Biochemical and structural studies of target lectin SapL1 from the emerging opportunistic microfungus Scedosporium apiospermum

Scedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in humans. Host–pathogen interactions often implicate lectins that have become therapeutic targets for the development of carbohydrate mimics for antiadhesive therapy. Here, we present the first report on the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 was found using bioinformatics as a homolog to the conidial surface lectin FleA from Aspergillus fumigatus known to play a role in the adhesion to host glycoconjugates present in human lung epithelium. In our strategy to obtain recombinant SapL1, we discovered the importance of osmolytes to achieve its expression in soluble form in bacteria. Analysis of glycan arrays indicates specificity for fucosylated oligosaccharides as expected. Submicromolar affinity was measured for fucose using isothermal titration calorimetry. We solved SapL1 crystal structure in complex with α-methyl-L-fucoside and analyzed its structural basis for fucose binding. We finally demonstrated that SapL1 binds to bronchial epithelial cells in a fucose-dependent manner. The information gathered here will contribute to the design and development of glycodrugs targeting SapL1.


Results
Production and purification of SapL1. The hypothetical protein XP_016640003.1 (EMBL accession number), encoded by SAPIO_CDS9261 and from now on referred as SapL1, was identified through data mining using the FleA (pdb entry 4D4U 34 ) sequence as bait into the genome of the reference strain S. apiospermum IHEM 14462 33 . During the sequence analysis, we found that the first 74 amino acids of the putative protein (Uniprot A0A084FYP2) represent a very disordered region that is not present in any other related protein. We suspected that this peptide could be due to an error during the genome annotation. To verify it, we tried to get the gene from total mRNAs extracted from mycelium by 5' Rapid Amplification of cDNA Ends (5' RACE) using the following primers GSP1-SapL1 5'-TTA AGC AGG GGG CAG AAC AGC-3' and GSP2-SapL1 5'-CCA ACG ACC CAG CCA GAG TTCC-3' but we were unsuccessful. This could be due to the fact that the gene was not expressed in the conditions tested. We did not have access to total mRNAs from other morphological stages of the microfungus. Thus, we decided to focus on the identified carbohydrate recognition domain (CRD) of SapL1 starting from methionine-75 (Fig. S1). The SapL1 coding sequence (75-369) was fused to an N-terminal 6xHis tag cleavable by the Tobacco Etch virus protease (TEV) under regulation of trc and T7 promoters into pProNde and pET-TEV vectors, respectively (Fig. 1A). Expression was performed in Escherichia coli and purification was carried out using immobilized metal affinity chromatography (IMAC). Unfortunately, the original expression yield with both vectors (~ 0.35 mg·L −1 of culture) was too low to proceed with characterization studies. Therefore, we explored new alternatives to enhance the expression. First, the thioredoxin protein (Trx) as well as 6-His tag and TEV cleavage site were fused at the N-terminus of SapL1 by subcloning into the pET32-TEV vector (Fig. 1A). This strategy substantially increased the SapL1 production yield but most of the protein remained insoluble as part of inclusion bodies (Fig. S2).
A wide range of expression conditions were subsequently assayed to achieve sufficient soluble expression by modification of various parameters such as growth temperature, host strain, inducer concentration, optical density of the culture at induction, culture duration, etc. Sixty-nine different sets of parameters were assayed (Table S1) and it was possible to improve the yield up to 4 mg·L −1 (Fig. 1B). The best set of conditions for SapL1 expression was using E. coli strain TRX, pProNde vector, LB medium, growth at 37 °C and 160 rpm until OD 600 = 0.4, before a switch of the temperature to 16 °C and overnight induction at OD 0.8 with 0.05 mM IPTG and 1% L-rhamnose (Rh). www.nature.com/scientificreports/ Rhamnose influence on SapL1 solubility. Interestingly, during the optimization of the expression conditions, we found that rhamnose (Rh) plays an essential role in the solubility and stability of SapL1. Therefore, we performed a new set of experiments to demonstrate the correlation between the amount of protein recovered after purification and the Rh concentration in the media. For this, we investigated SapL1 expression under the previously described conditions modified by supplementation of the culture medium with different concentrations of Rh (0.1%, 0.15%, 0.2%, 0.5% and 1%). Purification parameters were set to obtain high purity product and kept identical for all experiments. Figure 2 shows the chromatograms obtained for representative concentrations accompanied by their respective SDS-PAGE profile. To quantify SapL1 expression in these experiments, we integrated the area under peaks corresponding to the protein (Fig. 2, black bars). Thus, we confirmed that SapL1 recovery is directly proportional to the Rh concentration in the culture medium. Then, the experiment was repeated at three different concentrations (0.1%, 0.15%, 0.2%) using 20 °C as the induction temperature, and production behavior was similar to that found at 16 °C, but with lower performance. Additional experiments also showed that SapL1 expression can be induced at high concentration of rhamnose in a dose-dependent manner, even without addition of IPTG when the pProNde vector is used (data not shown).
Biochemical characterization. We assayed the thermal stability of SapL1 in 26 different buffers in a pH range of 5 to 10 through Thermal Shift Assay (TSA). The most suitable condition for this protein was MES buffer 100 mM pH 6.5, where a single denaturing event at T m of 55 °C was observed (Fig. S3). Then, to estimate the molecular size and oligomerization state of the native protein, we performed size exclusion chromatography using an ENrich™ SEC 70 column (Bio-Rad) and 20 mM MES, 100 mM NaCl, pH 6.5 as mobile phase. However, the protein displayed strong non-specific interactions with the matrix of the column and SapL1 could not be eluted even using 5 M of NaCl. Interestingly, it could be recovered when the buffer was supplemented with 20 mM α-methyl fucoside or L-rhamnose, evidencing similar effects on SapL1 elution for both sugars. Due to the impossibility to estimate the molecular weight of SapL1 by size exclusion chromatography on this resin, we performed measurements in solution using Dynamic Light Scattering (DLS). We obtained a monodisperse peak corresponding to a protein of 72 ± 29.4 kDa corroborating that SapL1 forms dimers as FleA and other proteins of this family (monomer MW: 40 kDa, data not shown) 27 . The range of the standard deviation also suggested an ellipsoidal shape, which is characteristic for the dimers in this lectin family 27,35 . Carbohydrate binding properties. A hemagglutination assay showed that recombinant SapL1 agglutinated rabbit red blood cells at 0.97 µg·mL −1 (Fig. 3A). It confirms that the recombinant lectin is active and that its heterologous production in E. coli did not alter its hemagglutinating properties.
To identify the potential ligands of SapL1 on epithelial cell surfaces, we submitted SapL1 to the glycan array version 5.4 of the Consortium for Functional Glycomics (USA) consisting of 585 mammalian glycans. It was labelled with Fluorescein Isothiocyanate (FITC) in a molar ratio of 0.426 and its binding properties were analysed at two different concentrations (5 and 50 μg·mL −1 ). As expected from its homology with FleA, SapL1 recognizes fucosylated oligosaccharides independently of the fucose linkage. The α1,2 and α1,3/4 linked fucosides displayed the highest affinity whilst the lowest was seen with the α1,6 linked ones. The weakest interactions with fucosylated compounds were reported for branched oligosaccharides (Figs. 3C and S4).
The affinity of SapL1 for L-fucose and α-methyl-fucoside was determined by Isothermal Titration Calorimetry (ITC) and the K d was found to be 225 ± 1.52 µM and 190 ± 1.44 µM, respectively with stoichiometry fixed to 1 since the measurements were done in the presence of an excess of ligand (Fig. 3B). These values are in agreement www.nature.com/scientificreports/ with the affinity constant (around 110 µM) reported for FleA for α-methyl-fucoside 36 . No binding interaction was observed for SapL1 with rhamnose by ITC (data not shown). Analysis of the glycans constituting blood group determinants revealed that SapL1 binds to all epitopes with a preference for H type 2 blood group (Fucα1-2Galβ1-4GlcNAcβ) then Lewis a (Galβ1-3(Fucα1-4)GlcNAcβ) and Lewis X (Galβ1-4(Fucα1-3)GlcNAcβ). However, most of the recognized branched oligosaccharides contained the core fucose Fucα1-6. Epitopes with two fucose units, such as Lewis b and fucosylated polylactosamine, were also well recognized. Addition of a galactose or a GalNAc as in blood group B or A antigens did not impair Fucα1-2 recognition (Fig. 3C). Spacers used to join the carbohydrates to the chip also display a strong influence on binding. It is remarkable that 70% of the 90 positive binders contained either the spacer Sp0 (CH 2 CH 2 NH 2 ) or Sp8 (CH 2 CH 2 CH 2 NH 2 ). Those spacers also display a strong influence on binding, especially for small glycans such as Galα1-3(Fucα1-2)Galβ1-4(Fucα1-3)GlcNAcβ which was recognized when attached to Sp0 but not to Sp8. This may be due to a steric hindrance caused by the modification of carbohydrate presentation on the surface of the chip.
Overall structure of SapL1. SapL1 was co-crystallized with α-methyl-fucoside and the structure of the complex was solved by molecular replacement at 2.3 Å resolution in the P2 1 space group using the coordinates of FleA (PDB code 4D4U 34 ) as the search model. The asymmetric unit contained two monomers, assembled as a dimer with all 295 amino acids visible apart of the N-terminal methionine. See data collection and refinement statistics in Table 1. SapL1 folds into the canonical six-bladed β-propeller with six-binding sites at the interface between blades typical for this family of lectin (Fig. 4). A fucose moiety was found in 5 and 4 of the six binding pockets of chains A and B, respectively while glycerol originating from the crystallizing solution was found in the other binding sites.
The overall fold of SapL1 and FleA as well as the overall dimer are very similar with a rsmd of 1.2 and 1.26 Å, respectively. They share 43% of sequence identity and both proteins present almost the same distribution of β-strands except for an extra strabd in blade 2 (β10), the lack of the last strand of blade 4 in SapL1, and the www.nature.com/scientificreports/   www.nature.com/scientificreports/ external face of blade 5, in which FleA displays an elongated β-strand (β22) that is split in two in SapL1 (β22/23). It is remarkable that the structure of the first 3 blades is highly conserved in both proteins, while the second half (blades 3-6) displays the largest discrepancies. Furthermore, FleA also presents two additional small α-helices (ή3 and ή4), located in the loops between sheets that serve as connectors for blades (2/3 and 5/6, respectively, Fig. 5). Similar conclusions can be drawn when comparing SapL1 to its homologue AOL in Aspergillus oryzae 37 .
Protein-ligand interactions. Due to divergence in the tandem repeat sequence forming each blade of the propeller, the six binding sites of SapL1 monomer are not equivalent, but they share important conserved features. Hydrophobic interactions are observed between the C6 of the fucose and at least three residues of the protein (mainly isoleucine, tryptophan/tyrosine and leucine). The O2 and O3 hydroxyls make strong hydrogen bonds with a conserved triad of amino acids consisting of an arginine, a glutamic acid and a tryptophan. In the cases where glycerol was found in the binding pocket, its own hydroxyls mimic the interactions of fucose with these same residues (Fig. 6). It is to be noted that SapL1 binding sites are more conserved than FleA binding sites where a glutamine can replace the glutamic acid and a tyrosine the tryptophan in the triad (Fig. S5). The O2 hydroxyl seemed to be the most versatile position, since it established interactions with an adjacent loop only present in four of the six binding sites (BPs 2, 4, 5 and 6). This is particularly interesting since the binding pockets that do not contain this loop (BPs 1 and 3) were mostly occupied by glycerol instead of fucose, indicating that those interactions could be responsible for enhancing the affinity and should be explored for development of inhibitors.
Binding to epithelial cells. In order to investigate the role of SapL1 in host-pathogen interactions and especially in adhesion, fluorescence microscopy was used. FITC-labelled SapL1 was incubated at two different concentrations with BEAS-2B human bronchial epithelial cells. The microscopy images, obtained for two separate experiments, clearly show binding of the lectin to those cells (Fig. 7). Fluorescence was observed all around the cell surface but also concentrated in some part of the nucleus. A strong fluorescence signal was already observed at the lowest concentration used (5 µg mL −1 ). The binding of SapL1 was inhibited in the presence of its cognate ligand: α-methyl-fucoside ( Fig. 7E-G). Some background signal was still observed in the first experiment (Fig. 7E, G) whilst it was totally abolished in the duplicate (Fig. 7F, H). These data show that SapL1 binding to the cells is dependent on the recognition of fucosylated carbohydrates. www.nature.com/scientificreports/

Discussion
In comparison with pathogenic bacteria, which have received particular attention for several years, there is a lack of information about the virulence factors and host recognition systems of pathogenic fungi 38 . This hinders the development of new drugs against these microorganisms, while their resistance to current antifungals is progressing quickly 9,21,39 . Therefore, there is a rush in obtaining information that may contribute to the development of new antifungal agents.
In this study, we present the identification, production and characterization of a new carbohydrate-binding protein from the emerging microfungus S. apiospermum, whose host-anchoring mechanisms are completely unknown. To the best of our knowledge, this is the first studied lectin for this opportunistic pathogen. SapL1 is homologous to the conidial surface lectin FleA from A. fumigatus known to be involved in adhesion to the host glycoconjugates present in bronchial mucus and human lung epithelium 28 . When we analyzed the amino acid sequence deduced from SAPIO_CDS9261, we found at its N-terminus, a peptide sequence of 74 amino acids that was lacking in any other related protein. We believed in a possible error in the annotation of the open reading frame (ORF) of SapL1 since this peptide, predicted to be disordered, cannot be found in other protein and was not found in any other lectin from this family. We failed, however, to amplify the gene from total mRNAs from mycelium but recent transcriptomics data revealed that SapL1 is not usually expressed in mycelium apart in some conditions at low pH and in hypercapnia explaining our negative results 40 . By analogy to FleA, it is probably expressed in conidia and new amplification trials should be done on total mRNAs from conidia, but we did not have access to some at that time and there are no transcriptomic data available for this morphological stage to date. When looking at the transcript KEZ40204 for SAPIO_CDS9261, we noticed that the first exon containing the actual initial methionine is only 8 bases long followed by a very long intron of 686 bases which is quite unusual in fungi. After alignment of this transcript sequence from all genome sequences available for Scedosporium species, we saw that the proposed initial methionine is not conserved contrary to the one for Met75 (Fig. S6). Whilst waiting for experimental data, this strengthens our hypothesis for a gene misannotation and provides additional information that supports our decision to study the construct of SapL1 comprising only the predicted CRD.
In our initial attempts to produce recombinant SapL1, we observed that the conventional parameters of expression in E. coli, resulted in the production of insoluble protein that was mainly found in inclusion bodies. Later, experimental data indicated that the presence of rhamnose or glycerol was required for SapL1 solubilization during bacterial expression and that the production yield was directly proportional to their concentration in the culture medium. We initially considered that the higher production rates of SapL1 could be attributed to the dissociation of the LacI-DNA complex caused by an increase in cytosolic potassium concentration. The accumulation of this ion is one of the main adaptive responses of E. coli to hypertonic shocks and its concentration has been correlated to the loss of interactions between DNA and proteins in in vitro assays [41][42][43] . However, previous reports have demonstrated that, in vivo, the effects of macromolecular crowding caused by plasmolysis (the reduction of intracellular water content as an adaptive response to osmotic shock) on the activity of cytoplasmic proteins is sufficient to buffer the kinetics of association of DNA-protein against changes in [K + ] 43,44 . Thus, it seems that the higher recovery rates of SapL1 actually were caused by a combination of two main parameters: 1) the leak of genetic repression by dissociation of the LacI-DNA complex, that is characteristic of lac promoters and 2) a positive effect on the solubility and folding of the protein due to the increased concentration of organic osmoregulators, whose synthesis is induced in E. coli as an adaptive response to the external hypertonicity caused www.nature.com/scientificreports/ here by the addition of rhamnose to the culture medium [41][42][43][44][45][46][47][48][49] . This hypothesis was later supported by the finding that the addition of glycerol, instead of rhamnose, displays the same effect on production and solubility of SapL1 (data not shown). Plasmolysis may also play an essential role in SapL1 stabilization, since it has been shown that macromolecular crowding has positive effects on protein folding and in some cases it can drive self-association of improper folded proteins into functional oligomers 43 . These findings, and the extensive research carried out to produce SapL1 in its soluble form, provide important insights for the heterologous expression of eukaryotic lectins tending to be produced as insoluble proteins in E. coli. Besides, our findings highlight the useful role of organic osmoregulators during heterologous expression of proteins to favour their proper folding and to avoid the formation of inclusion bodies. We demonstrated that SapL1 is strictly specific for fucosylated carbohydrates and recognized all blood group types present in the glycan array screening. These results are particularly interesting since it has been shown that there is six times more fucose α1,3/4 linked in glycoproteins in the CF airways 31 . This phenomenon is mainly due to an increased expression of the α1,3-fucosyltransferase, which is involved in the synthesis of sialyl-Lewis X and Lewis X determinants attached to bronchial mucins 32 . Therefore, the fact that SapL1 recognizes equally the α1,2 and α1,3/4-linked fucosides may explain the high incidence of scedosporiosis in CF patients. Besides, it correlates with the presence of SapL1 gene in all the pathogenic strains of Scedosporium whose genome has been sequenced to date 33,50,51 . However, there is no evidence suggesting that blood group phenotypes may have an influence in this recognition conversely to previous reports on other pathogens like Pseudomonas aeruginosa and Haemophilus influenzae [52][53][54] .
SapL1 adopts a the 6-bladed β-propeller fold and forms dimer leading to two fucose binding surfaces. It belongs to ProLec6A lectin family whose biological role is still unknown, in particular for the mushroom members 35 . The first member structurally characterized in this lectin family was AAL from the orange peel mushroom Aleuria aurantia 55 . It presents differences on both sides of the β-propeller with the microfungal members of this family: SapL1, AOL and FleA mainly at the level of the surface loops. On one side, this leads to a different dimerization interface and the dimer cannot overlay. On the other side, where sugar binding occurs, the differences in size and sequence of the surface loops lead to change in the architecture of the related binding sites and affect fucose binding since AAL has only five and not six functional binding sites 27,37,55 . The specificity and affinity of each one of the six binding pockets of SapL1 have been deeply analyzed to highlight the features that must be explored for the design of efficient inhibitors. Within our analysis, we found that the binding pockets are non-equivalent but they all share the features necessary for fucose recognition. This example of divergence can also be found in the other lectins of this family with FleA exhibiting the greatest differences between pockets known to date 34 . It is remarkable that although SapL1 has a relatively low sequence identity with other members of this family, its specificity and affinity are very similar to those reported for FleA from A. fumigatus 36 , AAL from A. aurantia 55 and the bacterial lectins BambL and RSL from Burkholderia ambifaria and Ralstonia solanacearum, respectively 56,57 .
Finally, fluorescence microscopy experiments allowed us to get preliminary results on SapL1 function. The lectin is able to recognize and to bind to bronchial epithelial cells, and we demonstrated that this binding is dependent on the recognition of fucosylated glycoconjugates at the surface of the target cells as it is abolished in the presence of fucose. These results corroborate a role of SapL1 in mediating the recognition and adhesion of S. apiospermum to host cells as found for other lectins from pathogenic microorganisms and in particular for its homolog FleA 22,27 . Localisation of the lectin in the microfungus would also help to better understand its role but no SapL1 antibodies are yet available. Together with the crystallographic data, our study shows that the structure and function of lectins belonging to the 6-bladed β-propeller fucose specific family recently named PropLec6A are highly conserved, settling the possibility for development of a broad-spectrum therapy 35 .
Overall, our research has revealed the first insights about the recognition of fucosylated human glycoconjugates by S. apiospermum lectin SapL1 and contributes to the general understanding of the host-binding process during the early stages of infection. The lectin is found in all Scedosporium strains sequenced. The detailed information exposed here places SapL1 as a promising target for treatment of Scedosporium infections although it is clear that more functional data are still required. It will be of great value to guide the development of antiadhesive glycodrugs against this pathogen.

Methods
Production. The coding sequence for SapL1 (75-369) was optimized for expression in Escherichia coli and ordered at Eurofins Genomics (Ebersberg, Germany) before cloning into the expression vectors pET-TEV 58 , pET32-TEV 36 and pProNde. pProNde is a homemade vector where the NcoI restriction site of pProEx HTb (EMBL, Heidelberg) has been replaced by a NdeI restriction site by PCR. Then, plasmids were introduced into E. coli strains by thermal shock at 42 °C and different expression parameters were assayed (Table S1) until soluble expression was achieved. Finally, SapL1 was expressed using pProNde vector in E. coli BL21trxB (DE3) strain with the cells grown at 37 °C and 160 rpm until an OD 600 of 0.4. The temperature was then lowered to 16 °C and when OD 600nm reached 0.8, the induction was carried out overnight by the addition 0.05 mM of IPTG and 1% L-rhamnose.
Purification. Cells were subsequently harvested by centrifugation at 5000 g for 10 min and resuspended in buffer A (50 mM Tris-HCl, 500 mM NaCl, pH 8.5). After addition of 1 µL of Denarase (C-LEcta GmbH, Leipzig, Germany) and moderate agitation during 30 min at room temperature, cells were disrupted at 1.9 kbar using a cell disruptor (Constant Systems Ltd, UK). Cell debris were removed by centrifugation at 22,000 g for 30 min. The supernatant was filtered using 0.45 µm membranes (PES, ClearLine) and the protein purification was carried out by IMAC using 1 mL His-Trap FF columns (Cytiva) and a NGC chromatography system (Bio-Rad). www.nature.com/scientificreports/ After loading the supernatant, the column was rinsed thoroughly with buffer A until stabilization of the baseline. Bound proteins were eluted through the addition of buffer B (50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.5) in an 0-500 mM imidazole gradient over 20 mL. Fractions containing SapL1 were pooled and concentrated by ultrafiltration (Pall, 10 kDa cut-off) prior buffer exchange to 50 mM Tris-HCl, 100 mM NaCl, pH 8.5 using PD10 desalting columns (Cytiva). Then, the fusion was cleaved off overnight at 19 °C using the TEV protease produced in the lab in 1:50 ratio and addition of 0.5 mM EDTA and 0.25 mM TCEP. The sample was loaded on the His-trap column to separate the cleaved protein collected in the flow-through from the TEV protease and potential uncleaved sample retained and eluted with imidazole. Sapl1 containing fractions were concentrated by centrifugation to the desired concentration.
Size exclusion chromatography. Size exclusion chromatography (SEC) was performed using a High-Resolution ENrich SEC 70 column (Bio-Rad) on NGC chromatography system (Bio-Rad). Column was equilibrated with 50 mL of buffer D (20 mM MES, 100 mM NaCl, pH 6.5) and 200 µL of sample at 10 mg mL −1 were injected into the system followed by 40 mL isocratic elution on buffer D supplemented with 20 mM α-methyl-Lfucoside (TCI Europe), 0.1% L-rhamnose, 5 M NaCl, 2 M NaCl or 500 mM NaCl, according to the experiment. Fractions were monitored by the absorbance at 280 nm and 0.5 mL fractions were collected in the resolving region of the column. Labelled lectin was sent to the Consortium for Functional Glycomics (CFG; Boston, MA, USA) and its binding properties were assayed at 5 and 50 μg·mL −1 on a "Mammalian Glycan Array version 5.4" which contain 585 glycans in replicates of 6. The highest and lowest signal of each set of replicates were eliminated and the average of the remaining data was normalized to the percentages of the highest RFU value for each analysis. Finally, the percentages for each glycan were averaged at different lectin concentrations.

Dynamic light scattering (DLS
Crystallization and data collection. Crystal  www.nature.com/scientificreports/ mercial screenings of Molecular Dimensions (Newmarket, UK). 2 µL drops were incubated at 19°C until crystals appeared. A subsequent optimization of positive conditions for SapL1 crystallization was carried out and crystals suitable for X-ray diffraction analysis were obtained under solution containing 100 mM Bicine pH 8.5, 1.5 M ammonium sulfate (NH 4 ) 2 SO 4 and 12% v/v glycerol. Crystals were soaked in mother liquor supplemented with 10% (v/v) glycerol, prior to flash cooling in liquid nitrogen. Data collection was performed on PX1 beamline at SOLEIL Synchrotron (Saint Aubin, France) using an Eiger2 X 9M pixel detector (Dectris Ltd, Switzerland).
Structure determination. Data were processed using XDS 59 software and were converted to structure factors using the CCP4 program package v.6.1 60 , with 5% of the data reserved for Rfree calculation. The structure was determined using the molecular-replacement method with Phaser v.2.5 61 , using the structure of FleA dimer (PDB entry 4D4U 34 ) as starting model. Model refinement was performed using REFMAC 5.8 62 alternated with manual model building in Coot v.0.7 63 . Sugar residues and other compounds that were present were placed manually using Coot and validated using Privateer 64