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- PDB-8trz: Cryo-EM structure of N-glycosylated Pomacea canaliculata hemocyanin -

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Basic information

Entry
Database: PDB / ID: 8trz
TitleCryo-EM structure of N-glycosylated Pomacea canaliculata hemocyanin
ComponentsHemocyanin
KeywordsOXYGEN TRANSPORT / Glycoprotein / Respiratory Pigment / Cooper Protein / Carbohydrates
Biological speciesPomacea canaliculata (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsBrola, T.R. / Chiumiento, I.R. / Gragera, M. / Carazo, J.M. / Heras, H. / Dreon, M.S. / Otero, L.H.
Funding support Argentina, 2items
OrganizationGrant numberCountry
Agencia Nacional de Promocion Cientifica y Tecnologica (FONCYT)PICT-2021-00158 Argentina
Agencia Nacional de Promocion Cientifica y Tecnologica (FONCYT)PICT-2020- SERIEA-03047 Argentina
CitationJournal: FEBS J / Year: 2026
Title: Cryo-EM structure of N-glycosylated Pomacea canaliculata hemocyanin provides insights into its role in the immune response of gastropods.
Authors: Tabata Romina Brola / Ignacio Rafael Chiumiento / Santiago Ituarte / Guillermo Ignacio Benítez / Marcos Gragera / Jose-Maria Carazo / Horacio Heras / Lisandro Horacio Otero / Marcos Sebastián Dreon /
Abstract: Hemocyanins are multifunctional soluble proteins found in most mollusks and some arthropods that may turn into a phenoloxidase-like enzyme (PO) associated with innate immune functions. To expand the ...Hemocyanins are multifunctional soluble proteins found in most mollusks and some arthropods that may turn into a phenoloxidase-like enzyme (PO) associated with innate immune functions. To expand the structural understanding of gastropod hemocyanins and their physiological implications, here we report the single-particle cryogenic electron microscopy (cryo-EM) structure of Pomacea canaliculata snail hemocyanin (PcH) at 4.4 Å resolution along with its intrinsic and proteolytically induced PO activity. PcH shows a Megathura crenulata (giant keyhole limpet) hemocyanin (KLH)-type structure with a cylindrical shape, comprising 20 protomers assembled as di-pentamers of antiparallel asymmetric dimers, organized in a D5 symmetry. Each protomer comprises eight paralogous functional units (FUs) sharing conserved structural features typical of hemocyanins. The achieved map resolution allowed delineation of interaction networks among adjacent subunits across quaternary structure tiers-dimer, pentamer of dimers (decamer), and di-pentamer of dimers (di-decamer). Additionally, we identified six N-glycosylation sites per protomer, totaling 120 glycan trees in the overall structure. Kinetic analysis of intrinsic PO activity using catechol as substrate revealed a Michaelis constant (K) of 45.3 mm and a catalytic rate constant (k) of 2.87 min. This specific activity was enhanced by limited proteolysis using digestive and bacterial proteases. Potential protease cleavage sites were identified in silico, mapped onto the PcH model, and their accessibility assessed. Combined with molecular dynamics simulations, these findings suggest a structural basis for the PO induction mechanism. This study expands our knowledge of KLH-type hemocyanins and provides clues into their PO activation, which is triggered by endogenous and/or pathogen-associated proteases. This further underscores the role of molluscan hemocyanins in the innate immune system.
History
DepositionAug 10, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hemocyanin
B: Hemocyanin
C: Hemocyanin
D: Hemocyanin
E: Hemocyanin
F: Hemocyanin
G: Hemocyanin
H: Hemocyanin
I: Hemocyanin
J: Hemocyanin
K: Hemocyanin
L: Hemocyanin
M: Hemocyanin
N: Hemocyanin
O: Hemocyanin
P: Hemocyanin
Q: Hemocyanin
R: Hemocyanin
S: Hemocyanin
T: Hemocyanin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)7,831,135140
Polymers7,774,54320
Non-polymers56,593120
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Hemocyanin


Mass: 388727.125 Da / Num. of mol.: 20 / Source method: isolated from a natural source / Source: (natural) Pomacea canaliculata (invertebrata)
#2: Polysaccharide...
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 40
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Polysaccharide...
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-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.: 20 / 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
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: Didecameric complex of Pomacea canaliculata hemocyanin
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Pomacea canaliculata (invertebrata)
Buffer solutionpH: 7.4
Buffer component
IDConc.FormulaBuffer-ID
120 mMTris-HCl1
220 mMCaCl21
320 mMMgCl21
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal magnification: 59000 X / Nominal defocus max: 1900 nm / Nominal defocus min: 1100 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 36.75 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 4584

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Processing

EM software
IDNameCategory
1crYOLOparticle selection
4GctfCTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
13cryoSPARC3D reconstruction
14Scipion3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 64970
SymmetryPoint symmetry: D5 (2x5 fold dihedral)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29935 / Symmetry type: POINT
Atomic model buildingB value: 110 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 103.84 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034565232
ELECTRON MICROSCOPYf_angle_d0.6307768674
ELECTRON MICROSCOPYf_chiral_restr0.044682498
ELECTRON MICROSCOPYf_plane_restr0.0046100688
ELECTRON MICROSCOPYf_dihedral_angle_d6.032776126

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