ジャーナル: J Mol Biol / 年: 2008 タイトル: Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy. 著者: Jayati Sengupta / Jakob Nilsson / Richard Gursky / Morten Kjeldgaard / Poul Nissen / Joachim Frank / 要旨: In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-A cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum ...In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-A cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the gamma-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.
ダウンロード / ファイル: emd_5015.map.gz / 形式: CCP4 / 大きさ: 8.2 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Cryo-EM reconstruction of 80S.eEF2 complex captured in transition state
ボクセルのサイズ
X=Y=Z: 2.82 Å
密度
表面レベル
登録者による: 65.200000000000003 / ムービー #1: 65.2
最小 - 最大
-115.040878300000003 - 227.622665409999996
平均 (標準偏差)
4.20576191 (±25.471893309999999)
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
130
130
130
Spacing
130
130
130
セル
A=B=C: 366.6 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
2.82
2.82
2.82
M x/y/z
130
130
130
origin x/y/z
0.000
0.000
0.000
length x/y/z
366.600
366.600
366.600
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-127
-127
-127
NX/NY/NZ
255
255
255
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
130
130
130
D min/max/mean
-115.041
227.623
4.206
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添付データ
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試料の構成要素
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全体 : The eEF2-bound 80S ribosome in the presence of aluminum fluoride ...
全体
名称: The eEF2-bound 80S ribosome in the presence of aluminum fluoride (AlF4-) and GDP
要素
試料: The eEF2-bound 80S ribosome in the presence of aluminum fluoride (AlF4-) and GDP
複合体: 80S ribosome
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超分子 #1000: The eEF2-bound 80S ribosome in the presence of aluminum fluoride ...
超分子
名称: The eEF2-bound 80S ribosome in the presence of aluminum fluoride (AlF4-) and GDP タイプ: sample / ID: 1000 / 詳細: AlF4- mimicking the g-phosphate during hydrolysis / Number unique components: 4
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超分子 #1: 80S ribosome
超分子
名称: 80S ribosome / タイプ: complex / ID: 1 / 組換発現: No / データベース: NCBI / Ribosome-details: ribosome-eukaryote: ALL
由来(天然)
生物種: Thermomyces lanuginosus (菌類)
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実験情報
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構造解析
手法
クライオ電子顕微鏡法
解析
単粒子再構成法
試料の集合状態
particle
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試料調製
緩衝液
pH: 7.2 / 詳細: 20 mM Hepes-NH3, 100 mM KCl, 20 mM MgCl2
グリッド
詳細: Quantifoil grid
凍結
凍結剤: NITROGEN / チャンバー内湿度: 90 % / チャンバー内温度: 93 K / 装置: OTHER / 詳細: Vitrification instrument: Vitrobot
試料ホルダー: Single tilt cyro-holder / 試料ホルダーモデル: GATAN LIQUID NITROGEN
実験機器
モデル: Tecnai F20 / 画像提供: FEI Company
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画像解析
詳細
The image processing using SPIDER included a 3D projection alignment procedure with correction of the contrast transfer function and enhancement of the high-resolution Fourier amplitudes based on X-ray solution scattering data.
CTF補正
詳細: Segregation in defocus groups and correction in volumes
最終 再構成
アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 12.6 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: Spider / 詳細: Supervised Classification was used / 使用した粒子像数: 28242