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- EMDB-47565: Cryo-EM structure of Maackia amurensis seed Leukoagglutinin (lect... -

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Basic information

Entry
Database: EMDB / ID: EMD-47565
TitleCryo-EM structure of Maackia amurensis seed Leukoagglutinin (lectin), MASL
Map data
Sample
  • Complex: Cryo-EM map of Maackia amurensis seed Leukoagglutinin (lectin), MASL
    • Protein or peptide: Seed leukoagglutinin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
  • Ligand: MANGANESE (II) ION
KeywordsSeed Lectin / Maackia amurensis / N-linked glycosylation / Intersubunit Disulphide bridge / leukoagglutinin / SUGAR BINDING PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsNayak AR / Goldberg GS / Temiakov D / Sedmen J / Zamudio-Ochoa A
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateMay 7, 2025-
Current statusMay 7, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47565.png

Downloads & links

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Map

FileDownload / File: emd_47565.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 216 pix.
= 200.88 Å		0.93 Å/pix.
x 216 pix.
= 200.88 Å		0.93 Å/pix.
x 216 pix.
= 200.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.93 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.21755707 - 0.48770124
Average (Standard dev.)-0.00004510898 (±0.017650181)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 200.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened map

Fileemd_47565_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_47565_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_47565_half_map_2.map
Projections & Slices
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Sample components

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Entire : Cryo-EM map of Maackia amurensis seed Leukoagglutinin (lectin), MASL

EntireName: Cryo-EM map of Maackia amurensis seed Leukoagglutinin (lectin), MASL
Components
  • Complex: Cryo-EM map of Maackia amurensis seed Leukoagglutinin (lectin), MASL
    • Protein or peptide: Seed leukoagglutinin
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Cryo-EM map of Maackia amurensis seed Leukoagglutinin (lectin), MASL

SupramoleculeName: Cryo-EM map of Maackia amurensis seed Leukoagglutinin (lectin), MASL
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Maackia amurensis (plant)
Molecular weightTheoretical: 125 KDa

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Macromolecule #1: Seed leukoagglutinin

MacromoleculeName: Seed leukoagglutinin / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Maackia amurensis (plant)
Molecular weightTheoretical: 31.32148 KDa
SequenceString: MATSNSKPTQ VLLATFLTFF FLLLNNVNSS DELSFTINNF VPNEADLLFQ GEASVSSTGV LQLTRVENGQ PQQYSVGRAL YAAPVRIWD NTTGSVASFS TSFTFVVKAP NPDITSDGLA FYLAPPDSQI PSGSVSKYLG LFNNSNSDSS NQIVAVEFDT Y FGHSYDPW ...String:
MATSNSKPTQ VLLATFLTFF FLLLNNVNSS DELSFTINNF VPNEADLLFQ GEASVSSTGV LQLTRVENGQ PQQYSVGRAL YAAPVRIWD NTTGSVASFS TSFTFVVKAP NPDITSDGLA FYLAPPDSQI PSGSVSKYLG LFNNSNSDSS NQIVAVEFDT Y FGHSYDPW DPNYRHIGID VNGIESIKTV QWDWINGGVA FATITYLAPN KTLIASLVYP SNQTTFSVAA SVDLKEILPE WV RVGFSAA TGYPTEVETH DVLSWSFTST LEANCDAATE NNVHIARYTA

UniProtKB: Seed leukoagglutinin

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 8 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #6: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 4 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 7.9
Details: 20 mM Tris-HCL (pH=7.9), 100 mM NaCL, 0.1 mM CaCl2, 0.1 mM MnCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details3 uM MASL monodisperse solution

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Electron microscopy

MicroscopeTFS GLACIOS
Specialist opticsDetails: No Energy filter was used
SoftwareName: EPU (ver. 3.6)
DetailsCalibrated pixel size -0.93
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 8543 / Average electron dose: 60.0 e/Å2
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)
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 8343216
CTF correctionDetails: CTFFIND 4.0 / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: Ab -initio reconstruction in cryoSPARC 4.4.
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 2656977
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 4.4)
Details: Particles lacking organization, and missing subunits were separated
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1dbn


Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: Coot (ver. 0.9.8.95)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9e6h:
Cryo-EM structure of Maackia amurensis seed Leukoagglutinin (lectin), MASL

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