ジャーナル: Nat Struct Mol Biol / 年: 2015 タイトル: Structure of a yeast 40S-eIF1-eIF1A-eIF3-eIF3j initiation complex. 著者: Christopher H S Aylett / Daniel Boehringer / Jan P Erzberger / Tanja Schaefer / Nenad Ban / 要旨: Eukaryotic translation initiation requires cooperative assembly of a large protein complex at the 40S ribosomal subunit. We have resolved a budding yeast initiation complex by cryo-EM, allowing ...Eukaryotic translation initiation requires cooperative assembly of a large protein complex at the 40S ribosomal subunit. We have resolved a budding yeast initiation complex by cryo-EM, allowing placement of prior structures of eIF1, eIF1A, eIF3a, eIF3b and eIF3c. Our structure highlights differences in initiation-complex binding to the ribosome compared to that of mammalian eIF3, demonstrates a direct contact between eIF3j and eIF1A and reveals the network of interactions between eIF3 subunits.
pH: 7.6 / 詳細: 25 mM K-HEPES, 10 mM MgCl2, 75 mM KCl, 0.5 mM TCEP
グリッド
詳細: 200 mesh Quantifoil R 2/2 holey carbon grids with a thin continuous carbon support film
凍結
凍結剤: ETHANE / チャンバー内湿度: 75 % / チャンバー内温度: 120 K / 装置: HOMEMADE PLUNGER 詳細: carbon floated on the sample prior to recovery onto the grid and flash-freezing. 手法: 5 seconds blotting before plunging.
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電子顕微鏡法 #1
Microscopy ID
1
顕微鏡
FEI TITAN KRIOS
温度
平均: 100 K
アライメント法
Legacy - 非点収差: Objective lens astigmatism was corrected at 150,000 times magnification
日付
2014年4月6日
撮影
カテゴリ: CCD フィルム・検出器のモデル: FEI FALCON II (4k x 4k) 実像数: 7200 / 平均電子線量: 25 e/Å2 詳細: Single movie frame readout. 7 frames per exposure. 4 images per hole. ビット/ピクセル: 16
Legacy - 非点収差: Objective lens astigmatism was corrected at 150,000 times magnification
日付
2014年7月16日
撮影
カテゴリ: CCD フィルム・検出器のモデル: FEI FALCON II (4k x 4k) 実像数: 8828 / 平均電子線量: 25 e/Å2 詳細: Single movie frame readout. 7 frames per exposure. Drift corrected in post-processing. 4 images per hole. ビット/ピクセル: 16
For full details see the supplemental materials and methods in the accompanying publication which provides processing details and the classification schema.
CTF補正
詳細: By image
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: 6.47 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: CTFFIND3, RELION, 1.2 / 使用した粒子像数: 27354