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データを開く
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基本情報
登録情報 | データベース: EMDB / ID: EMD-3542 | |||||||||
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タイトル | Structure of a spliceosome remodeled for exon ligation | |||||||||
![]() | Map of Saccharomyces cerevisiae spliceosome C-star complex generated by focussed refinement on U2 snRNP peripheral regions. | |||||||||
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生物種 | ![]() ![]() ![]() ![]() | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 4.6 Å | |||||||||
![]() | Fica SM / Oubridge C / Galej WP / Wilkinson ME / Newman AJ / Bai X-C / Nagai K | |||||||||
![]() | ![]() タイトル: Cryo-EM structure of the spliceosome immediately after branching. 著者: Wojciech P Galej / Max E Wilkinson / Sebastian M Fica / Chris Oubridge / Andrew J Newman / Kiyoshi Nagai / ![]() 要旨: Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the ...Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. | |||||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | EMマップ: ![]() ![]() ![]() |
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ダウンロードとリンク
-EMDBアーカイブ
マップデータ | ![]() | 243.9 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 59.3 KB 59.3 KB | 表示 表示 | ![]() |
FSC (解像度算出) | ![]() | 14.5 KB | 表示 | ![]() |
画像 | ![]() | 62.6 KB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
関連構造データ | ![]() 3539C ![]() 3541C ![]() 5mpsC ![]() 5mq0C C: 同じ文献を引用 ( |
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類似構造データ | |
電子顕微鏡画像生データ | ![]() Data #1: Unaligned movies of C-complex spliceosome with 3' splice site AG to AC mutation (Dataset 1) [micrographs - multiframe] Data #2: Unaligned movies of C and C*-complex spliceosomes with 3' splice site AG to AdG mutation (Dataset 2) [micrographs - multiframe] Data #3: Unaligned movies of C and C*-complex spliceosomes with 3' splice site AG to AdG mutation (Dataset 3) [micrographs - multiframe] Data #4: Aligned movies of C-complex spliceosomes with cold-sensitive prp16-302 mutation, purified with Cwc25 (Dataset 4) [micrographs - multiframe] Data #5: Unaligned movies of C-complex spliceosomes with cold-sensitive prp16-302 mutation, purified with Cwc25 and incubated with ATP and Mg (Dataset 5) [micrographs - multiframe] Data #6: Unaligned movies of C, C*, and P-complex spliceosomes with dominant-negative Prp22 mutation K512A, purified with Slu7 (Dataset 6) [micrographs - multiframe] Data #7: Unaligned movies of P-complex spliceosomes with dominant-negative Prp22 mutation K512A, treated with anti-3'exon RNaseH oligo, purified in presence of Mg (Dataset 9) [micrographs - single frame] Data #8: Selected C-complex particles after polishing [picked particles - single frame - processed] Data #9: Selected P-complex particles after polishing [picked particles - single frame - processed] Data #10: Various signal subtractions for C- and P-complex spliceosomes [picked particles - single frame - processed]) |
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リンク
EMDBのページ | ![]() ![]() |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Map of Saccharomyces cerevisiae spliceosome C-star complex generated by focussed refinement on U2 snRNP peripheral regions. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.43 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
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試料の構成要素
+全体 : Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-...
+超分子 #1: Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-...
+分子 #1: Intron of UBC4 pre-mRNA
+分子 #2: 5'exon of UBC4 pre-mRNA
+分子 #3: U2 snRNA
+分子 #4: U6 snRNA
+分子 #5: U5 snRNA
+分子 #34: 3'exon of UBC4 pre-mRNA bound to Prp22 helicase
+分子 #6: Pre-mRNA-splicing factor 8
+分子 #7: Pre-mRNA-splicing factor SNU114
+分子 #8: Pre-mRNA-splicing factor CWC22
+分子 #9: Pre-mRNA-splicing factor PRP46
+分子 #10: Pre-mRNA-processing protein 45
+分子 #11: Pre-mRNA-splicing factor BUD31
+分子 #12: Pre-mRNA-splicing factor CEF1
+分子 #13: Pre-mRNA-splicing factor CWC15
+分子 #14: Pre-mRNA-splicing factor CWC21
+分子 #15: Pre-mRNA-splicing factor CLF1
+分子 #16: Pre-mRNA-splicing factor SYF1
+分子 #17: Pre-mRNA-splicing factor 18
+分子 #18: Pre-mRNA-splicing factor SLU7
+分子 #19: Pre-mRNA-splicing factor SYF2
+分子 #20: Small nuclear ribonucleoprotein-associated protein B
+分子 #21: Small nuclear ribonucleoprotein Sm D3
+分子 #22: Small nuclear ribonucleoprotein E
+分子 #23: Small nuclear ribonucleoprotein F
+分子 #24: Small nuclear ribonucleoprotein G
+分子 #25: Small nuclear ribonucleoprotein Sm D1
+分子 #26: Small nuclear ribonucleoprotein Sm D2
+分子 #27: Pre-mRNA-splicing factor CWC2
+分子 #28: Pre-mRNA-splicing factor SLT11
+分子 #29: Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22
+分子 #30: Pre-mRNA-processing factor 17
+分子 #31: U2 small nuclear ribonucleoprotein A'
+分子 #32: U2 small nuclear ribonucleoprotein B''
+分子 #33: Unknown protein
+分子 #35: MAGNESIUM ION
+分子 #36: POTASSIUM ION
+分子 #37: INOSITOL HEXAKISPHOSPHATE
+分子 #38: GUANOSINE-5'-TRIPHOSPHATE
+分子 #39: ZINC ION
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | 単粒子再構成法 |
試料の集合状態 | particle |
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試料調製
濃度 | 0.3 mg/mL | ||||||||||||||||||
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緩衝液 | pH: 7.9 構成要素:
詳細: NP-40 is also called IGEPAL CA-630 | ||||||||||||||||||
グリッド | モデル: Quantifoil R1.2/1.3 / 材質: COPPER / メッシュ: 400 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY ARRAY / 支持フィルム - Film thickness: 6.0 nm / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 雰囲気: AIR / 前処理 - 気圧: 20.0 kPa | ||||||||||||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK III 詳細: 3.5 microlitres sample were applied to the grid, left for 25 seconds and then blotted for 3.0-3.5 seconds before plunging.. |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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特殊光学系 | エネルギーフィルター - 名称: GIF Quantum energy filter, 20 eV slit width |
撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: SUPER-RESOLUTION / 実像数: 3596 / 平均露光時間: 0.8 sec. / 平均電子線量: 2.0 e/Å2 詳細: Total dose: 40 electrons/Angstrom^2 over 16 seconds. 20 movie frames collected at 1.25 frames per second. |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 4.5 µm / 最小 デフォーカス(公称値): 0.5 µm / 倍率(公称値): 81000 |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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画像解析
-原子モデル構築 1
詳細 | Used secondary structure restraints generated in ProSMART and LibG for Refmac refinement. |
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精密化 | 空間: RECIPROCAL / プロトコル: FLEXIBLE FIT / 温度因子: 330 / 当てはまり具合の基準: Fourier Shell Correlation |