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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-4980 | |||||||||
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タイトル | Cryo-EM structure of an MCM loading intermediate | |||||||||
![]() | Full map - complete MCM-ORC (MO) origin licensing intermediate | |||||||||
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![]() | DNA Replication / Origin licensing / MCM2-7 helicase / Origin Recognition Complex / REPLICATION | |||||||||
機能・相同性 | ![]() CDC6 association with the ORC:origin complex / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / Assembly of the ORC complex at the origin of replication / premeiotic DNA replication ...CDC6 association with the ORC:origin complex / Cul8-RING ubiquitin ligase complex / maintenance of rDNA / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / Assembly of the ORC complex at the origin of replication / premeiotic DNA replication / nuclear origin of replication recognition complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / single-stranded 3'-5' DNA helicase activity / nuclear pre-replicative complex / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / replication fork protection complex / mitotic DNA replication checkpoint signaling / mitotic DNA replication initiation / double-strand break repair via break-induced replication / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / single-stranded DNA helicase activity / DNA strand elongation involved in DNA replication / Orc1 removal from chromatin / DNA unwinding involved in DNA replication / regulation of DNA replication / nuclear replication fork / 3'-5' DNA helicase activity / DNA replication origin binding / subtelomeric heterochromatin formation / DNA replication initiation / nucleosome binding / heterochromatin formation / helicase activity / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA damage response / chromatin binding / ATP hydrolysis activity / nucleoplasm / ATP binding / nucleus / metal ion binding / cytoplasm 類似検索 - 分子機能 | |||||||||
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: 4.4 Å | |||||||||
![]() | Miller TCR / Locke J | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM. 著者: Thomas C R Miller / Julia Locke / Julia F Greiwe / John F X Diffley / Alessandro Costa / ![]() 要旨: In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double- ...In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data. | |||||||||
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: ![]() ![]() ![]() |
添付画像 |
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ダウンロードとリンク
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マップデータ | ![]() | 9.9 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 62.1 KB 62.1 KB | 表示 表示 | ![]() |
FSC (解像度算出) | ![]() ![]() ![]() | 12.1 KB 12.1 KB 12.1 KB | 表示 表示 表示 | ![]() |
画像 | ![]() | 131.5 KB | ||
マスクデータ | ![]() ![]() ![]() | 149.9 MB 149.9 MB 149.9 MB | ![]() | |
Filedesc metadata | ![]() | 13.7 KB | ||
その他 | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | 96.8 MB 96.8 MB 8.4 MB 108.5 MB 6.5 MB 108.5 MB 118.1 MB 118.2 MB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-検証レポート
文書・要旨 | ![]() | 414.3 KB | 表示 | ![]() |
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文書・詳細版 | ![]() | 413.5 KB | 表示 | |
XML形式データ | ![]() | 12.5 KB | 表示 | |
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
EMDBのページ | ![]() ![]() |
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「今月の分子」の関連する項目 |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Full map - complete MCM-ORC (MO) origin licensing intermediate | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.38 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
+マスク #1
+マスク #2
+マスク #3
+追加マップ: Half map - MCM-Orc6N lobe of MCM-ORC (MO)...
+追加マップ: Half map - MCM-Orc6N lobe of MCM-ORC (MO)...
+追加マップ: Full map - MCM-Orc6N lobe of MCM-ORC (MO)...
+追加マップ: Half map - Orc1-5-Orc6C lobe of MCM-ORC (MO)...
+追加マップ: Full map - Orc1-5-Orc6C lobe of MCM-ORC (MO)...
+追加マップ: Half map – Orc1-5-Orc6C lobe of MCM-ORC (MO)...
+ハーフマップ: Half map - complete MCM-ORC (MO) origin licensing intermediate
+ハーフマップ: Half map - complete MCM-ORC (MO) origin licensing intermediate
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試料の構成要素
+全体 : The MCM-ORC (MO) loading intermediate
+超分子 #1: The MCM-ORC (MO) loading intermediate
+超分子 #2: MCM-Orc6N lobe of the MCM-ORC (MO) origin licensing intermediate.
+超分子 #3: Orc1-5-Orc6C lobe of the MCM-ORC (MO) origin licensing intermediate
+超分子 #4: The MCM-ORC (MO) loading intermediate protein complex
+超分子 #5: DNA
+分子 #1: Origin recognition complex subunit 1
+分子 #2: Origin recognition complex subunit 2
+分子 #3: Origin recognition complex subunit 3
+分子 #4: Origin recognition complex subunit 4
+分子 #5: Origin recognition complex subunit 5
+分子 #6: Origin recognition complex subunit 6
+分子 #7: DNA replication licensing factor MCM2
+分子 #8: DNA replication licensing factor MCM3
+分子 #9: DNA replication licensing factor MCM4
+分子 #10: Minichromosome maintenance protein 5
+分子 #11: DNA replication licensing factor MCM6
+分子 #12: DNA replication licensing factor MCM7
+分子 #13: DNA (88-MER)
+分子 #14: DNA (88-MER)
+分子 #15: ADENOSINE-5'-TRIPHOSPHATE
+分子 #16: MAGNESIUM ION
+分子 #17: ADENOSINE-5'-DIPHOSPHATE
+分子 #18: 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
緩衝液 | pH: 7.6 構成要素:
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グリッド | 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: LACEY / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 雰囲気: AIR | ||||||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 90 % / チャンバー内温度: 288 K / 装置: LEICA EM GP 詳細: 10 second incubation, 3.5 seconds single side blotting.. |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / デジタル化 - 画像ごとのフレーム数: 1-30 / 撮影したグリッド数: 1 / 平均電子線量: 1.68 e/Å2 |
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 / Cs: 2.7 mm |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
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
初期モデル |
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精密化 | 空間: REAL / プロトコル: RIGID BODY FIT | ||||||||||
得られたモデル | ![]() PDB-6rqc: |