EMDB-2023: Three-dimensional reconstruction of a targeted individual 17nm na...

基本情報

• 登録情報: データベース: EMDB / ID: EMD-2023
• タイトル: Three-dimensional reconstruction of a targeted individual 17nm nascent high-density lipoprotein (HDL) particle by individual-particle electron tomography (IPET). The reconstruction displayed a ring-shaped structure of containing three protein - apoipoprotein A-Is (28 kDa each).
• マップデータ: 17nm Nascent HDL particle Number 1 by cryo-ET

試料

• 試料: a 17nm nascent HDL particle
• タンパク質・ペプチド: 17 nm nascent HDL

• キーワード: reconstituted 17nm nascent high-density lipoprotein (HDl)
• 生物種: Homo sapiens (ヒト)
• 手法: サブトモグラム平均法 / クライオ電子顕微鏡法 / ネガティブ染色法 / 解像度: 42.0 Å
• データ登録者: Zhang L / Ren G
• 引用: ジャーナル: PLoS One / 年: 2012; タイトル: IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure.; 著者: Lei Zhang / Gang Ren / ; 要旨: The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a "focused electron tomography reconstruction" (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single "snapshot" molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.

履歴

登録	2012年1月4日	-
ヘッダ(付随情報) 公開	2012年1月20日	-
マップ公開	2012年1月27日	-
更新	2016年3月9日	-
現状	2016年3月9日	処理サイト: PDBe / 状態: 公開

マップ

• ファイル: ダウンロード / ファイル: emd_2023.map.gz / 形式: CCP4 / 大きさ: 3.7 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• 注釈: 17nm Nascent HDL particle Number 1 by cryo-ET
• ボクセルのサイズ: X=Y=Z: 3.46 Å

密度

表面レベル	登録者による: 0.08 / ムービー #1: 0.08
最小 - 最大	-0.06166719 - 0.24149512
平均 (標準偏差)	0.000000000028398 (±0.02020695)

• 対称性: 空間群: 1

詳細

EMDB XML:

マップ形状	Axis order X Y Z Origin 0 0 0 サイズ 100 100 100 Spacing 100 100 100
セル	A=B=C: 346.0 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	3.46	3.46	3.46
M x/y/z	100	100	100
origin x/y/z	0.000	0.000	0.000
length x/y/z	346.000	346.000	346.000
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	100	100	100
D min/max/mean	-0.062	0.241	0.000

添付データ

試料の構成要素

全体 : a 17nm nascent HDL particle

• 全体: 名称: a 17nm nascent HDL particle

要素

• 試料: a 17nm nascent HDL particle
• タンパク質・ペプチド: 17 nm nascent HDL

超分子 #1000: a 17nm nascent HDL particle

• 超分子: 名称: a 17nm nascent HDL particle / タイプ: sample / ID: 1000 / 詳細: fresh sample was prepared with 2 days before using. / 集合状態: monomer / Number unique components: 1
• 分子量: 実験値: 200 KDa / 理論値: 200 KDa

分子 #1: 17 nm nascent HDL

• 分子: 名称: 17 nm nascent HDL / タイプ: protein_or_peptide / ID: 1 / Name.synonym: 17 nm nascent HDL; 詳細: HDL contains lipids and three apoA-I molecules. Each A-I molecular weight is about 28kDa.; コピー数: 1 / 集合状態: monomer / 組換発現: No / データベース: NCBI
• 由来(天然): 生物種: Homo sapiens (ヒト) / 株: nascent HDL / 別称: Human / 細胞中の位置: Plasma
• 分子量: 実験値: 200 KDa / 理論値: 200 KDa

実験情報

構造解析

• 手法: ネガティブ染色法, クライオ電子顕微鏡法
• 解析: サブトモグラム平均法

試料調製

• 濃度: 0.01 mg/mL
• 緩衝液: pH: 7.4 ; 詳細: 1X Dulbeccos phosphate-buffered saline (Invitrogen, La Jolla, CA), 2.7 mM KCl, 1.46 mM KH2PO4, 136.9 mM NaCl, and 8.1 mM Na2HPO4
• 染色: タイプ: NEGATIVE; 詳細: EM Specimens were prepared by standard cryo-EM protocol.
• グリッド: 詳細: 200 mesh glow-discharged holey thin carbon-coated EM grids (Cu-200HN, Pacific Grid-Tech, USA)
• 凍結: 凍結剤: NITROGEN / チャンバー内湿度: 90 % / チャンバー内温度: 103 K / 装置: HOMEMADE PLUNGER / 詳細: Vitrification instrument: manual / 手法: Blot for 2 seconds before plunging

電子顕微鏡法

• 顕微鏡: FEI TECNAI 12
• 電子線: 加速電圧: 120 kV / 電子線源: LAB6
• 電子光学系: 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / 最大 デフォーカス（公称値）: 2.0 µm / 最小 デフォーカス（公称値）: 1.0 µm / 倍率（公称値）: 67000
• 試料ステージ: 試料ホルダー: Gatan / 試料ホルダーモデル: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
• 温度: 平均: 100 K
• 詳細: Tilt step is 1.5 degree
• 撮影: カテゴリ: CCD; フィルム・検出器のモデル: GATAN ULTRASCAN 4000 (4k x 4k); デジタル化 - サンプリング間隔: 1.73 µm / 実像数: 81 / 平均電子線量: 140 e/Ų / ビット/ピクセル: 16

画像解析

• CTF補正: 詳細: TOMOCTF
• 最終 角度割当: 詳細: Tomography tilt angle from -60 to 60 in step of 1.5
• 最終 再構成: アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 42.0 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: IPET, and, FETR; 詳細: Map was reconstructed by individual-particle electron tomography (IPET)and Focus ET Reconstruction Algorithm.
• 詳細: Single targeted particle"s images were reconstructed by focus ET reconstruction (FETR) algorithm of individual particle electron tomography (IPET) method. Average number of tilts used in the 3D reconstructions: 81. Average tomographic tilt angle increment: 1.5.