ジャーナル: PLoS One / 年: 2011 タイトル: Three-dimensional cryoEM reconstruction of native LDL particles to 16Å resolution at physiological body temperature. 著者: Vibhor Kumar / Sarah J Butcher / Katariina Öörni / Peter Engelhardt / Jukka Heikkonen / Kimmo Kaski / Mika Ala-Korpela / Petri T Kovanen / 要旨: 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.
ダウンロード / ファイル: emd_2180.map.gz / 形式: CCP4 / 大きさ: 33.5 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Reconstruction of native LDL at 37C using cryo-EM based single particle reconstruction
ボクセルのサイズ
X=Y=Z: 1.4 Å
密度
表面レベル
By EMDB: 0.3 / ムービー #1: 0.3
最小 - 最大
-1.93679738 - 2.4810586
平均 (標準偏差)
0.0 (±0.42754436)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
-104
-104
-104
サイズ
208
208
208
Spacing
208
208
208
セル
A=B=C: 291.19998 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.4
1.4
1.4
M x/y/z
208
208
208
origin x/y/z
0.000
0.000
0.000
length x/y/z
291.200
291.200
291.200
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-184
-184
-183
NX/NY/NZ
368
368
368
MAP C/R/S
1
2
3
start NC/NR/NS
-104
-104
-104
NC/NR/NS
208
208
208
D min/max/mean
-1.937
2.481
0.000
-
添付データ
-
試料の構成要素
-
全体 : LDL at 37C
全体
名称: LDL at 37C
要素
試料: LDL at 37C
タンパク質・ペプチド: apoB-100
-
超分子 #1000: LDL at 37C
超分子
名称: LDL at 37C / タイプ: sample / ID: 1000 詳細: 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 ...詳細: 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
Initially 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
CTF補正
詳細: Each particle
最終 再構成
想定した対称性 - 点群: C1 (非対称) / アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 16.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: EMAN / 使用した粒子像数: 29844