ジャーナル: J Struct Biol / 年: 2014 タイトル: Chemically functionalized carbon films for single molecule imaging. 著者: Marc C Llaguno / Hui Xu / Liang Shi / Nian Huang / Hong Zhang / Qinghua Liu / Qiu-Xing Jiang / 要旨: Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and ...Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and chemical linkage to introduce a high density of bioactive ligands onto nanometer-thick carbon films and enable selective enrichment of individual macromolecular complexes at subnanogram levels. The introduced ligands are physically separated. Ni-NTA, Protein G and DNA/RNA oligonucleotides were covalently linked to the carbon surface. They embody negligible mass and their stability makes the functionalized films able to survive long-term storage and tolerate variations in pH, temperature, salts, detergents, and solvents. We demonstrated the application of the new method to the electron microscopic imaging of the substrate-bound C3PO, an RNA-processing enzyme important for the RNA interference pathway. On the ssRNA-linked carbon surface, the formation of C3PO oligomers at subnanomolar concentrations likely mimics their assembly onto ssRNA substrates presented by their native partners. Interestingly, the 3D reconstructions by negative stain EM reveal a side port in the C3PO/ssRNA complex, and the 15Å cryoEM map showed extra density right above the side port, which probably represents the ssRNA. These results suggest a new way for ssRNAs to interact with the active sites of the complex. Together our data demonstrate that the surface-engineered carbon films are suitable for selectively enriching low-abundance biological complexes at nanomolar level and for developing novel applications on a large number of surface-presented molecules.
ダウンロード / ファイル: emd_5892.map.gz / 形式: CCP4 / 大きさ: 3.3 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
C3PO on let7 ssRNA ChemiC grids
ボクセルのサイズ
X=Y=Z: 2.33 Å
密度
表面レベル
登録者による: 0.068 / ムービー #1: 0.068
最小 - 最大
-0.41981968 - 1.28919888
平均 (標準偏差)
0.00334152 (±0.06032538)
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
96
96
96
Spacing
96
96
96
セル
A=B=C: 223.68 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
2.33
2.33
2.33
M x/y/z
96
96
96
origin x/y/z
0.000
0.000
0.000
length x/y/z
223.680
223.680
223.680
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-95
-75
153
NX/NY/NZ
200
200
200
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
96
96
96
D min/max/mean
-0.420
1.289
0.003
-
添付データ
-
試料の構成要素
-
全体 : Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA pre...
全体
名称: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA presented by ChemiC grids
要素
試料: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA presented by ChemiC grids
タンパク質・ペプチド: Component 3 Promoter of RISC
RNA: let7 ssRNA
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超分子 #1000: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA pre...
超分子
名称: Complex 3 Promoter of RISC (C3PO) assembled on the let7 ssRNA presented by ChemiC grids タイプ: sample / ID: 1000 / 詳細: The sample was monodisperse. / 集合状態: One C3PO octamer binds to one let7 ssRNA / Number unique components: 2
分子量
実験値: 240 KDa / 理論値: 240 KDa / 手法: Gel filtration and ultra-centrifugation
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分子 #1: Component 3 Promoter of RISC
分子
名称: Component 3 Promoter of RISC / タイプ: protein_or_peptide / ID: 1 / Name.synonym: C3PO 詳細: C3PO was incubated on let7 ssRNA presented on ChemiC films. コピー数: 1 / 集合状態: Octamer / 組換発現: Yes
由来(天然)
生物種: Homo sapiens (ヒト) / 別称: Human / 細胞中の位置: cytoplasm