.03 Å, respectively; however, they differ in length (Figs. 2,3). The majority of these extra residues present in both Tup1p fragment (487 amino acids) and Ski8p (397 amino acids) as compared with the G protein (340 amino acids) are found in the loops connecting the strands rather than interrupting the regularity of the general blade structure (Sprague et al. 2000). Most variability among the G protein and Ski8p occurs on the DA loop joining adjacent blades. This high degree of structural conservation can be seen in an overlay of a G blade (Asp 267–Gly 310) with all seven Ski8p blades. The overlap yields r.m.s. deviations between 0.38 Å and 1.06 Å, similar to the range of 0.6–1.2 Å resulting from overlapping individual G blades (Wall et al. 1995). Comparison of the G blade with blades 5 and 6 from Ski8p (the two unpredicted from sequence) results in r.m.s. deviations of 0.67 Å and 0.61 Å, respectively, indicating that they are authentic.

View larger version (68K): [in this window] [in a new window]   Figure 2. (A) A stereo view of structural alignments of the WD repeats in Ski8p demonstrate that all are authentic. Repeats one through seven are colored red through purple, respectively. (B) Structure-based sequence alignments of the conserved regions of the WD repeats in Ski8p, consisting of strands A, B, and C. Included are those previously identified by sequence (designated WD1–WD5) and additional repeats found in the structure (S1 and S2). Larger numbers of nonhomologous residues, which are inserted into the repeat loop regions, are abbreviated with square brackets. The consensus sequence as described by Neer et al. (1994) is shown below the alignments in descending frequency.

View larger version (51K): [in this window] [in a new window]   Figure 3. A superimposition of Ski8p (blue), G (green), and Tup1p (red) in an orientation similar to that shown in Figure 1A indicate that the core fold is preserved. Structural divergence is concentrated in the loops, particularly those on the top of the propeller (directed out of the page).

G exhibits no such variations except where important in binding (Sondek et al. 1996). In G , there is a four-residue insertion between strands C and D of blade 2, providing part of the binding interaction with G-, hinting that the exposed Ski8p loops may function in binding its known partner, Ski3p.

Structural divergence in the blade
In general, the existence of well-ordered blades with large or disordered D-A loops and C-D turns suggest that it is possible to maintain the blade structure despite variations in the traditional WD repeat pattern. More specifically, the presence of large or disordered loops within the WD repeats that were not predicted (repeats four and five) with low r.m.s.d. suggests that there may be more variability within these regions than that allowed by current algorithms (Fig. 4). We also see that the sixth and seventh WDs are actually much larger than predicted, with the differences forming large loop regions. Further, because current algorithms underestimated the size of WDs six and seven, as well as overlooking the fourth and fifth repeats, it appears that methods of identifying WD motifs in sequence can be improved.

View larger version (36K): [in this window] [in a new window]   Figure 4. Confirmed and potential WD repeats in other representative budding yeast proteins. (A) Repeats that had been previously located in sequences are shown in red. Additional potential repeats are shown in yellow. In the cases of Aip1p and Ski8p, these additional repeats have been confirmed by structures. (B) Sequence alignments of additional repeats described in A. Shown are the conserved regions in strands A, B, and C, and consensus sequences as described in Figure 2.

View larger version (27K): [in this window] [in a new window]   Figure 5. Divergence and disappearance of the structural tetrad in representative Ski8p blades. (A) Blade 1 contains a nearly conventional tetrad involving hydrogen bonding between the side chains of Trp 41, Ser 31, and His 15. The aspartate completes the tetrad in the canonical structure. (B) Blade 3 has Trp 153 and Asp 145 at the expected positions, but other residues are divergent. The core of this blade is stabilized via a mixture of hydrogen bonding and hydrophobic interactions. (C) Blade 7 contains residues that are homologous in sequence to a conventional tetrad. The positions are structurally divergent, however.

Almost all known proteins with WD-repeat domains are involved in protein–protein interactions, but have extremely diverse cellular functions (Smith et al. 1999). Currently, only a relatively small number have been structurally characterized. With the exception of Cdc4p, all have been found to fold into domains containing circular seven-bladed propellers. Whereas G is involved in cell–cell signaling, Tup1p is believed to be a general transcription inhibitor in yeast. Despite the extreme difference in the physiological role of these individual proteins, there could be a similarity in how they carry out the function of reversible protein–protein binding. It is possible that a seven-bladed propeller is common to most WDs and has been evolutionarily groomed to act as a protein adapter, bringing different proteins within close proximity of one another, perhaps by using the top surface of the protein formed by the DA and BC loops, a hypothesis that has been previously put forth (Smith et al. 1999). This would allow modulation of protein-binding properties without significant perturbation of the core structure. Structures of G (Wall et al. 1995; Loew et al. 1998), p40 (Robinson et al. 2001), and Cdc4 (Orlicky et al. 2003) in complex with partner proteins or peptides demonstrate full or partial involvement of this region in binding.

Only further study can tell whether the latter is a general property of this fold. In the future, this can be studied by examining how Ski8p binds to the TPR-repeat protein, Ski3p. Proteins containing TPRs are often found associated with WD proteins, suggesting the possibility of a common function for TPR–WD binding partners. The seven-bladed Tup1p tetramer binds Ssn6p, also a TPR repeat, forming a transcriptional corepressor complex (Sprague et al. 2000). In this case, specificity is conferred by a third DNA-binding protein. One of these, Mat2 contains a carboxy-terminal domain that binds the TPR of Ssn6 and DNA, as well as an amino-terminal domain that binds Tup1p. Ski8p has a similar arrangement, in which a complex formed between Ski8p and TPR-containing Ski3p, binds Ski2p, a member of the superfamily II RNA helicase family.

The discovery that the G-protein, Ski7p, binds both the exosome and the Ski8–Ski3–Ski2 complex lends itself to comparisons with the G subunit of the signal-transducing G-protein. Both Ski8p and G are seven-bladed propellers that bind the G-proteins, Ski7p and the G subunit, respectively. It is tempting to guess that Ski8p may accomplish reversible binding through a nucleotide-exchange mechanism similar to that of G.

Implications for other WD-repeat protein structures
The pseudo sevenfold symmetry seen in Ski8p, along with similar structures seen for G, the Tup1p carboxy-terminal domain, p40 of the Arp2/3 complex, and the carboxy-terminal domain of Groucho, might suggest the possibility that all, or perhaps most, WD-repeat proteins fold into seven-bladed propellers or have multiple seven-bladed domains for those found to contain more than seven in the sequence. There are many structural precedents for common core folds, such as the Rossmann fold for nucleotide binding and the (/)₈ barrel for enzymes (Branden 1991). We have determined that the structure of Bub3p, a mitotic checkpoint protein that was thought to have four WD repeats (Taylor et al. 1998), actually folds into a seven-bladed propeller (D. Wilson and D. Cerna, unpubl.). Similarly, Aip1p was believed to have as many as 10 of these repeats (Ono 2001). Subsequent crystallographic analysis revealed the presence of two-propeller domains, each consisting of seven structurally authentic WD repeats (Voegtli et al. 2003). This provides evidence that these proteins may have a propensity to form smaller propeller domains rather than a single large domain when larger numbers of repeats occur in tandem. In addition, we have manually aligned a number of sequences that contain an irregular number of repeats and found plausible, yet divergent, WD sequences to bring the total number to seven (Fig. 4). There is no possibility that the seven-bladed propeller is the universal fold for WD-repeat proteins. Proteins that have only six repeats and no extra sequence to accommodate another repeat have been identified previously (Pryer et al. 1993). Moreover, the structure of Cdc4p reveals an eight-bladed propeller (Orlicky et al. 2003).

Modeling studies are consistent with the idea of seven-bladed core structure for these proteins, but do not preclude numbers close to seven. Attempts to generate a three-dimensional model for the Hat protein from the cyanobacterium Synechocystis PCC6803, a WD-repeat protein believed to contain 11 repeats have been described (Hisbergues et al. 2001). An 11-bladed model did not allow for efficient interactions between blades, which likely led to structural instability. The possibility of a two-domain model was also considered with a four-bladed and a seven-bladed domain. Although experimental precedents exist for non-WD-containing, four-bladed propellers (Faber et al. 1995), the four-bladed model for the Hat protein was not entirely satisfactory. In lieu of such strained models, we suggest that extra repeats may be hidden in the sequence that could lead to a protein that is more stable. The difficulty in identifying these repeats is underscored by the lack of sequence conservation seen in the Ski8p repeats. Even at the most highly conserved residue in the repeat, an aspartate located at the eighth position from the end of the consensus sequence, which is present in ~90% of the repeats identified, considerable divergence is seen.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Cloning, expression, and purification
SKI8 was amplified from yeast genomic DNA and inserted into the NdeI and SmaI restriction sites in the multiple cloning site of the pTYB2 vector (New England Biolabs) containing a T7 promoter by use of the forward primer 5'-GCTAGTGCATATGTC CAAAGTGTTTATTGCC and 5'-ACTGCCCGGGTTTACCGC CAGCTTCTCTAAA. The fusion protein resulting from this construct contained a self-cleavable intein tag as well as a chitin-binding domain, simplifying initial protein purification.

Wild-type Ski8p was expressed by transforming the Escherichia coli strain ER2566 (DE3) with this construct, followed by overnight growth in LB medium with 100 µg/mL ampicillin. The overnight culture was centrifuged at 5000 rpm for 5 min, washed, and resuspended in water. These cells were used to inoculate Luria Bertani medium with 100 µg/mL ampicillin. Upon reaching an optical density of .6 at 600 nm, the cells were spun down and washed with water. Following resuspension, the cells were cooled down to 15°C and induced by adding 0.5 mM isopropyl thiogalactoside. After 12 h, the cells were harvested by centrifugation and immediately resuspended in lysis buffer (100 mM Tris, 500 mM NaCl, 0.5 mM EDTA, 0.1% [v/v] Triton X-100 [pH 8.0]). A protease inhibitor cocktail was also added to stabilize the protein after lysis. The cells were lysed using a microfluidizer (Microfluidics, Inc.) at 15,000 p.s.i., followed by sonication and clarification by centrifugation. The supernatant was filtered over a 0.2-µm filter and loaded onto a chitin column (New England Biolabs) at 1 mL/min. The column was washed overnight with 1 L of lysis buffer at 1 mL/min. Intein cleavage was induced with 50 mM 2-mercaptoethanol for 15 h. The buffer was changed to 50 mM Hepes (pH 7.5), and the protein was concentrated to 15 mg/mL using a 15-mL concentrator with a 10-kDa cutoff (Millipore). The concentrated protein was run over a quaternized polyethyl-eneimine anion-exchange column (PerSeptive Biosystems) with 25 mM Bis-Tris-propane, 25 mM Tris buffer (pH 6.5). Ski8p eluted at ~300 mM NaCl in a 0–1 M NaCl gradient. The protein was then concentrated and exchanged with a 50 mM Hepes (pH 7.5) buffer to a final concentration of 15 mg/mL for crystallization.

Selenomethionine-substituted Ski8p was produced using a previously described protocol with slight modifications (Doublie 1997). Sterilized solutions of lysine, phenylalanine, and threonine were also added to a final concentration of 1 mg/mL. Additionally, isoleucine, leucine, and valine were added to a final concentration of 0.5 mg/mL. The cells were induced at an OD₆₀₀ of 0.85 by adding 500 µM isopropyl thio galactoside for 7 h. The cells from the selenomethionine prep were lysed and loaded onto a chitin column in the same manner as the wild-type prep. The soluble protein was concentrated and changed into a 50-mM Hepes (pH 7.5) buffer. The protein was run under identical conditions as wild-type eluting at 25 msec in a continuous 0–1 M Nacl gradient. The protein was changed into a buffer consisting of 20 mM Hepes, 20 mM NaCl, 10 mM 2-mercaptoethanol (pH 7.5), and concentrated to a final concentration of 15 mg/mL.

Crystallization and structure determination
Native crystals were grown overnight by hanging drop vapor diffusion in 30% PEG 8000, 150 mM ammonium sulfate, and 100 mM cacodylic acid (pH 6.5). However, the protein degraded in solution over time, and the crystals often looked cracked or lasted only a few weeks. To ensure longer lasting crystals, subsequent native protein preps were stored in 20 mM NaCl, 10 mM 2-mercaptoethanol, 20 mM Hepes (pH 7.5); however, subsequent crystals diffracted no better than 2.2 Å, regardless of the presence or absence of NaCl and 2-mercaptoethanol. Many attempts at molecular replacement were made using a variety of approaches and search models, but none of these were successful. Efforts were then directed to determine phase information using multiwave-length anomalous dispersion.

Selenomethionine crystals were grown in 18%–25% PEG 8K, 150–200 mM ammonium sulfate, 10 mM BME, 50 mM MES (pH 8.0) in 3–5 d. Both native and selenomethionine crystals were cryoprotected by briefly streaking them through a drop of Para-tone-N oil to remove surface buffer before cooling and collecting data at 100°K. The resulting crystals were thin-rod shaped and grew to approximate dimensions of 0.1 mm x 0.1 mm x 0.6 mm. Data collection indicated P2₁2₁2₁ diffraction symmetry and lattice parameters of a = 65.54 Å, b = 67.64 Å, c = 80.31 Å. The sele-nomethionine derivative was used for phasing by collecting a 2.0 Å MAD data set at the Stanford Synchrotron Radiation Laboratory (SSRL). The data were processed with DENZO and SCALEPACK (Otwinowski and Minor 1997). SOLVE was used to locate four of the seven expected selenium sites (including the initiator methionine) at residue numbers 119, 183, 294, and 331, and was then used to refine phases to yield a figure of merit of 0.64–2.06 Å resolution (Terwilliger and Berendzen 1999). The map was further improved using RESOLVE to yield a figure of merit of 0.71. The initial structure was obtained using ARP/wARP, which yielded 268 of the expected 397 amino acids fit to the electron-density map (Perrakis et al. 1999). The final model was produced by several cycles of refitting, addition of water molecules using the graphics program O (Jones et al. 1991), and refinement using CNS (Brünger et al. 1998). Statistics describing the quality of the final structure are given in Table 2.

	Acknowledgments

 
This work was supported by grant GM66135 from the NIH. The data collection facilities at Stanford Synchrotron Radiation Laboratory are funded by the U.S. Department of Energy and the NIH. Coordinates have been deposited in the Protein Data Bank under accession no. 1SQ9.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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