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- EMDB-4400: CryoEM reconstruction of full-length, fully-glycosylated human bu... -

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Basic information

Entry
Database: EMDB / ID: EMD-4400
TitleCryoEM reconstruction of full-length, fully-glycosylated human butyrylcholinesterase tetramer
Map datacryo-EM map of tetrameric full-length, glycosylated human butyrylcholinesterase (HuBChE) purified from human plasma
Sample
  • Complex: heteropentameric complex consisting of four copies of butyrylcholinesterase and one copy of a lamellipodin-derived polyproline peptide
    • Protein or peptide: Cholinesterase
    • Protein or peptide: lamellipodin-derived polyproline peptide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordscholinesterase / tetramer / HYDROLASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.7 Å
AuthorsLeung MR / van Bezouwen LS
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, 2018-
Header (metadata) releaseNov 14, 2018-
Map releaseDec 19, 2018-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6i2t
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4400.png

Downloads & links

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Map

FileDownload / File: emd_4400.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM map of tetrameric full-length, glycosylated human butyrylcholinesterase (HuBChE) purified from human plasma
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 300 pix.
= 308.55 Å		1.03 Å/pix.
x 300 pix.
= 308.55 Å		1.03 Å/pix.
x 300 pix.
= 308.55 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0285 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.03921342 - 0.0840524
Average (Standard dev.)0.0002668748 (±0.0023981098)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 308.55 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.02851.02851.0285
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z308.550308.550308.550
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0390.0840.000

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Supplemental data

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Sample components

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Entire : heteropentameric complex consisting of four copies of butyrylchol...

EntireName: heteropentameric complex consisting of four copies of butyrylcholinesterase and one copy of a lamellipodin-derived polyproline peptide
Components
  • Complex: heteropentameric complex consisting of four copies of butyrylcholinesterase and one copy of a lamellipodin-derived polyproline peptide
    • Protein or peptide: Cholinesterase
    • Protein or peptide: lamellipodin-derived polyproline peptide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: heteropentameric complex consisting of four copies of butyrylchol...

SupramoleculeName: heteropentameric complex consisting of four copies of butyrylcholinesterase and one copy of a lamellipodin-derived polyproline peptide
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human) / Tissue: plasma
Molecular weightTheoretical: 340 KDa

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Macromolecule #1: Cholinesterase

MacromoleculeName: Cholinesterase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: cholinesterase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.1495 KDa
SequenceString: EDDIIIATKN GKVRGMNLTV FGGTVTAFLG IPYAQPPLGR LRFKKPQSLT KWSDIWNATK YANSCCQNID QSFPGFHGSE MWNPNTDLS EDCLYLNVWI PAPKPKNATV LIWIYGGGFQ TGTSSLHVYD GKFLARVERV IVVSMNYRVG ALGFLALPGN P EAPGNMGL ...String:
EDDIIIATKN GKVRGMNLTV FGGTVTAFLG IPYAQPPLGR LRFKKPQSLT KWSDIWNATK YANSCCQNID QSFPGFHGSE MWNPNTDLS EDCLYLNVWI PAPKPKNATV LIWIYGGGFQ TGTSSLHVYD GKFLARVERV IVVSMNYRVG ALGFLALPGN P EAPGNMGL FDQQLALQWV QKNIAAFGGN PKSVTLFGES AGAASVSLHL LSPGSHSLFT RAILQSGSFN APWAVTSLYE AR NRTLNLA KLTGCSRENE TEIIKCLRNK DPQEILLNEA FVVPYGTPLS VNFGPTVDGD FLTDMPDILL ELGQFKKTQI LVG VNKDEG TAFLVYGAPG FSKDNNSIIT RKEFQEGLKI FFPGVSEFGK ESILFHYTDW VDDQRPENYR EALGDVVGDY NFIC PALEF TKKFSEWGNN AFFYYFEHRS SKLPWPEWMG VMHGYEIEFV FGLPLERRDN YTKAEEILSR SIVKRWANFA KYGNP NETQ NNSTSWPVFK STEQKYLTLN TESTRIMTKL RAQQCRFWTS FFPKVLEMTG NIDEAEWEWK AGFHRWNNYM MDWKNQ FND YTSKKESCVG L

UniProtKB: Cholinesterase

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Macromolecule #2: lamellipodin-derived polyproline peptide

MacromoleculeName: lamellipodin-derived polyproline peptide / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.183393 KDa
SequenceString:
PPPPPPPPPP PP

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.3 mg/mL
BufferpH: 8 / Component - Concentration: 10.0 mM / Component - Formula: C4H11NO3 / Component - Name: Tris
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details: blot for either 2s or 3s at blot force -2 or 0, respectively.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 4518 / Average exposure time: 6.3 sec. / Average electron dose: 49.5 e/Å2
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: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 414189
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 5.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 111986
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

4aqd

source_name: PDB, initial_model_type: experimental model

1vzj

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6i2t:
CryoEM reconstruction of full-length, fully-glycosylated human butyrylcholinesterase tetramer

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