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- EMDB-2180: 3D reconstruction of LDL at 37C (human body temperature) using cr... -

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Basic information

Entry
Database: EMDB / ID: EMD-2180
Title3D reconstruction of LDL at 37C (human body temperature) using cryo-EM techniques
Map dataReconstruction of native LDL at 37C using cryo-EM based single particle reconstruction
Sample
  • Sample: LDL at 37C
  • Protein or peptide: apoB-100
KeywordsLDL / native LDL / human body temperature
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsKumar V / Butcher SJ / Oorni K / Engelhardt P / Heikkonen J / Kaski K / Ala-Korpela M / Kovanen PT
CitationJournal: PLoS One / Year: 2011
Title: Three-dimensional cryoEM reconstruction of native LDL particles to 16Å resolution at physiological body temperature.
Authors: Vibhor Kumar / Sarah J Butcher / Katariina Öörni / Peter Engelhardt / Jukka Heikkonen / Kimmo Kaski / Mika Ala-Korpela / Petri T Kovanen /
Abstract: BACKGROUND: Low-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. ...BACKGROUND: Low-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. The most prominent structural components in LDL are the core-forming cholesteryl esters (CE) and the particle-encircling single copy of a huge, non-exchangeable protein, the apolipoprotein B-100 (apoB-100). The shape of native LDL particles and the conformation of native apoB-100 on the particles remain incompletely characterized at the physiological human body temperature (37 °C).
METHODOLOGY/PRINCIPAL FINDINGS: To study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles ...METHODOLOGY/PRINCIPAL FINDINGS: To study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles vitrified at 6 °C and 37 °C resulted in reconstructions at ~16 Å resolution at both temperatures. 3D variance map analysis revealed rigid and flexible domains of lipids and apoB-100 at both temperatures. The reconstructions showed less variability at 6 °C than at 37 °C, which reflected increased order of the core CE molecules, rather than decreased mobility of the apoB-100. Compact molecular packing of the core and order in a lipid-binding domain of apoB-100 were observed at 6 °C, but not at 37 °C. At 37 °C we were able to highlight features in the LDL particles that are not clearly separable in 3D maps at 6 °C. Segmentation of apoB-100 density, fitting of lipovitellin X-ray structure, and antibody mapping, jointly revealed the approximate locations of the individual domains of apoB-100 on the surface of native LDL particles.
CONCLUSIONS/SIGNIFICANCE: Our study provides molecular background for further understanding of the link between structure and function of native LDL particles at physiological body temperature.
History
DepositionAug 23, 2012-
Header (metadata) releaseAug 29, 2012-
Map releaseAug 29, 2012-
UpdateAug 29, 2012-
Current statusAug 29, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with section colored by density value
  • Surface level: 1.24
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2180.tif

Downloads & links

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Map

FileDownload / File: emd_2180.map.gz / Format: CCP4 / Size: 33.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of native LDL at 37C using cryo-EM based single particle reconstruction
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy EMDB: 0.3 / Movie #1: 0.3
Minimum - Maximum-1.93679738 - 2.4810586
Average (Standard dev.)0.0 (±0.42754436)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-104-104-104
Dimensions208208208
Spacing208208208
CellA=B=C: 291.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z208208208
origin x/y/z0.0000.0000.000
length x/y/z291.200291.200291.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-104-104-104
NC/NR/NS208208208
D min/max/mean-1.9372.4810.000

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Supplemental data

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Sample components

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Entire : LDL at 37C

EntireName: LDL at 37C
Components
  • Sample: LDL at 37C
  • Protein or peptide: apoB-100

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Supramolecule #1000: LDL at 37C

SupramoleculeName: LDL at 37C / type: sample / ID: 1000
Details: Samples were vitrified on Quantifoil holey carbon grids (Quantifoil Micro Tools, GmbH) either at 6C or 37C, allowing pre-equilibration of the sample at the desired temperature for at least ...Details: Samples were vitrified on Quantifoil holey carbon grids (Quantifoil Micro Tools, GmbH) either at 6C or 37C, allowing pre-equilibration of the sample at the desired temperature for at least 45 minutes prior to plunging.
Number unique components: 1

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Macromolecule #1: apoB-100

MacromoleculeName: apoB-100 / type: protein_or_peptide / ID: 1 / Name.synonym: apoB / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: Blood

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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

Concentration0.3 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: OTHER
DateDec 30, 2005
Image recordingDigitization - Scanner: ZEISS SCAI / Number real images: 26083
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 29844
DetailsInitially only phase-flipping was carried out to correct for the contrast transfer function. To reduce the effect of image noise and artefacts, an initial single particle reconstruction was made on images de-noised with an information theory [Minimum Description Length (MDL)] based method which we have introduced earlier. Fifty initial class averages were obtained using a reference-free classification of 3000 images by multivariate statistical analysis using parameters, as suggested in EMAN . Angles were assigned using the cross common lines method prior to reconstruction using weighted-back projection. The initial 3D models were low-pass filtered. The 2D image dataset was increased to include all of the particles and iterative refinement continued. After each iteration of the single-particle-reconstruction process only 75% of the images with the highest correlation values were chosen. In order to avoid any bias in 3D reconstruction due to the de-noising, the final reconstructions were made using raw images after being assigned to classes using their de-noised equivalents

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