+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-3541 | ||||||||||||
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タイトル | Structure of a spliceosome remodeled for exon ligation | ||||||||||||
マップデータ | Map of the spliceosomal C-star complex resulting from focussed classification on Prp22 | ||||||||||||
試料 |
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機能・相同性 | 機能・相同性情報 U2-type post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / mRNA branch site recognition / spliceosomal complex disassembly / cellular bud site selection / pre-mRNA 3'-splice site binding / post-mRNA release spliceosomal complex / nuclear mRNA surveillance / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome ...U2-type post-spliceosomal complex / U2-type post-mRNA release spliceosomal complex / mRNA branch site recognition / spliceosomal complex disassembly / cellular bud site selection / pre-mRNA 3'-splice site binding / post-mRNA release spliceosomal complex / nuclear mRNA surveillance / generation of catalytic spliceosome for first transesterification step / cis assembly of pre-catalytic spliceosome / splicing factor binding / U4/U6 snRNP / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / 7-methylguanosine cap hypermethylation / Prp19 complex / pICln-Sm protein complex / small nuclear ribonucleoprotein complex / pre-mRNA binding / U2-type catalytic step 1 spliceosome / SMN-Sm protein complex / spliceosomal tri-snRNP complex / U2-type spliceosomal complex / mRNA cis splicing, via spliceosome / poly(U) RNA binding / commitment complex / U2-type prespliceosome assembly / U2-type catalytic step 2 spliceosome / U4 snRNP / U2 snRNP / U1 snRNP / U2-type prespliceosome / precatalytic spliceosome / Formation of TC-NER Pre-Incision Complex / generation of catalytic spliceosome for second transesterification step / spliceosomal complex assembly / Gap-filling DNA repair synthesis and ligation in TC-NER / DNA replication origin binding / mRNA 5'-splice site recognition / protein K63-linked ubiquitination / mRNA 3'-splice site recognition / Dual incision in TC-NER / DNA replication initiation / spliceosomal tri-snRNP complex assembly / U5 snRNA binding / U5 snRNP / U2 snRNA binding / U6 snRNA binding / spliceosomal snRNP assembly / positive regulation of cell cycle / pre-mRNA intronic binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / nuclear periphery / positive regulation of RNA splicing / spliceosomal complex / RING-type E3 ubiquitin transferase / mRNA splicing, via spliceosome / ubiquitin-protein transferase activity / metallopeptidase activity / ubiquitin protein ligase activity / RNA helicase activity / RNA helicase / DNA repair / GTPase activity / mRNA binding / chromatin binding / chromatin / GTP binding / ATP hydrolysis activity / mitochondrion / DNA binding / RNA binding / zinc ion binding / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm 類似検索 - 分子機能 | ||||||||||||
生物種 | Saccharomyces cerevisiae (パン酵母) / Baker's yeast (パン酵母) | ||||||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 4.17 Å | ||||||||||||
データ登録者 | Fica SM / Oubridge C / Galej WP / Wilkinson ME / Newman AJ / Bai X-C / Nagai K | ||||||||||||
資金援助 | 英国, 3件
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引用 | ジャーナル: Nature / 年: 2016 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 |
ムービービューア |
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構造ビューア | EMマップ: SurfViewMolmilJmol/JSmol |
添付画像 |
-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_3541.map.gz | 248.2 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-3541-v30.xml emd-3541.xml | 64.9 KB 64.9 KB | 表示 表示 | EMDBヘッダ |
FSC (解像度算出) | emd_3541_fsc_1.xml emd_3541_fsc_2.xml | 14.5 KB 14.5 KB | 表示 表示 | FSCデータファイル |
画像 | emd_3541_1.png emd_3541_2.png | 63.7 KB 62.6 KB | ||
その他 | emd_3541_additional.map.gz | 243.9 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-3541 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3541 | HTTPS FTP |
-関連構造データ
関連構造データ | 5mq0MC 3539C 3542C 5mpsC M: このマップから作成された原子モデル C: 同じ文献を引用 (文献) |
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類似構造データ | |
電子顕微鏡画像生データ | EMPIAR-10687 (タイトル: Yeast C, Ci, C*, and P complex spliceosomes / Data size: 8.9 TB 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]) |
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_3541.map.gz / 形式: CCP4 / 大きさ: 266.8 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Map of the spliceosomal C-star complex resulting from focussed classification on Prp22 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
-追加マップ: Map of the spliceosomal C-star complex resulting from...
ファイル | emd_3541_additional.map | ||||||||||||
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注釈 | Map of the spliceosomal C-star complex resulting from focussed classification on peripheral regions of U2 snRNP | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-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: Yeast UBC4 gene for ubiquitin-conjugating enzyme
+分子 #2: 5'-EXON OF UBC4 PRE-MRNA
+分子 #3: S.cerevisiae chromosome II reading frame ORF YBR230c
+分子 #4: Saccharomyces cerevisiae strain T.52_2H chromosome XII sequence
+分子 #5: Saccharomyces cerevisiae strain WI_C_MBSP_4 chromosome VII sequence
+分子 #34: 3'-EXON OF UBC4 PRE-MRNA, BOUND BY 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 CWC2
+分子 #13: Pre-mRNA-splicing factor SLT11
+分子 #14: Pre-mRNA-splicing factor CEF1
+分子 #15: Pre-mRNA-splicing factor CWC15
+分子 #16: Pre-mRNA-splicing factor CWC21
+分子 #17: Pre-mRNA-splicing factor CLF1
+分子 #18: Pre-mRNA-splicing factor SYF1,PRE-MRNA-SPLICING FACTOR SYF1
+分子 #19: Pre-mRNA-splicing factor 18
+分子 #20: Pre-mRNA-splicing factor SLU7
+分子 #21: Pre-mRNA-processing factor 17
+分子 #22: UNKNOWN PROTEIN
+分子 #23: Pre-mRNA-splicing factor SYF2
+分子 #24: Small nuclear ribonucleoprotein-associated protein B
+分子 #25: Small nuclear ribonucleoprotein Sm D3
+分子 #26: Small nuclear ribonucleoprotein E
+分子 #27: Small nuclear ribonucleoprotein F
+分子 #28: Small nuclear ribonucleoprotein G
+分子 #29: Small nuclear ribonucleoprotein Sm D1
+分子 #30: Small nuclear ribonucleoprotein Sm D2
+分子 #31: Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22
+分子 #32: U2 small nuclear ribonucleoprotein A'
+分子 #33: U2 small nuclear ribonucleoprotein B''
+分子 #35: Pre-mRNA-splicing factor SNT309
+分子 #36: Pre-mRNA-processing factor 19
+分子 #37: MAGNESIUM ION
+分子 #38: POTASSIUM ION
+分子 #39: INOSITOL HEXAKISPHOSPHATE
+分子 #40: GUANOSINE-5'-TRIPHOSPHATE
+分子 #41: 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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解析 | 単粒子再構成法 |
試料の集合状態 | particle |
-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.. |
-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 |
詳細 | 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: FIELD EMISSION GUN |
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 |
+画像解析
-原子モデル構築 1
詳細 | Used secondary structure restraints generated in ProSMART and LibG. |
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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 |
得られたモデル | PDB-5mq0: |