Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its Quaternary Interactions

Abstract

Many members of the monocot mannose-binding lectin family, characterized by specificity towards mannose have been characterized and cloned. A majority of these lectins molecules contain 1-4 polypeptides of about 110 residues each. From the previously solved crystal structures of a few such lectins, mostly from non-edible plants, these lectins are thought to possess a common β-prism II fold structure. The major tuber storage protein of Colocasia esculenta is a monocot mannose-binding, widely used, dietary lectin. This tuber agglutinin contains two polypeptides of 12.0 and 12.4 kDa by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. By gel filtration at pH 7.2, the purified lectin has a α2β2 form of apparent molecular mass of 48.2 kDa in solution but at pH 3, it has the heterodimeric αβ form. Lectin crystals were obtained by hanging-drop, vapor-diffusion method at room temperature and high-resolution X-ray diffraction data were collected using a home X-ray source. Among previously solved crystal structures of this family are garlic, Solomon’s seal, snowdrop, daffodil and Spanish blue-bell lectins, but the protein sequence of the Colocasia esculenta tuber agglutinin was found to be closest to that of the Remusatia vivipara lectin having no simple mannose-binding property. Using the previously solved 2.4Å crystal structure of the Remusatia vivipara lectin, that of Colocasia esculenta has been solved by molecular replacement and subsequent crystallographic refinement and root mean square deviations between various lectins are tabulated and rationalized. The asymmetric unit in our lectin crystal structure contains four β-prism II domains or two αβ heterodimers, each forming a α2β2 heterotetramer with a symmetry related unit. The tetrameric interface obtained from our crystal structure is used to explain the conversion to dimers in acidic pH. Five ordered magnesium ions were located in the asymmetric unit and the presence of magnesium verified by atomic absorption spectroscopy.

Full text

Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
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Journal of Glycobiology
Chattopadhyaya et al., J Glycobiol 2017, 6:2
DOI: 10.4172/2168-958X.1000126
Research Article OMICS International
Crystal Structure of
Colocasia esculenta
Tuber Agglutinin at 1.74 Å
Resolution and Its Quaternary Interactions
Rajagopal Chattopadhyaya*, Himadri Biswas and Apurba Sarkar
Department of Biochemistry, Bose Institute, Calcutta, India
Abstract
Many members of the monocot mannose-binding lectin family, characterized by specicity towards mannose
have been characterized and cloned. A majority of these lectins molecules contain 1-4 polypeptides of about 110
residues each. From the previously solved crystal structures of a few such lectins, mostly from non-edible plants,
these lectins are thought to possess a common β-prism II fold structure. The major tuber storage protein of Colocasia
esculenta is a monocot mannose-binding, widely used, dietary lectin. This tuber agglutinin contains two polypeptides
of 12.0 and 12.4 kDa by matrix assisted laser desorption ionisation time-of-ight mass spectrometry. By gel ltration
at pH 7.2, the puried lectin has a α2β2 form of apparent molecular mass of 48.2 kDa in solution but at pH 3, it
has the heterodimeric αβ form. Lectin crystals were obtained by hanging-drop, vapor-diffusion method at room
temperature and high-resolution X-ray diffraction data were collected using a home X-ray source. Among previously
solved crystal structures of this family are garlic, Solomon’s seal, snowdrop, daffodil and Spanish blue-bell lectins,
but the protein sequence of the Colocasia esculenta tuber agglutinin was found to be closest to that of the Remusatia
vivipara lectin having no simple mannose-binding property. Using the previously solved 2.4Å crystal structure of the
Remusatia vivipara lectin, that of Colocasia esculenta has been solved by molecular replacement and subsequent
crystallographic renement and root mean square deviations between various lectins are tabulated and rationalized.
The asymmetric unit in our lectin crystal structure contains four β-prism II domains or two αβ heterodimers, each
forming a α2β2 heterotetramer with a symmetry related unit. The tetrameric interface obtained from our crystal
structure is used to explain the conversion to dimers in acidic pH. Five ordered magnesium ions were located in the
asymmetric unit and the presence of magnesium veried by atomic absorption spectroscopy.
*Corresponding author: Rajagopal Chattopadhyaya, Department of Biochemistry,
Bose Institute, Calcutta-700054, India, Tel: 91-33-2569-3239; Fax: 91-33-2355-
3886; E-mail: raja@jcbose.ac.in
Received February 10, 2017; Accepted April 10, 2017; Published April 17, 2017
Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of
Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its Quaternary
Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Copyright: © 2017 Chattopadhyaya R, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited.
Keywords: Monocot mannose-binding lectins; β-prism II fold;
Edible Colocasia esculenta tuber agglutinin; Crystal structure
Abbreviations: CEA: Colocasia esculenta Agglutinin; CVL: Crocus
vernus Lectin; F.O.M.: Figure of Merit; MALDI-TOF: Matrix Assisted
Laser Desorption Ionisation Time-of-Flight Mass Spectrometry; PCL:
Polygonatum cyrtonema Agglutinin; RVL: Remusatia vivipara Lectin;
Scafet: Scilla campanulata Heterodimeric Agglutinin
Introduction
e recognition of carbohydrate moieties by lectins has important
applications in a number of biological processes such as cell-cell
interaction, signal transduction, cell growth and dierentiation [1]. e
functionality of lectin molecules depend on the specic carbohydrate
recognition domain, a part of the three dimensional structure of the
protein, normally held together by non-covalent interactions and
disulde linkages.
Among the twelve new families of plant lectins [2] or the seven
families by the older classication [3], the monocot mannose-binding
lectin family comprises lectins with an exclusive specicity towards
mannose, also called GNA-related lectins. Numerous members of this
family of lectins have been characterized and cloned from Alliaceae,
Amaryllidaceae, Araceae, Bromeliaceae, Liliaceae and Orchidaceae
species, as summarized by Barre et al. [4]. e majority of all these
lectins consist of one, two or four identical polypeptide(s) of about
110 amino-acid residues. Each subunit possesses a novel pseudo-
3-fold symmetry having three 4-stranded anti parallel β-sheets
oriented as 3 sides of a trigonal prism forming a 12-stranded β-barrel,
referred to as the β-prism II fold, rst reported in the homotetrameric
Gallanthus nivalis crystal structure in complex with methyl-α-D-
mannoside (PDB ID 1MSA) [5]. e core of this β-barrel is lined with
conserved hydrophobic side chains, which stabilize the fold. Later
crystal structures of complexes of the heterodimeric Allium sativum
lectin (1BWU) [6], homotetrameric Narcissus pseudonarcissus lectin
(1NPL) [7], homodimeric Scilla campanulata lectin (1B2P) [8] and
homodimeric Polygonatum cyrtonema (3A0C) [9] all show the β-prism
II fold and are monocot mannose binding lectins. Except for the garlic
(A. sativum) and Solomon’s seal (P. cyrtonema) lectins which are from
edible/medicinal plants, other mannose binding lectins mentioned
above are from non-edible, gardening plants like snowdrop (G. nivalis),
daodil (N. pseudonarcissus) and Spanish blue-bells (S. campanulata),
the last two being highly poisonous. In view of possible intestinal or
immunological toxicity/distress, the study of lectins from edible plants
assumes greater importance than those from poisonous ones.
Colocasia esculenta of the Araceae family is a tuberous
monocotyledonous Asian plant growing in tropical and subtropical
climates; it is widely used for human consumption as a supplementary
food source [10]. Colocasia extracts (from taro, corm) possess
important pharmacological properties including anti-inammatory,
anti-cancer, anti-fungal, anti-viral [11], while the lectin has insecticidal
activities [12]. Another group reported several isoforms of the very
similar lectin tarin and its covalent modication [13], but in our study
of the storage protein, covalently bound sugar was never found [12].
We nd the intact protein is a α2β2 heterotetramer of 49 kDa composed
of two dierent polypeptides, with small subunits of 12.0 kDa and large
subunits of 12.4 kDa though slightly dierent masses were reported
earlier [14].

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 2 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
Our 1.74Å crystal structure of a β-prism II (BP2) fold of the
Colocasia lectin (PDB ID 5D5G) describes the dimeric and tetrameric
interactions.
Materials and Methods
Materials
Syringe-driven lters, 0.22 µm pore size, were purchased from
Merck Millipore (Mumbai, India). Boxes for setting hanging drop
crystals were bought from Nunc. (Roskilde, Denmark) and cover slips
from Blue Star (Mumbai, India). Most of the buering agents (Hepes,
Na-cacodylate, MOPS, etc.), precipitants (PEGs) and Sigmacote were
procured from the Sigma Chemical Company, Missouri, USA. All other
chemicals, obtained from Merck (Mumbai, India), were of molecular
biology or analytical grade.
Protein purication
CEA was puried to homogeneity following a modication of the
known protocol [14]. e tubers were homogenized in 0.2 M NaCl
containing 1 gl-1 ascorbic acid (5 ml per gram of fresh weight) at pH
7.0 using a Waring blender. e homogenates were ltered through
cheesecloth and centrifuged (12,000 rpm for 10 min). Aer it was
brought to 20 mM in CaCl2, the pH was adjusted to 9.0 (with 1 N NaOH),
kept overnight in the cold, and re-centrifuged at 12,000 rpm for 10 min.
en it was adjusted to pH 4.0 (with 1 N HCl) and re-centrifuged at
12,000 rpm for 10 min. Subsequently, the clear supernatant was adjusted
to pH 7.5 (with 1 N NaOH) and solid ammonium sulphate was added
to reach a nal concentration of 1.5 M. Aer standing overnight in the
cold room, the precipitate was removed by centrifugation at 12,000
rpm for 30 min. e nal supernatant was decanted, ltered through
lter paper (Whatman 3 MM). A column of mannose-Sepharose 4B
was equilibrated with 1.5 M ammonium sulphate (in 50 mM sodium
acetate, pH 6.5). Aer passing the extract through the column, it was
next washed with 1.5 M ammonium sulphate (in 50 mM sodium
acetate, pH 6.5) until the A280 decreased below 0.01. en, the lectin
was eluted by a gradient of ammonium sulphate decreasing from 1.5
M to 0 M. e elution prole was monitored online in a Waters 2489
detector at 280 nm. e concentrations of the protein solutions, stated
in our text, were determined from their heterodimeric extinction
coecient, ε280, of 46,660 M-1 cm-1. Hence all concentrations mentioned
here mean the heterodimeric concentration.
MALDI-TOF analysis
Matrix assisted laser desorption ionisation time-of-ight mass
spectrometry (MALDI-TOF-MS) analysis of the crystal was performed on
an Ultraex TOF/TOF (Bruker Daltonics, Germany) mass spectrometer
equipped with a UV nitrogen laser of 337 nm. A protein solution (1 μL)
was mixed with 1 μL matrix solution (saturated solution of sinapinic acid in
acetonitrile/0.1% aqueous triuoroacetic acid) and 1 μL of this mixture was
deposited on the probe plate. e spectra were recorded in the reectron
positive ion mode aer the evaporation of the solvent and were acquired
and analyzed by Bruker Daltonics Flex control soware.
pH treatment of CEA
To study the eect of pH on the secondary and tertiary structure
of CEA, the following buers were used, all having 150 mM NaCl: 10
mM Glycine-HCl (pH 1-3), 10 mM Na-acetate (pH 4-5), 10 mM Na-
phosphate (pH 6-7.2), 10 mM Tris-Cl (pH 8-9) and 10 mM Glycine-
NaOH (pH 10-12). Protein was incubated for 12 h at room temperature
at dierent pH values before recording various size exclusion
chromatography as described below.
Size-exclusion chromatography
e size-exclusion chromatography experiments were performed on
a Superdex-75 10/300 GL column attached to a Waters HPLC system. An
aliquot of 200 µl of protein samples (0.5 mg/ml) prepared by incubation
with varying pH was injected on to the column. e column was pre-
equilibrated with the appropriate buer of varying pH. e ow rate was
adjusted to 0.5 ml/min and elutant was detected on-line by Waters 2489
UV visible detector at 280 nm. To determine the size, the column was
calibrated with the following proteins in PBS, pH 7.2: Helix promita lectin
(79 kDa), Galanthus nivalis lectin (52 kDa), Narcissus pseudonarcissus
lectin (26 kDa), Pseudomonas aeruginosa lectin (13.7 kDa).
Crystallization and X-ray diraction data collection
CEA crystals were obtained by hanging-drop, vapor-diusion method
at room temperature, in about 3 to 4 weeks using 0.1 M Hepes pH 6.0
and 1.8 M ammonium sulfate as precipitant. Data collections at cryogenic
temperature (−170°C) were carried out at the home X-ray source. For
data collection, the crystals were soaked in the cryo-protectant solution
(respective buer containing additional 30% ethylene glycol). e collected
data were indexed, processed and scaled using Crystal Clear™ (Version 2.0)
and the implemented program d*TREK®.
Molecular replacement, renement and interface analysis
e structure solution and analyses were carried out using various
modules of CCP4i; the structures were solved using molecular
replacement [15] using the crystal structure of the two domain RVL
(PDB ID: 3R0E) [16] having 94.6% sequence identity to CEA. e
model was monitored and modied using the molecular graphics
program Coot within the CCP4i package, substituting the side chains
for the residues diering between CEA and RVL. e renement was
carried out mostly using Refmac5 [17] also within the CCP4i package,
switching to PHENIX 1.8.1 [18] at the very end.
e physicochemical features of the dimeric and tetrameric
interfaces were calculated using the PISA web server available within the
PDB [19], including listing of salt bridges, hydrogen bonds, interfacing
residues, etc. e program Coot [15] was used for monitoring the
progress of the crystallographic renement as well as for the display
of models superposed with electron density maps, as shown here.
Refer to Supplementary Table 1 for crystallography data collection and
renement statistics.
Magnesium detection by atomic absorption
Aer nding the ordered magnesium ions in our electron density
maps, the detection of magnesium (absorption at 285.4 nm) and
manganese (absorption at 279.7 nm) from some CEA samples dissolved
in 0.01 N hydrochloric acid were attempted by atomic absorption
spectroscopy in a ermo Scientic iCE3000 series.
Results
Mass spectrometric analysis
e mass spectrum (Figure 1) of the solubilised CEA crystal
ascertained that two chains of masses 11.9166 kDa and 12.5283 kDa
were crystallized and these correspond roughly with the masses
calculated from the sequences of the two protein chains in CEA,
11.999 kDa (109 aa) and 12.404 kDa (111 aa), respectively, dierences
attributed to either loss of terminal residues in the gas phase or addition
of hydrated magnesium ions particularly for chain B.

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 3 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
(45% and 50%) have lower sequence identity with CEA. is justies
our use of RVL as the search model for molecular replacement, though
RVL is supposedly not a simple sugar-binding lectin.
Structure solution by molecular replacement
e crystal was primitive orthorhombic, with unit cell parameters
a=122.01 Å, b=47.20 Å, c=82.25 Å and α=β=γ=90.00o. A listing of
the axial reections post-processing showed it belonged to P2221, by
noting the systematic absences. e website http://www.ruppweb.org
predicts a 93.2% probability of having two dimers in the asymmetric
unit, Vm=2.52 Å3/Da by submitting the cell parameters and an eective
resolution of 1.6 Å, by comparing with protein structures of similar
resolution. Most of the molecular replacement jobs were carried out
with chains A and B only from 3R0E as the input model. A total of
~60 separate molecular replacement jobs were carried out within the
program MOLREP, CCP4 package [15] by varying resolution ranges
of data used for the rotation function and translation function, and
dierent input models selected from entries 3R0E, 1KJ1 and 1MSA
either using a heterodimer or a tetramer (dimer of a heterodimer)
aer deletion of the water molecules/ligands, but were judged to be
ultimately unsuccessful as the nal R-value using 32,328 reections in
the 8.0-2.0 Å range varied in the narrow range of 0.5363 to 0.5417 for
ve dierent rotation and translation function solutions found using
MOLREP, with a common position of the rst dimer and dierent
positions of the second in the unit cell, aer restrained renement using
REFMAC5 [15].
At this point, the input model was rened aer making the
necessary amino acid substitutions for changing the protein sequence
Gel ltration chromatography
Gel permeation chromatography of the Colocasia lectin shows a
single peak corresponding to a calculated molecular mass of 48.2 kDa
at pH 7.2. ere is an appreciable change in the position of the CEA
peak at pH 3 relative to higher pH measurements and elution volume
increases progressively from 10.17 ml to 11.65 ml in the range of pH
7.2 to 3 (Figure 2). However, at pH 5, no change in elution prole is
observed with respect to pH 7.2. At pH 3.5, the presence of two peaks
eluting, ~10.17 ml and ~11.65 ml are noticed. Aer lowering pH to
3, only the peak eluting ~11.65 ml is observed. is implies, upon
lowering pH from 7.2 to 3, CEA converts from heterotetramer to
heterodimer (Figure 2).
Previous high resolution lectin crystal structures
e family of monocot mannose-binding lectins has been
described [2-4]. Among the few other high resolution (2 Å or better)
protein structures of the same family preceding our study are the
homodimeric Polygonatum cyrtonema agglutinin (PCL, 3A0C 28,647
rens, 2.0 Å) [9] and homodimeric Scilla campanulata lectin (ScaMan,
1B2P, 33,837 rens, 1.7 Å) [8]. Another high resolution structure was
the heterodimeric Crocus vernus Dutch bulb lectin from the chitinase
family (CVL, 3MEZ, 30,977 rens, 1.94 Å); for this a preliminary paper
was published [20].
Among previously solved crystal structures, Remusatia vivipara
lectin (RVL, PDB ID: 3R0E, 25,784 rens, 2.40 Å) [16] has the highest
sequence identity of 93.8% with the Colocasia esculenta agglutinin.
Other lectins like Scafet (32% or lower), PCL (45% or lower) and CVL
Figure 1: MALDI-TOF mass spectrometric analysis of the CEA crystal performed after dissolving it in water. The two major m/z peaks are at 11.9166 kDa and 12.5283
kDa, corresponding to the two polypeptides contained in the heterodimeric CEA crystal, roughly agreeing with their calculated molecular masses using protein sequence
alone, 11.999 kDa (109 aa, chain A) and 12.404 kDa (111 aa, chain B). For chain A, the 11.999 kDa is converted to the 11.9166 kDa peak probably through the loss
of the C-terminal Gly 109 in the gas phase. However, there is still a minor peak at 11.999 kDa corresponding to the intact chain A. The peak at ~12.142 kDa probably
occurs from the intact chain B around 12.404 kDa through the loss of both the N-terminal Asn and C-terminal Gln 111 in the gas phase. Other peaks at higher molecular
masses like 12.468 kDa, 12.528 kDa, 12.638 kDa could be due to one, two or three Mg2+ ions with hydroxyl ions and water molecules as ligands in addition to the
main chain carbonyls or due to additional residues at B Ala 112 and B Lys 113 (Figure 5). Enlargements of the peaks around 12.0 kDa and 12.5 kDa are also pasted
for clearer viewing.

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 4 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
from that in 3R0E to that of Colocasia esculenta agglutinin. Molecular
replacement was run afresh using the Phaser MR package [15]. is
run was successful and rigid body renement within REFMAC5
reduced the R-factor to 0.4196 using 12.0-3.0 Å data and a F.O.M. of
0.5519; using 16.0-2.5 Å data the R-factor became 0.4381 and F.O.M. of
0.4380. is new solution seemed even more probable/trusted as both
the dimers formed tetramers through their B chains (as in [16]) in the
arrangement resulting from these rotations and translations within the
P2221 unit cell (Figure 3). Only one of the two dimers had shown this
property in the previous solutions found by MOLREP.
By inspecting the 2Fo–Fc and Fo–Fc maps at the end of the rigid
body renement, the N-termini of the A and C chains were repositioned
into density, as they were sitting in negative density in the dierence
map for the trial model (Figure 4); the C-termini of the B and D chains
were adjusted manually using real space renement in Coot [15].
Several side chains were also manually positioned into densities visible
aer the rigid renement stage. Near the beginning of the restrained
renement, using 9,776 reections in the 16.0-3.0 Å range, the R-value
reduced to 0.2946, F.O.M 0.6113 using positional renement with
a constant overall B-factor of 20 Å2 within REFMAC5 and near the
end, using 49,194 reections in the 21.0-1.74 Å range R-value became
0.3441 and R-free remained at 0.4272. e Fo, Fc correlation coecient
is calculated to be 0.79 for the nal model. PDB calculated the Wilson
B of our dataset to be 12.40 Å2 using data till 1.74 Å but that calculated
in program Xtriage by the PDB yielded a Wilson B of 6.5 Å2 using all
data up to 1.536 Å.
Deviation from the Remusatia vivipara lectin structure
Indeed, the structure of CEA is also therefore expected to be close
to that of RVL, obtained from an ornamental monocotyledonous
owering plant (Araceae family) tuber. However, the RVL crystallized
in a tetragonal space group P41 and its 2.4 Å structure included 3510
protein atoms and 129 water molecules [16].
e comparison of 5D5G with the RVL structure which was used
as the input model in molecular replacement deserves special mention
(Figure 4). On superposing residues A:1-109 and B:1-110 of CEA with
those of 3R0E, including a total of 1,752 backbone atoms, the RMSD
between the structures is 0.8945 Å. However, if we omit a few residues
from the termini of chain A, including only A:3-106 and B:1-110
instead, the RMSD between the two structures in the 1,712 backbone
atoms is only 0.5872 Å (Table 1 and Figure 4). is was actually apparent
at the start of crystallographic renement, as maps had been calculated
aer rigid body renement and the termini of the A and C chains were
sitting in negative densities in the Fo–Fc map.
Colocasia esculenta crystal structure
e average temperature factor of an atom including waters and
ligands was 19.0 Å2 for our submitted structure, as noted in the PDB le
for 5D5G, but the backbone atoms like Cα have much lower average B
values (Figures 5 and 6). ese are much lower than those obtained for
3R0E [16], as shown.
Our crystal structure of mannose-free CEA also contains two
independent heterodimers in the asymmetric unit, each forming a
α2β2 tetramer by associating with its symmetry mate within the crystal.
Among the 3,577 non-hydrogen atoms in the asymmetric unit, were
the protein atoms, one Hepes molecule, one sulphate ion, 5 magnesium
ions and 134 waters located in the electron density maps (Figure 6).
Crystallization conditions, details of X-ray data and its collection,
renement, characteristics of nal model are described in some detail
in the header of our PDB entry 5D5G. e structure factor les are
also publicly available from the PDB. Whereas synchrotron X-ray data
collected at the ESRF beamline gave the 2.4 Å dataset consisting of
25,784 reections for RVL, our dataset was collected to 1.536 Å in a
home X-ray source, though our data was used till 1.74 Å as completeness
declined aerwards [21,22]. ough the Rmerge in intensities for our
1.536 Å dataset was on the high side (0.38) we decided to keep the high
resolution reections to 1.74Å (Rmerge=0.35) as this was to be a structure
solution by molecular replacement, neither by multiple isomorphous
Figure 2: Size-exclusion chromatography of CEA at neutral and acidic pH.
Gel permeation of the Colocasia lectin showed a single peak at elution volume
of 10.17 ml at both pH 7.2 and pH 5. However, at pH 3.5, a second minor
peak corresponding to a smaller molecular mass appears for CEA. The
bottom panel superposes results from CEA (solid curve, pH 3) and various
standard lectins of similar shape and known oligomerization status at pH 7.2
(dotted curves): H. promita lectin hexamer, 79 kDa (peak 1), G. nivalis lectin
homotetramer, 52 kDa (peak 2), N. pseudonarcissus lectin homodimer, 26 kDa
(peak 3), P. aeruginosa lectin monomer, 13.7 kDa (peak 4). Thus the higher
molecular mass CEA peak is close to the GNA homotetramer (peak 2) and
the lower molecular mass CEA peak is close to the N. pseudonarcissus lectin
homodimer (peak 3). These are interpreted to be the α2β2 heterotetramer and
αβ heterodimer respectively.

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 5 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
Figure 3: Packing diagram for correct molecular replacement solution obtained by Phaser MR Package [15], as Cα models in stereo. Chains A, B in yellow and red
comprise one heterodimer and chains C, D in blue and green comprise the other and together they make the asymmetric unit in this P2221 crystal; orientation of the
three crystallographic axes indicated. There is an exact 2-fold about vertical axis a, relating the two parts of a tetramer separately for each heterodimer shown in color.
Other symmetry related molecules lling up the unit cell are shown in grey. A copy of Chain B’ related by a unit translation along –c pairs with Chain B and a copy of
Chain D’ related by the same unit translation pairs with Chain D, forming the tetramers as shown in Figure 9.
Figure 4: New positions for residues 1-3 in Chains A and C relative to those in 3R0E, shown in stereo with 2Fo–Fc map (blue). The largest differences between the
CEA structure and that of RVL occur at the N-termini of the A- and C-chains. The molecular replacement solution shows Cα models of Chain A (green) and Chain C
(light blue), placed in a clashing position. In fact, these N-terminal residues were also sitting in negative density after rigid body renement. All non-hydrogen atoms in
the rened model are superimposed, with carbon atoms in yellow, nitrogen in blue and oxygen in red; 2Fo–Fc map is displayed at 1.5σ level, and Fo–Fc map at 3σ.
Distances in Å that these Cα atoms had to be moved to t the X-ray data are displayed for both Chains A and C, between their original and nal positions.

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 6 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
Figure 5: Cα-atom temperature factors compared with those in 3R0E and distances moved by crystallographic renement. The B-values are plotted for Chains A and C
in 5D5G along with those for Chain A in 3R0E (top panel) and for Chains B and D in 5D5G with those for Chain B in 3R0E (middle panel). The temperature factors are
generally much lower for 5D5G, as expected in a higher resolution structure. Residue numbers and protein sequences for Chains A, B, C, D appear below the plots, with
substitutions needed in red shown for RVL. Also, residues in β-sheets comprising the β-prism II fold possess lower B-values for 5D5G compared to loop regions (top and
middle panels). The intact carbohydrate recognition sites satisfying the requirement QXDXNXVXY [16] appear only before the third β-strand in domain III, for residues
A28-A36 and B31-B39, underlined in green, generally possess low to moderate B-values including the loop regions therein. Among distances moved for the Cα atoms for
Chains A (blue), B (green) and C (red) for residues 3-107 as a result of the crystallographic renement (bottom panel), they were highest for Chain C loop regions which
also have the highest B-values. In general, the movements were all within 0.8 Å. Exceptions are either in loop regions or at the termini (not shown here).
Regions from 5D5G used Other structure, regions used respectively, reference Atoms compared RMSD
3MEZ or CVL [20]
A3-A36, B45-B110 A2-A35, B48-B113 (remaining regions do not superpose well) 800 0.9310Å
1DLP or Scafet [27]
A3-A8, A10-A37, A40-A46, A49-A102; B7-B11, B13-B40, B44-B108 A1-A5, A15-A42, A48-A54, A56-A109; A125-A129, A139-A166, A171-A235 1,544 1.1140Å
3A0C or PCL [9]
A3-A21, A22-A36, A40-A105 A1-A19, A21-A35, A42-A107 800 1.0574Å
B8-B17, B19-B24, B26-B39, B51-B108 B3-B12, B14-B19, B22-B35, B48-B105 704 0.9567Å
When both the regions from A, B subunits are used to compare 1,504 1.6134Å
3R0E or RVL [16]
A:1-109, B:1-110 A:1-109, B:1-110 1,752 0.8945Å
A:3-106, B:1-110 A:3-106, B:1-110 1,712 0.5872Å
Table 1: Comparison of A, B dimer in 5D5G with previous heterodimeric β-prism II lectin crystal structures, using backbone atoms only.

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 7 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
replacement nor anomalous methods. Since the input model in 3R0E
was quite reliable for our molecular replacement solution, using data
till 1.74 Å is justied in order to maintain a high data-to-parameter
ratio of 49,194/(4 × 3,577) or roughly 3.5/1. We believe in the advice
of keeping as much X-ray data as possible as long as the shells are high
in completeness, though much of the data is weak, as this is benecial
regarding the accuracy of the structure [22]. As a result, our R value for
the 49,194 reections is 0.344, as opposed to 0.209 for RVL [16]. e
rened structure in 5D5G has no bond length, bond angle, chirality or
planarity outliers and has 0.9% Ramachandran outliers.
e mannose-free CEA structure solved using 49,194 reections in
the 20.99-1.74 Å resolution range contains four β-prism II domains,
each with a β-barrel made by three subdomains of amphipathic anti-
parallel β-sheets arranged around a pseudo 3-fold axis like three faces
of an equilateral prism (Figures 3, 4 and 6). Electron density for residue
Gln 111 was observed only in chain D, but it was not observed for chain
B having the same sequence. Side chain atoms for which density could
not be observed at all in the electron density map are given in our PDB
le for 5D5G as “missing” atoms, particularly from A Arg 49, B Ala 98,
C Leu 1, C Arg 49 and D Ala 98. It was particularly observed that some
aliphatic side chains at the core of these β-prisms had less than unit
occupancy, probably due to motion allowed inside the cores.
Cys 31, Cys 51 in the A, C subunits and Cys 34, Cys 56 in the B, D
subunits form disulphide bridges. Cis peptide bonds exist at Gly 97-Pro
98 in the A, C subunits and at Gly 102-Pro 103 in the B, D subunits near
the C-termini as found with RVL [16].
As previously observed in RVL and other β-prism II domains, the
two domains (A, B or C, D) have their C-terminal β-strands hydrogen
bonding with a β-sheet in their partner domain, called C-terminal
exchange [23].
Presence of magnesium ions in CEA
Colocasia esculenta tubers were homogenized in 0.2 M NaCl, 20
mM CaCl2 pH 9 adjusted with NaOH and nally adjusted to 1.5 M
ammonium sulfate pH 6.5 with 50 mM sodium acetate used for the
mannose-Sepharose B anity column. Prior to crystallization, the CEA
solution was made 20 mM in Tris pH 8, 150 mM NaCl. us the buers
used for purication or for crystal growth all lacked magnesium.
Magnesium was detected in CEA by atomic absorption spectroscopy,
conrming our nding of ordered magnesium in the electron density
maps (Figure 7). ough our sample was tested for manganese also, the
result was negative for that element.
It was found that for the ve Mg2+ ions located in electron density
map, both residues that contributed a carbonyl as a ligand to such
an ion had to possess B-values in the 5-10 Å2 range. Otherwise, the
magnesium ions could not be observed. We looked near the same
residues in subunits C (Leu 6) and D (Ala 85, Asn 6, r 22, Val 89, Ile
91, Phe 97, Val 99, Gly 102) but as those had B-values exceeding 10 Å2,
Figure 6: Model in 5D5G color coded according to temperature factors. The asymmetric unit is again shown as a Cα model in stereo, color coded here according to
the temperature factor at each residue, color-coded as follows: blue, less than 10 Å2; turquoise, 10 to 20 Å2; green, 20 to 30 Å2; yellow, 30 to 40 Å2; orange, 40 to 50 Å2.
The four domains A, B, C, D are labeled, as are the Hepes molecule, the sulfate ion and ve Mg2+ as white dots. On average, chains B and D have the lowest, C has
the highest and A intermediate in temperature factors. Also, regions comprising the dimeric or the tetrameric interfaces have the lowest temperature factors in all four
chains. The tetrameric interfaces are all generated around the vertical a axis (along x) as it is a crystallographic 2-fold, hence domains B, D are closest to the viewer
and A, C away. The highest B-values (>36 Å2) occur in labeled residues A66, A67, B110, region C43-C50, region C64-C70 and C109, seen in yellow/orange color,
accompanied by weaker electron densities especially for their side chains; these are mostly regions which do not press against a neighboring molecule but are free
to move in the disordered solvent in the crystal.

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 8 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
the densities corresponding to equivalent magnesium ions could not
be observed. However, a dierence density indicated a Mg2+ ion with
C Pro 98 (as with A Pro 98), but its other carbonyl partner was from D
Ile 100 (instead of B Gly 102) as here, D Ile 100 has the lower B-value
compared to D Gly 102.
Ordered sulfate in the crystal
A strong tetrahedral density sitting adjacent to B His 55 and B’
His 62 whose side chains stack with each other, became apparent at
the intermediate stages of our crystallographic renement. Since there
was no phosphate in the concentrated CEA stock solution (containing
20 mM Tri s -HCl) used for crystallization and the protein solution
eventually had at least ~0.5 M ammonium sulfate in the drop from
which crystals had resulted, we interpreted this as a sulfate anion and
this was incorporated in our renement thenceforth. Figure 6 shows
the position of this ordered sulfate in the unit cell and Figure 8 shows
how it explains the electron density map and justies its location by
hydrogen bonding to the two histidine side chains near the tetrameric
interface and a main chain amide in B Gly 17. Similarly, we located a
molecule of Hepes, used as a buer during crystal growth (Figure 6).
Tetramer and dimer formation
e presence of two distinct N-termini is a consequence of the
original polypeptide chain folding into two β-prism II domains with
two cleavages near the middle. e distance between the C-terminus
at A Gly 109 and the N-terminus at B Asn 1 negates a possibility of
the 7-residue linker remaining in a disordered form. e two domains
or subunits A and B have 44% sequence identity between them. Using
PYMOL [24], Figure 9 shows a view and a close up highlighting specic
interactions stabilizing the tetramer formation; Figure 10 shows the
various interactions stabilizing the dimer between subunits A and B.
Analyses of αβ and ββ’ interfaces in 5D5G
Using the PISA web server [19], both interfaces scored the maximum
possible complex formation signicance score of 1.00. For the interface
between chain B and its symmetry mate B’, 22 hydrogen bonds and
12 salt bridges were found (Figure 9); for that between chains A and
B, 21 hydrogen bonds and 1 salt bridge were found. Similarly, for the
interface between chains C and D, 29 hydrogen bonds and 1 salt bridge
were found; for that between D and D’, 20 hydrogen bonds and 10 salt
bridges were found. In lieu of the numerous salt bridges seen for the
B-B’ or D-D’ interfaces, the A-B and C-D interfaces having 1644.9 Å2
and 1813.5 Å2 interface areas respectively, are stabilized by interactions
between numerous hydrophobic residues (Figure 10), characteristic of
the β-prism II lectin structures [16].
Structural comparisons within the four chains in the
asymmetric unit
As each of the four subunits in the asymmetric unit exists in a
β-prism II structure, it is useful to record the similarity and dissimilarity
among all possible pairs between the subunits A, B, C, D as shown in
Tabl e 2. ese root mean square deviations were generally calculated
for the pairs for the backbone atoms only, but when the sequence is
identical, as for (A, C) and (B, D) pairs, it may also include the side
chains. us the structural similarity is the highest between the B, D
pair followed by that for the A, C pair, regardless of the residue ranges
Figure 7: One of the ve Mg2+ ions to be located at the nal stages of renement, bound between the carbonyls of A Pro 98 and B Gly 102, at distances of 1.88Å and
1.86Å from those oxygen atoms. These had between 5.14 σ to 5.86 σ levels in the Fo–Fc map. While this magnesium sits at the border of Chains A (carbons in yellow)
and B, three others are embedded between pairs of carbonyl oxygens within Chain B and one between those in Chain A. Though Mg2+ and Na+ are isoelectronic with
divalent oxygen, these difference densities cannot be explained by water molecules due to their proximity with the carbonyl oxygen atoms. Na+ has an average ligand
distance of 2.46 (14) Å with its oxygen ligands as observed from an analysis of the protein structures in the PDB [28], while Mg2+ exhibits smaller metal-ligand distances.
The presence of bound magnesium was also conrmed by atomic absorption spectroscopy.

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 9 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
Figure 8: Sulfate bridging parts of Chain B and also Chain B’. The ordered sulfate ion seen in the 2Fo–Fc density contoured at 1.6 σ level, forming hydrogen bonds with
nitrogen atoms in two histidine side chains belonging to two different subunits related by crystal symmetry and with the main chain amide of B Gly 17.
Pair used Regions superposed in InsightII No. atoms compared RMSD
A, B A2-A36 with B5-B39, A41-A102 with B46-B107 776 0.923 Å
A, C A1-A109 with C1-C109 (backbone only) 872 0.993 Å
A, C A3-A106 with C3-C106 (backbone only) 832 0.706 Å
A, C A2-A109 with C2-C109 (with side chains) 1,664 1.287 Å
A, D A2-A36 with D5-D39, A41-A102 with D46-D107 776 0.932 Å
B, C B5-B39 with C2-C36, B46-B107 with C41-C102 776 0.924 Å
B, D B1-B110 with D1-D110 (backbone only) 880 0.609 Å
B, D B1-B110 with D1-D110 (with side chains) 1,734 1.002 Å
B, D B1-B108 with D1-D108 (backbone only) 864 0.529 Å
B, D B1-B108 with D1-D108 (with side chains) 1,708 0.953 Å
C, D C2-C36 with D5-D39, C41-C102 with D46-D107 776 0.893 Å
(A, B), (C, D) A3-A106 with C3-C106, B1-B108 with D1-D108 1,696 0.664 Å
(A, B), (C, D) A3-A106 with C3-C106, B1-B108 with D1-D108 (with side chains) 3,326 1.061 Å
Table 2: RMSD between all possible pairs of CEA subunits in 5D5G.
compared, or whether side chains were included in the comparison
(Tabl e 2). e other pairs have dissimilar sequences, hence the greater
values of RMSD are expected. Some regions were kept out of the
superposition calculations as they show greater deviations and their
inclusion will needlessly worsen the deviation observed.
Pairwise comparisons with other lectin crystal structures
In the backbone comparisons with other β-prism II heterodimeric
lectins, the RMSD is lowest for 3MEZ (Crocus vernus lectin or CVL) as
there are only a few regions of overlap (Table 1); the rest of this pair do
not superpose well, though CVL has 45% and 50% sequence identity in
the two chains with CEA. Among the remaining two, overall RMSD is
lower for the 3.3 Å structure 1DLP (heterodimeric Scilla campanulata
agglutinin or Scafet) compared to that with 3A0C (Polygonatum
cyrtonema agglutinin or PCL) (Table 1). Individual domains in 3A0C
superpose better than the dimer as a whole, as PCL has higher sequence
identity with CEA compared to Scafet, but the dimer does not, due to
a rotation observed between the A and B subunits in 3A0C relative to
5D5G. All of these are however, less similar with 5D5G compared to
the RVL structure.
e RMSD values obtained in all these cases are close to 1 Å,
around the same values obtained in Ta b l e 2 for chains having dierent
sequences (like A, B or C, D or A, D or B, C), since the two dissimilar
chains within CEA share 44% sequence identity between them, about
the same level as with these other pairs of lectins.
Discussion
Gel ltration of the lectin suggested its presence in the α2β2 form
at neutral pH, eluting at the same place where the homotetrameric G.
nivalis standard elutes (peak 2), in agreement with gel ltration data
(Figure 2). ough our Colocasia esculenta crystal structure in 5D5G
was the rst such released by the PDB in September, 2015, two later
studies on the similar lectins of other variants [26,27] did not mention it,
their PDB entries released June and September, 2016, respectively. One
of these discusses a CEA crystal structure solved at 2.1 Å rened with
22,646 reections only to the level of the heterodimer [25], completely
ignoring the fact that in their P3121 crystal, an almost identical
heterotetramer is observed between αβ dimers related by symmetry,
just as in our study. Despite data collection at an Argonne Synchrotron,
the temperature factor average is 56.5 Å2 for that structure. For our
X-ray data, the Wilson B factor was calculated to be 12.4 Å2 to 1.74 Å by
our renement or 6.5 Å2 using data to 1.536 Å by the PDB. is lectin
has 7 amino acid substitutions and a 2 residue deletion relative to ours

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 10 of 12
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ISSN: 2168-958X
(positions 13, 75, 82, 87 in Chain A; positions 25, 85, 98 in Chain B, lack
of residues B112-113) [25].
Yet another recent study describes the CEA lectin crystallized in P21
space group diracting to 1.72 Å and its trimannoside-bound complex
in P1 space group diracting to 1.91 Å [26]. In this study, the X-ray
data was also collected at an Argonne Synchrotron, and has better data
statistics, in the Rsym of 6.7% or 8.5% [26]. For the native crystal, the cell
parameters are also quite close to those obtained for our crystal in P2221
space group. According to Figure 1D in that paper [26], we have solved
the structure of a slightly modied CESP here (6 substitutions relative
to our sequence), whereas they have TarinA and TarinB. ere are a
total of 11 amino acid substitutions and a deletion between our protein
sequence and that of the tarin (positions 13, 14, 15, 47, 77, 87 in Chain
A; positions 6, 25, 80, 85, 110 in Chain B, lack of residues B111-113).
So RVL is equally close to our CEA lectin with 11 substitutions and the
same deletion (Figure 5). However, tarin also exhibits ββ’ interactions
[26], like RVL [16] and our CEA lectin.
Our crystal structure shows there are several salt bridges between
side-chains in the ββ’ interface (Figure 9). Upon reaching pH 3, several
of these, e.g. B Glu 54, B Asp 71, B Asp 72 are protonated from both the
participating subunits, hence at least 6 of the salt bridges are broken,
thereby explaining the observed transition from heterotetramer to
Figure 9: Tetramer formation within the CEA crystal between two adjacent copies of the AB dimer using subunit B. Subunit A is in yellow, subunit B is in red. In the
second copy of the AB dimer, subunit A’ is in light blue and subunit B’ is in light green. (A) In the top half, we are looking down the crystallographic a axis, a perfect 2-fold,
passing through the centre in this diagram, between the red and green molecules. (B) In the bottom half, displaying both hydrogen bonds/salt bridges and hydrophobic
interactions important in the dimer-dimer interface using two copies of subunit B, looking down the a axis. Here the two copies of Sheet II are facing each other across
a crystallographic 2 fold. The crystallographically independent molecules C and D within the asymmetric unit also form an identical tetramer with a symmetry-related C’
and D’, though not shown here. Figure generated using PYMOL [24].

Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 11 of 12
Volume 6 • Issue 2 • 1000126
J Glycobiology, an open access journal
ISSN: 2168-958X
heterodimer. e pH dependence on the stability of CEA as seen in our
gel ltration chromatography (Figure 2) suggested that the electrostatic
interactions in the salt bridges make a signicant contribution to the
conformational stability of the heterotetramer.
e classical carbohydrate recognition site satisfying the sequence
requirement QXDXNXVXY [16] appears only once in each of Chains
A and B, both located on domain III (Figure 5, green underline). ese
are at 28-QDDCNLVLY-36 in Chain A and at 31-QGDCNLVLY-39
in Chain B, just as with RVL [16]. In both Chains A and B, domain
I is involved in the formation of the dimeric interface and domain
II from two B chains are involved in the formation of the tetrameric
interface (Figures 9 and 10). So domain III containing the carbohydrate
recognition site is not directly involved in these interfaces.
Legume lectins are known to contain Mn2+ and Ca2+, but there is
probably no previous report of Mg2+ in plant lectins. However, we did
not test for lectin activity as a function of increasing amounts of EDTA
concentration. e previous study on tarin showed its activity does not
depend on metal ions [13]. However the existence of several isoforms
with a heterogeneous population of dierent covalently attached glycans
[13], usually does not yield such highly resolution X-ray diraction as
reported by almost the same authors [26]. eir purication scheme
[26] shows they used the natural tarin for crystallography.
Since the heterodimeric Scilla campanulata agglutinin or Scafet
diracted only to 3.3 Å [27], the CEA becomes the rst heterodimeric
structure to be studied at such a high resolution, the comparable
resolution being for Polygonatum cyrtonema agglutinin or PCL [9]
and the tarin [26]. Our biophysical study correlating with the crystal
structure described here is expected to be published shortly.
Acknowledgement
We thank Mr. Pallab Chakraborty for maintaining our Institute X-ray diffraction
data collection facility and for helping with crystallographic data collection, Mr.
Avisek Mondal of our department for help with various crystallographic programs.
Figure 10: Dimer formation between subunits A and B seen in the CEA crystal is shown above, with the top half emphasizing the hydrophobic residues important in
that interface, displayed as stick models. Subunit A is mainly to the right, in dark green or grey color; subunit B is mainly to the left in green. The sheet I of subunit A
faces and interacts with the sheet I of subunit B, and this is the stabilizing interaction between the two subunits. As in other lectins in this category, the C-terminal tail of
each subunit (top right side for subunit B, lower left for subunit A) crosses over to the other side and hydrogen bonds with the other subunit, thereby completing sheet I
and spreading the volume over which the two subunits interact [23]. Oxygen atoms are in red and nitrogen atoms in blue displayed for some residues in this interface.
Numerous hydrophobic side chains are involved in this interaction, most of them marked here. There is a similar interface between independent subunits C and D.
Funding
This study was funded by the Department of Science and Technology,
Government of India through Bose Institute.
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Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its
Quaternary Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126
Page 12 of 12
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J Glycobiology, an open access journal
ISSN: 2168-958X
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Citation: Chattopadhyaya R, Biswas H, Sarkar A (2017) Crystal Structure of
Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its Quaternary
Interactions. J Glycobiol 6: 126. doi:10.4172/2168-958X.1000126