Calpain-3 is an intracellular Ca2+-dependent cysteine protease abundant in skeletal muscle. Its physiological role in the sarcomere is thought to include removing damaged muscle proteins after exercise. Loss-of-function mutations in its single-copy gene cause a dystrophy of the limb-girdle muscles. These mutations, of which there are over 500 in humans, are spread all along this 94-kDa multi-domain protein that includes three 40+-residue sequences (NS, IS1, and IS2). The latter sequences are unique to this calpain isoform and are hypersensitive to proteolysis. To investigate the whole enzyme structure and how mutations might affect its activity, we produce the proteolytically more stable 85-kDa calpain-3 ΔNS ΔIS1 form with a C129A inactivating mutation as a recombinant protein in E. coli. During size-exclusion chromatography, this calpain-3 was consistently eluted as a much larger 0.5-MDa complex rather than the expected 170-kDa dimer. Its size, which was confirmed by SEC-MALS, Blue Native PAGE, and AUC, made the complex amenable to single-particle cryo-EM analysis. From two data sets, we obtained a 3.85-Å reconstruction map that shows the complex is a trimer of calpain-3 dimers with six penta-EF-hand domains at its core. Calpain-3 has been reported to bind the N2A region of the giant muscle protein titin. When this 37-kDa region of titin was co-expressed with calpain-3 the multimer was reduced to a 320-kDa particle, which appears to be the calpain dimer bound to several copies of the titin fragment. We suggest that newly synthesized calpain-3 is kept as an inactive hexamer until it binds the N2A region of titin in the sarcomere, whereupon it dissociates into functional dimers.

ABSTRACT Bacteria often make initial contact with their hosts through the ligand-binding domains of large adhesin proteins. Recent analyses of repeats-in-toxin (RTX) adhesins in Gram-negative bacteria suggest that ligand-binding domains can be identified by the way they emerge from “split” domains within the adhesin. Here, using this criterion and an AlphaFold3 model of a 5047-residue RTX adhesin from Aeromonas hydrophila, we identified three different ligand-binding domains in this fibrillar protein. The crystal structures of the two novel domains were solved to 1.4 and 1.95 Å resolution, respectively, and demonstrate excellent agreement with their modeled structures. The other domain was recognized as a carbohydrate-binding module based on its beta-strand topology and confirmed by its micromolar affinity for fucosylated glycans, including the Lewis B and Y antigens. This lectin-like module, which was recombinantly produced with its companion split domain and nearby extender domain, bound to a wide variety of cells including yeasts, diatoms, erythrocytes, and human endothelial cells. In each case, 50 mM free fucose prevented this binding and may offer some protection from infection. The carbohydrate-binding module with its neighboring domains also caused aggregation of yeast and erythrocytes, which was again blocked by the addition of free fucose. The second putative ligand-binding domain has a beta-roll structure supported by a parallel alpha-helix, and the third is a homolog of a von Willebrand Factor A domain. These two domains bind to a more limited range of cell types, and their ligands have yet to be identified.IMPORTANCECharacterizing the ligand-binding domains of fibrillar adhesins is important for understanding how bacteria can colonize host surfaces and how this colonization might be blocked. Here, we show that the opportunistic pathogen, Aeromonas hydrophila, uses a carbohydrate-binding module (CBM) to attach to several different cell types. The CBM is one of three ligand-binding domains at the distal tip of the adhesin. Identifying the glycans bound by the CBM as Lewis B and Y antigens has helped explain the range of cell types that the bacterium will bind and colonize, and it has suggested sugars that might interfere with these processes. Indeed, fucose, which is a constituent of the Lewis B and Y antigens, is effective at 50 mM concentrations in blocking the attachment of the CBM to host cells. This will lead to the design of more effective inhibitors against bacterial infections.

Bacterial adhesins attach their hosts to surfaces that the bacteria will colonize. This surface adhesion occurs through specific ligand-binding domains located towards the distal end of the long adhesin molecules. However, recognizing which of the many adhesin domains are structural and which are ligand binding has been difficult up to now. Here we have used the protein structure modeling program AlphaFold2 to predict structures for these giant 0.2- to 1.5-megadalton proteins. Crystal structures previously solved for several adhesin regions are in good agreement with the models. Whereas most adhesin domains are linked in a linear fashion through their N- and C-terminal ends, ligand-binding domains can be recognized by budding out from a companion core domain so that their ligand-binding sites are projected away from the axis of the adhesin for maximal exposure to their targets. These companion domains are "split" in their continuity by projecting the ligand-binding domain outwards. The "split domains" are mostly β-sandwich extender modules, but other domains like a β-solenoid can serve the same function. Bioinformatic analyses of Gram-negative bacterial sequences revealed wide variety ligand-binding domains are used in their Repeats-in-Toxin adhesins. The ligands for many of these domains have yet to be identified but known ligands include various cell-surface glycans, proteins, and even ice. Recognizing the ligands to which the adhesins bind could lead to ways of blocking colonization by bacterial pathogens. Engineering different ligand-binding domains into an adhesin has the potential to change the surfaces to which bacteria bind.

RTX adhesins are long, multi-domain proteins present on the outer membrane of many Gram-negative bacteria. From this vantage point, adhesins use their distal ligand-binding domains for surface attachment leading to biofilm formation. To expand the reach of the ligand-binding domains, RTX adhesins maintain a central extender region of multiple tandem repeats, which makes up most of the proteins' large molecular weight. Alignments of the 10-15-kDa extender domains show low sequence identity between adhesins. Here we have produced and structurally characterized protein constructs of four tandem repeats (tetra-tandemers) from two different RTX adhesins. In comparing the tetra-tandemers to each other and already solved structures from Marinomonas primoryensis and Salmonella enterica, the extender domains fold as diverse beta-sandwich structures with widely differing calcium contents. However, all the tetra-tandemers have at least one calcium ion coordinated in the linker region between beta-sandwich domains whose role appears to be the rigidification of the extender region to help the adhesin extend its reach.

Antifreeze proteins (AFPs) are characterized by their ability to adsorb to the surface of ice crystals and prevent any further crystal growth. AFPs have independently evolved for this purpose in a variety of organisms that encounter the threat of freezing, including many species of polar fish, insects, plants and microorganisms. Despite their diverse origins and structures, it has been suggested that all AFPs can organize ice-like water patterns on one side of the protein (the ice-binding site) that helps bind the AFP to ice. Here, to test this hypothesis, we have solved the crystal structure at 2.05 Å resolution of an AFP from the longhorn beetle, Rhagium mordax with five molecules in the unit cell. This AFP is hyperactive, and its crystal structure resembles that of the R. inquisitor ortholog in having a β-solenoid fold with a wide, flat ice-binding surface formed by four parallel rows of mainly Thr residues. The key difference between these structures is that the R. inquisitor AFP crystallized with its ice-binding site (IBS) making protein–protein contacts that limited the surface water patterns. Whereas the R. mordax AFP crystallized with the IBSs exposed to solvent enabling two layers of unrestricted ordered surface waters to be seen. These crystal waters make close matches to ice lattice waters on the basal and primary prism planes.

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca2+, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.

Limb-girdle muscular dystrophy type 2a arises from mutations in the Ca2+-activated intracellular cysteine protease calpain-3. This calpain isoform is abundant in skeletal muscle and differs from the main isoforms, calpain-1 and -2, in being a homodimer and having two short insertion sequences. The first of these, IS1, interrupts the protease core and must be cleaved for activation and substrate binding. Here, to learn how calpain-3 can be regulated and inhibited, we determined the structures of the calpain-3 protease core with IS1 present or proteolytically excised. To prevent intramolecular IS1 autoproteolysis, we converted the active-site Cys to Ala. Small-angle X-ray scattering (SAXS) analysis of the C129A mutant suggested that IS1 is disordered and mobile enough to occupy several locations. Surprisingly, this was also true for the apo version of this mutant. We therefore concluded that IS1 might have a binding partner in the sarcomere and is unstructured in its absence. After autoproteolytic IS1 removal from the active Cys129 calpain-3 protease core, we could solve its crystal structures with and without the cysteine protease inhibitors E-64 and leupeptin covalently bound to the active-site cysteine. In each structure, the active state of the protease core was assembled by the cooperative binding of two Ca2+ ions to the equivalent sites used in calpain-1 and -2. These structures of the calpain-3 active site with residual IS1 and with bound E-64 and leupeptin may help guide the design of calpain-3-specific inhibitors.

Antifreeze proteins (AFPs) are characterized by their capacity to adsorb to the surface of ice crystals and prevent their growth. This adsorption lowers the freezing temperature of a solution below its melting point. AFPs have independently evolved in a variety of organisms that may encounter the threat of freezing, including many species of polar fish, insects, plants and microorganisms. Despite their diverse origins and structures we suggest that all AFPs organize ice-like water patterns on one side of the protein (the icebinding site) that then bind the AFP to ice. Here, to help test this hypothesis, we have solved two AFP crystal structures. One is of Lake Ontario midge (Chironomidae) AFP, which has intermediate antifreeze activity. Previously our group modelled the midge AFP based on its sequence characteristics with the crystal structure of Tenebrio molitor AFP as a template. The midge crystal structure at 1.9 Å-resolution is a close match to the modelled structure and shows a 10-residue repeated solenoid fold, with 8 disulfide-bonds stabilizing the coils and 7 Tyr pointing outward from one side of the solenoid structure as a potential ice-binding site. The second protein crystal structure is from Rhagium mordax, a longhorn beetle, solved at 2.05-Å resolution. This AFP is hyperactive and its crystal structure resembles that of the Rhagium inquisitor ortholog in having a β-solenoid fold with a wide, flat, ice-binding surface formed by four parallel rows of mainly Thr residues. The key difference between these structures is that the Rhagium mordax AFP has crystallized with its ice-binding site exposed to solvent providing the opportunity to see the organization of surface waters. In contrast the Rhagium inquisitor AFP crystallized with its ice-binding site making protein-protein contacts that obscure the surface water patterns. Supported by CIHR and the Canada Research Chair program

The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain.

The alpha-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the alpha-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A- CAT). Crystallization of A- CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the beta- and alpha-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A- CAT hydrolyzed ATP, ADP, and AMP with k(cat) values of 1.9, 0.6, and 0.32 min(-1), respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2'/3'-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded K-d values for ATP, ADP, AMP, and adenosine of 20 +/- 3, 60 +/- 20, 160 +/- 60, and 45 +/- 15 +/- mu M, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg2+ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased k(cat) values for kinase and ATPase activities by 3-6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.

Skeletal muscle-specific protein calpain-3 (CAPN3/p94) is a member of the calcium-dependent cysteine protease family. CAPN3 is indispensable for the maintenance and survival of functional skeletal muscles, and defects in the CAPN3 gene that cause loss of enzyme activity lead to the development of limb-girdle muscular dystrophy type 2A. The CAPN3 protease core (domains PC1 and 2) is particularly rich in these inactivating mutations. The whole enzyme, a homodimer of CAPN3, undergoes very rapid autoproteolysis. It has two insertion sequences, IS1 and IS2 not found in other calpains, and a long N-terminal region, NS. IS1 is present in the protease core. Previous biochemistry studies from our lab have shown that IS1 contributes to the instability of CAPN3 by acting as a readily cleavable internal pro-peptide that occupies the active site of the protease core. Here we report the 2.3Å resolution crystal structure of CAPN3 (ΔNS, C129S) protease core with Ca2+ ions present. Although the architecture of this protease core is similar to those of the CAPN1 and CAPN2, with two Ca2+ bound, the crystal structure of the CAPN3 core exhibits several novel features. The N-terminal anchor is more flexible than that of CAPN1 and CAPN2 even with the N-terminal 45 residues removed. Residues in the mutation-rich region of PC1 (207 to 234) form van der Waals and H-bond interactions with residues of both IS1 and helix10. The active site is blocked by part of IS1 that forms H-bonds through the OH groups of Tyr274 and Tyr322 to Trp360 from the active site, as well as a hydrophobic interaction between Tyr274 of IS1 to His334 of the catalytic triad. This aromatic stacking will hinder substrates and inhibitors from entering the active site. The crystal structure further confirms that IS1 is a critical region governing CAPN3 activation and the binding of substrates and inhibitors. Supported by funds from CIHR and the CRC program.

Calcineurin is the only known calmodulin (CaM) activated protein phosphatase, which is involved in the regulation of numerous cellular and developmental processes and in calcium-dependent signal transduction. Although commonly assumed that CaM displaces the autoinhibitory domain (AID) blocking substrate access to its active site, the structural basis underlying activation remains elusive. We have created a fused ternary complex (CBA) by covalently linking three polypeptides: CaM, calcineurin regulatory B subunit (CnB) and calcineurin catalytic A subunit (CnA). CBA catalytic activity is comparable to that of fully activated native calcineurin in the presence of CaM. The crystal structure showed virtually no structural change in the active site and no evidence of CaM despite being covalently linked. The asymmetric unit contains four molecules; two parallel CBA pairs are packed in an antiparallel mode and the large cavities in crystal packing near the calcineurin active site would easily accommodate multiple positions of AID-bound CaM. Intriguingly, the conformation of the ordered segment of AID is not altered by CaM; thus, it is the disordered part of AID, which resumes a regular α-helical conformation upon binding to CaM, which is displaced by CaM for activation. We propose that the structural basis of calcineurin activation by CaM is through displacement of the disordered fragment of AID which otherwise impedes active site access.

Dictyostelium myosin heavy chain kinase A (MHCK‐A) is a member of the alpha‐kinase family of atypical Ser/Thr protein kinases. The X‐ray crystal structure of the alpha‐kinase domain of MHCK A (A‐CAT: residues 552–841) shows that an aspartylphosphate residue is present within the active site, indicating that the catalytic mechanism may proceed through the formation of a phosphoenzyme intermediate. Further structural analysis reveals that A‐CAT can hydrolyze ATP to adenosine. Regulation of A‐CAT is predicted to involve the binding of a Mg2+ ion to a loop, termed, the N/D‐loop, that regulates access to the active site cleft. In addition, A‐CAT contains a positively‐charged phosphate‐binding pocket that functions to potently activate kinase and ATPase activity. A critical autophosphorylation site (T825) located in a disordered sequence C‐terminal to the alpha‐kinase domain is predicted to activate A‐CAT by providing a ligand for the phosphate‐binding pocket. A fluorescent probe has been used to analyze nucleotide binding to the A‐CAT active site. In addition, a fluorescent probe has been linked to the N/D‐loop in order to directly measure the ability of the loop to undergo a divalent cation‐induced conformational change. The effects of altering the position of the phosphothreonine within the C‐terminal tail on A‐CAT activity is also examined.

Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A) disrupts the assembly and cellular activity of bipolar filaments of myosin II by phosphorylating sites within its alpha-helical, coiledcoil tail. MHCK A is a member of the atypical alpha-kinase family of serine and threonine protein kinases and displays no sequence homology to typical eukaryotic protein kinases. We report the crystal structure of the alpha-kinase domain (A-CAT) of MHCK A. When crystallized in the presence of adenosine triphosphate (ATP), A-CAT contained adenosine monophosphate (AMP) at the active site. However, when crystallized in the presence of ATP and a peptide substrate, which does not appear in the structure, adenosine diphosphate (ADP) was found at the active site and an invariant aspartic acid residue (Asp(766)) at the active site was phosphorylated. The aspartylphosphate group was exposed to the solvent within an active-site pocket that might function as a docking site for substrates. Access to the aspartylphosphate was regulated by a conformational switch in a loop that bound to a magnesium ion (Mg2+), providing a mechanism that allows alpha-kinases to sense and respond to local changes in Mg2+.

Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A), a member of the atypical α-kinase family, phosphorylates sites in the myosin II tail that block filament assembly. Here we show that the catalytic activity of A-CAT, the α-kinase domain of MHCK A (residues 552–841), is severely inhibited by the removal of a disordered C-terminal tail sequence (C-tail; residues 806–841). The key residue in the C-tail was identified as Thr825, which was found to be constitutively autophosphorylated. Dephosphorylation of Thr825 using shrimp alkaline phosphatase decreased A-CAT activity. The activity of a truncated A-CAT lacking Thr825 could be rescued by Pi, phosphothreonine, and a phosphorylated peptide, but not by threonine, glutamic acid, aspartic acid, or an unphosphorylated peptide. These results focused attention on a Pi-binding pocket located in the C-terminal lobe of A-CAT. Mutational analysis demonstrated that the Pi-pocket was essential for A-CAT activity. Based on these results, it is proposed that autophosphorylation of Thr825 activates ACAT by providing a covalently tethered ligand for the Pi-pocket. Ab initio modeling studies using the Rosetta FloppyTail and FlexPepDock protocols showed that it is feasible for the phosphorylated Thr825 to dock intramolecularly into the Pi-pocket. Allosteric activation is predicted to involve a conformational change in Arg734, which bridges the bound Pi to Asp762 in a key active site loop. Sequence alignments indicate that a comparable regulatory mechanism is likely to be conserved in Dictyostelium MHCK B-D and metazoan eukaryotic elongation factor-2 kinases.

Calcineurin (CN) is the only serine/threonine protein phosphatase under the control of Ca2+/calmodulin (CaM). CaM up-regulates the phosphatase activity of CN by binding to CaM-binding domain (CBD) of calcineurin subunit A. However, the disordered structure of CBD has not been resolved in all crystal structures of CN determined so far. To gain insight into the structural basis by which CaM binds to CBD of CN and exerts its interaction, we have created a fusion construction in which CBD is covalently linked to CaM via a 5-glycine flexible linker and solved the crystal structure. However, the structure that displays a novel CaM binding conformation is still debatable because of the linker between CaM and CBD and/or crystallization conditions, which may affect protein packing. To investigate whether or not the novel CaM-CBD structure could potentially represent an artifact, we have attempted cocrystallization of CaM in complex with CBD and have now obtained cocrystals in two crystal forms. Comparison of crystallization conditions, crystal morphologies, and crystal parameters indicated that both CaM in complex with CBD peptide and CaM-CBD peptide chimera have the same crystal characteristics and behaviors in crystal growth under the similar crystallization conditions. The results provide direct evidence that the glycine linker did not affect the protein structure and the crystallization conditions did not result in different conformations. The advantage of using the fusion construct includes a higher yield of the CBD peptide driven by robust expression of CaM and the ease to purify both components simultaneously.

Calcineurin is a calmodulin-binding protein in brain and the only serine/threonine protein phosphatase under the control of Ca2+/calmodulin (CaM), which plays a critical role in coupling Ca2+ signals to cellular responses. CaM up-regulates the phosphatase activity of calcineurin by binding to the CaM-binding domain (CBD) of calcineurin subunit A. Here, we report crystal structural studies of CaM bound to a CBD peptide. The chimeric protein containing CaM and the CBD peptide forms an intimate homodimer, in which CaM displays a native-like extended conformation and the CBD peptide shows (x-helical structure. Unexpectedly, the N-terminal lobe from one CaM and the C-terminal lobe from the second molecule form a combined binding site to trap the peptide. Thus, the dimer provides two binding sites, each of which is reminiscent of the fully collapsed conformation of CaM commonly observed in complex with, for example, the myosin light chain kinase (MLCK) peptide. The interaction between the peptide and CaM is highly specific and similar to MLCK.

BsSco is a membrane-associated protein from Bacillus subtilis characterized by the sequence CXXXCP, which is conserved in yeast and human mitochondrial Sco proteins, and their bacterial homologues. BsSco is involved in the assembly of the Cu(A) center in cytochrome c oxidase and may play a role in the transfer of copper to this site. We have characterized the soluble domain of BsSco by biochemical, spectroscopic, and structural approaches. Soluble BsSco is monomeric in solution, and the two conserved cysteines are involved in an intramolecular cystine bridge. The cystine bridge is easily reduced, and circular dichroism spectroscopy shows no large-scale changes in BsSco's secondary structure upon reduction. The crystal structure of soluble BsSco, determined at 1.7 A resolution, reveals typical elements of a thioredoxin fold. The CXXXCP motif, in which Cys45 and Cys49 are conserved, is located in a turn structure on the surface of the protein. In various native and His135Ala mutant structures, both disulfide-bonded and non-disulfide-bonded forms of CXXXCP are observed. However, despite extensive attempts, copper has not been found near or beyond the CXXXCP motif, a presumptive copper-binding site. Another potential copper binding residue, His135, is located in a highly flexible loop parallel to the CXXXCP loop but is more than 10 A from Cys45 and Cys49. If these three residues are to coordinate copper, a conformational change is necessary. The structural identification of a disulfide switch demonstrates that BsSco has the capability to fill a redox role in Cu(A) assembly.

Bri3 is a recently identified proline-rich transmembrane polypeptide up-regulated during TNF-mediated inflammation and immunity. The polyproline-rich N-terminal (residues 1-60) domain of Bri3 was affinity-purified to homogeneity as a glutathione-S-transferase (GST) fusion protein. Crystals were obtained in approximately 3 d by the equilibrium vapour-diffusion method from a solution containing 1.5-2.2 M ammonium sulfate and 0.1 M bis-tris pH 6.0. The crystals belong to space group P4(3)2(1)2, with unit-cell parameters a = b = 91.66, c = 57.53 A. An X-ray data set was collected to 1.6 A resolution using synchrotron radiation, with an Rsym of 0.058 and a completeness of 95.3%. There is one molecule of the fusion protein in the asymmetric unit, which corresponds to approximately 35% solvent content.