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- EMDB-5347: 3D map of CETP-HDL complex at 14 Angstrom by optimized negative-s... -

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Basic information

Entry
Database: EMDB / ID: EMD-5347
Title3D map of CETP-HDL complex at 14 Angstrom by optimized negative-staining EM and single-particle reconstruction
Map dataThis is 3D reconstruction of HDL-CETP by negative-staining EM
Sample
  • Sample: Recombinant human CETP (about 53 kDa before post-translational modifications) was expressed in the dihydrofolate reductase-deficient Chinese hamster ovary cell line DG44. The spherical HDL were converted from discoidal reconstituted HDL (rHDL)by incubation with fatty acid-free bovine serum albumin, beta-mercaptoethanol, ultracentrifugally isolated LDL and purified lecithin-cholesterol acyltransferase (LCAT), and then isolated by sequential ultracentrifugation in the density range of 1.07-1.21 g/ml.
  • Protein or peptide: Cholesteryl ester transfer protein
KeywordsCholesteryl ester transfer protein / lipoprotein / CETP / LDL / VLDL / HDL / electron microscopy / CETP mechanism
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 14.0 Å
AuthorsZhang L / Yan F / Zhang S / Lei D / Charles MA / Cavigiolio G / Oda M / Krauss RM / Weisgraber KH / Rye KA ...Zhang L / Yan F / Zhang S / Lei D / Charles MA / Cavigiolio G / Oda M / Krauss RM / Weisgraber KH / Rye KA / Pownall HJ / Qiu X / Ren G
CitationJournal: Nat Chem Biol / Year: 2012
Title: Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein.
Authors: Lei Zhang / Feng Yan / Shengli Zhang / Dongsheng Lei / M Arthur Charles / Giorgio Cavigiolio / Michael Oda / Ronald M Krauss / Karl H Weisgraber / Kerry-Anne Rye / Henry J Pownall / Xiayang Qiu / Gang Ren /
Abstract: Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an ...Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.
History
DepositionOct 5, 2011-
Header (metadata) releaseNov 28, 2011-
Map releaseMay 21, 2012-
UpdateMay 30, 2012-
Current statusMay 30, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5347_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5347.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is 3D reconstruction of HDL-CETP by negative-staining EM
Voxel sizeX=Y=Z: 2.812 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 3.5
Minimum - Maximum-1.40113115 - 19.586109159999999
Average (Standard dev.)0.15034387 (±1.09604871)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 359.936 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.8122.8122.812
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z359.936359.936359.936
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-1.40119.5860.150

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Supplemental data

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Sample components

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Entire : Recombinant human CETP (about 53 kDa before post-translational mo...

EntireName: Recombinant human CETP (about 53 kDa before post-translational modifications) was expressed in the dihydrofolate reductase-deficient Chinese hamster ovary cell line DG44. The spherical HDL were ...Name: Recombinant human CETP (about 53 kDa before post-translational modifications) was expressed in the dihydrofolate reductase-deficient Chinese hamster ovary cell line DG44. The spherical HDL were converted from discoidal reconstituted HDL (rHDL)by incubation with fatty acid-free bovine serum albumin, beta-mercaptoethanol, ultracentrifugally isolated LDL and purified lecithin-cholesterol acyltransferase (LCAT), and then isolated by sequential ultracentrifugation in the density range of 1.07-1.21 g/ml.
Components
  • Sample: Recombinant human CETP (about 53 kDa before post-translational modifications) was expressed in the dihydrofolate reductase-deficient Chinese hamster ovary cell line DG44. The spherical HDL were converted from discoidal reconstituted HDL (rHDL)by incubation with fatty acid-free bovine serum albumin, beta-mercaptoethanol, ultracentrifugally isolated LDL and purified lecithin-cholesterol acyltransferase (LCAT), and then isolated by sequential ultracentrifugation in the density range of 1.07-1.21 g/ml.
  • Protein or peptide: Cholesteryl ester transfer protein

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Supramolecule #1000: Recombinant human CETP (about 53 kDa before post-translational mo...

SupramoleculeName: Recombinant human CETP (about 53 kDa before post-translational modifications) was expressed in the dihydrofolate reductase-deficient Chinese hamster ovary cell line DG44. The spherical HDL were ...Name: Recombinant human CETP (about 53 kDa before post-translational modifications) was expressed in the dihydrofolate reductase-deficient Chinese hamster ovary cell line DG44. The spherical HDL were converted from discoidal reconstituted HDL (rHDL)by incubation with fatty acid-free bovine serum albumin, beta-mercaptoethanol, ultracentrifugally isolated LDL and purified lecithin-cholesterol acyltransferase (LCAT), and then isolated by sequential ultracentrifugation in the density range of 1.07-1.21 g/ml.
type: sample / ID: 1000
Details: The CETP sample was thawed from storage at -70 degrees Celcius before being used, the HDL sample was freshly prepared.
Oligomeric state: one CETP bound to one HDL / Number unique components: 2
Molecular weightExperimental: 74 KDa / Theoretical: 53 KDa / Method: Sedimentation

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Macromolecule #1: Cholesteryl ester transfer protein

MacromoleculeName: Cholesteryl ester transfer protein / type: protein_or_peptide / ID: 1 / Name.synonym: CETP
Details: optimized negative stain method was used for the CETP.HDL complex reconstruction
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human / Tissue: plasma
Molecular weightExperimental: 74 KDa / Theoretical: 53 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.005 mg/mL
BufferpH: 7.4 / Details: DPBS buffer
StainingType: NEGATIVE
Details: CETP.HDL complex specimens were prepared for EM by the optimized NS protocol (reported in Zhang, L., J. Lipid Research, 2010, 51,5, 1228-36, and 2011, 52,1, 175-84.)
GridDetails: thin-carbon-coated 300 mesh copper grid (Cu-300CN, Pacific Grid-Tech, USA)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.7 µm / Nominal defocus min: 0.1 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 94 K / Max: 99 K / Average: 96 K
DetailsLose dose mode
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 50 e/Å2

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Image processing

CTF correctionDetails: ctffind3 in the FREALIGN software package, and corrected by EMAN
Final two d classificationNumber classes: 317
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 8879
DetailsThe particles were initially selected using an automatic selection program, boxer, then manually adjusted by deleted the obviously poor quality particles.

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Atomic model buiding 1

Initial modelPDB ID:

2obd

SoftwareName: Chimera
DetailsProtocol: Chimera Rigid Body Docking
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation

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