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- PDB-9e6h: Cryo-EM structure of Maackia amurensis seed Leukoagglutinin (lect... -

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Basic information

Entry
Database: PDB / ID: 9e6h
TitleCryo-EM structure of Maackia amurensis seed Leukoagglutinin (lectin), MASL
ComponentsSeed leukoagglutinin
KeywordsSUGAR BINDING PROTEIN / Seed Lectin / Maackia amurensis / N-linked glycosylation / Intersubunit Disulphide bridge / leukoagglutinin
Function / homology
Function and homology information


carbohydrate binding / metal ion binding
Similarity search - Function
Legume lectin / Legume lectin, alpha chain, conserved site / Legume lectins alpha-chain signature. / Legume lectins beta-chain signature. / Legume lectin domain / Legume lectin, beta chain, Mn/Ca-binding site / Legume lectin domain / : / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
: / Seed leukoagglutinin
Similarity search - Component
Biological speciesMaackia amurensis (plant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsNayak, A.R. / Goldberg, G.S. / Temiakov, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM131832 United States
CitationJournal: J Biol Chem / Year: 2025
Title: Maackia amurensis seed lectin structure and sequence comparison with other M. amurensis lectins.
Authors: Ashok R Nayak / Cayla J Holdcraft / Ariel C Yin / Rachel E Nicoletto / Caifeng Zhao / Haiyan Zheng / Dmitry Temiakov / Gary S Goldberg /
Abstract: Maackia amurensis lectins, including MASL, MAA, and MAL2, are widely utilized in biochemical and medicinal research. However, the structural and functional differences between these lectins have not ...Maackia amurensis lectins, including MASL, MAA, and MAL2, are widely utilized in biochemical and medicinal research. However, the structural and functional differences between these lectins have not been defined. Here, we present a high-resolution cryo-EM structure of MASL revealing that its tetrameric assembly is directed by two intersubunit disulfide bridges. These bridges, formed by C272 residues, are central to the dimer-of-dimers assembly of a MASL tetramer. This cryo-EM structure also identifies residues involved in stabilizing the dimer interface, multiple glycosylation sites, and calcium and manganese atoms in the sugar-binding pockets of MASL. Notably, our analysis reveals that Y250 in the carbohydrate-binding site of MASL adopts a flipped conformation, likely acting as a gatekeeper that obstructs access to noncognate substrates, a feature that may contribute to MASL's substrate specificity. Sequence analysis suggests that MAA is a truncated version of MASL, while MAL2 represents a homologous isoform. Unlike MASL, neither MAL2 nor MAA contains a cysteine residue required for disulfide bridge formation. Accordingly, analysis of these proteins using reducing and nonreducing SDS-PAGE confirms that the C272 residue in MASL drives intermolecular disulfide bridge formation. These findings provide critical insights into the unique structural features of MASL that distinguish it from other M. amurensis lectins, offering a foundation for further exploration of its biological and therapeutic potential.
History
DepositionOct 30, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2May 7, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Seed leukoagglutinin
B: Seed leukoagglutinin
C: Seed leukoagglutinin
D: Seed leukoagglutinin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,07428
Polymers125,2864
Non-polymers8,78824
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Seed leukoagglutinin / Leukoagglutinating lectin MAL / Seed leucoagglutinin


Mass: 31321.480 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Maackia amurensis (plant) / References: UniProt: P0DKL3

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Sugars , 3 types, 16 molecules

#2: Polysaccharide
alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#3: Polysaccharide
alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 8 molecules

#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#6: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cryo-EM map of Maackia amurensis seed Leukoagglutinin (lectin), MASL
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.125 MDa / Experimental value: YES
Source (natural)Organism: Maackia amurensis (plant)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
Details: 20 mM Tris-HCL (pH=7.9), 100 mM NaCL, 0.1 mM CaCl2, 0.1 mM MnCl2
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 3 uM MASL monodisperse solution
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS / Details: Calibrated pixel size -0.93
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8543
Details: Total number of frames - 40, Defocus range - -0.3 to -1.2 um in 0.1 increments, Output mode - TIFF, Autofocus recurrence - after centering, Drift measurement - once per grid square (Threshold - 0.4 nm/sec)
EM imaging opticsDetails: No Energy filter was used

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4particle selection
2EPU3.6image acquisition
7Coot0.9.8.95model fitting
9PHENIX1.21.2.5419model refinement
12cryoSPARC4.4classification
13cryoSPARC4.43D reconstruction
CTF correctionDetails: CTFFIND 4.0 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8343216
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2656977 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1DBN

1dbn


Accession code: 1DBN / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028306
ELECTRON MICROSCOPYf_angle_d0.60911394
ELECTRON MICROSCOPYf_dihedral_angle_d19.6161322
ELECTRON MICROSCOPYf_chiral_restr0.0471426
ELECTRON MICROSCOPYf_plane_restr0.0051410

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