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- PDB-6i2t: CryoEM reconstruction of full-length, fully-glycosylated human bu... -

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Basic information

Entry
Database: PDB / ID: 6i2t
TitleCryoEM reconstruction of full-length, fully-glycosylated human butyrylcholinesterase tetramer
Components
  • Cholinesterase
  • lamellipodin-derived polyproline peptide
KeywordsHYDROLASE / cholinesterase / tetramer
Function / homology
Function and homology information


cholinesterase / cocaine metabolic process / neuroblast differentiation / Neurotransmitter clearance / cholinesterase activity / choline binding / response to folic acid / acetylcholine catabolic process / response to alkaloid / peptide hormone processing ...cholinesterase / cocaine metabolic process / neuroblast differentiation / Neurotransmitter clearance / cholinesterase activity / choline binding / response to folic acid / acetylcholine catabolic process / response to alkaloid / peptide hormone processing / negative regulation of synaptic transmission / choline metabolic process / hydrolase activity, acting on ester bonds / acetylcholinesterase activity / Aspirin ADME / nuclear envelope lumen / Synthesis of PC / catalytic activity / Synthesis, secretion, and deacylation of Ghrelin / response to glucocorticoid / xenobiotic metabolic process / learning / amyloid-beta binding / blood microparticle / negative regulation of cell population proliferation / endoplasmic reticulum lumen / enzyme binding / extracellular space / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
Acetylcholinesterase, tetramerisation domain / Acetylcholinesterase tetramerisation domain / : / Cholinesterase / Carboxylesterase type B, conserved site / Carboxylesterases type-B signature 2. / Carboxylesterase type B, active site / Carboxylesterases type-B serine active site. / Carboxylesterase, type B / Carboxylesterase family / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.7 Å
AuthorsLeung, M.R. / van Bezouwen, L.S. / Schopfer, L.M. / Sussman, J.L. / Silman, I. / Lockridge, O. / Zeev-Ben-Mordehai, T.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Cryo-EM structure of the native butyrylcholinesterase tetramer reveals a dimer of dimers stabilized by a superhelical assembly.
Authors: Miguel Ricardo Leung / Laura S van Bezouwen / Lawrence M Schopfer / Joel L Sussman / Israel Silman / Oksana Lockridge / Tzviya Zeev-Ben-Mordehai /
Abstract: The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localization and function. Of practical importance, BChE is a ...The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localization and function. Of practical importance, BChE is a promising therapeutic candidate for intoxication by organophosphate nerve agents and insecticides, and for detoxification of addictive substances. Efficacy of the recombinant enzyme hinges on its having a long circulatory half-life; this, in turn, depends strongly on its ability to tetramerize. Here, we used cryoelectron microscopy (cryo-EM) to determine the structure of the highly glycosylated native BChE tetramer purified from human plasma at 5.7 Å. Our structure reveals that the BChE tetramer is organized as a staggered dimer of dimers. Tetramerization is mediated by assembly of the C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit as a superhelical assembly around a central lamellipodin-derived oligopeptide with a proline-rich attachment domain (PRAD) sequence that adopts a polyproline II helical conformation and runs antiparallel. The catalytic domains within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely shielded by the catalytic domains, which may contribute to the stability of the human BChE (HuBChE) tetramer. Our cryo-EM structure reveals the basis for assembly of the native tetramers and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases but may provide a promising template for designing other proteins with improved circulatory residence times.
History
DepositionNov 1, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 19, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 26, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jan 9, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.5Nov 13, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4400
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
B: Cholinesterase
A: Cholinesterase
C: Cholinesterase
D: Cholinesterase
J: lamellipodin-derived polyproline peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)263,55113
Polymers261,7815
Non-polymers1,7708
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10710 Å2
ΔGint-51 kcal/mol
Surface area94730 Å2
MethodPISA

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Components

#1: Protein
Cholinesterase / Acylcholine acylhydrolase / Butyrylcholine esterase / Choline esterase II / Pseudocholinesterase


Mass: 65149.500 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P06276, cholinesterase
#2: Protein/peptide lamellipodin-derived polyproline peptide


Mass: 1183.393 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
#3: 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: isolated from a genetically manipulated source
Formula: C8H15NO6
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 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: heteropentameric complex consisting of four copies of butyrylcholinesterase and one copy of a lamellipodin-derived polyproline peptide
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightValue: 0.34 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human) / Tissue: plasma
Buffer solutionpH: 8
Buffer componentConc.: 10 mM / Name: Tris / Formula: C4H11NO3
SpecimenConc.: 3.3 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 R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K
Details: blot for either 2s or 3s at blot force -2 or 0, respectively

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 6.3 sec. / Electron dose: 49.5 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4518
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
19PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 414189
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111986 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
14AQD

4aqd

14AQD1PDBexperimental model
21VZJ

1vzj

11VZJ2PDBexperimental model

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