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基本情報
登録情報 | データベース: EMDB / ID: EMD-10227 | |||||||||
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タイトル | Cryo-EM structure of the CMG Fork Protection Complex at a replication fork - Conformation 1 | |||||||||
![]() | Conformation1 CMG-Csm3-Tof1-Mrc1-Ctf4 and a DNA fork | |||||||||
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![]() | protein-DNA complex / replisome / AAA+ helicase / CMG / GINS / fork DNA / MCM / fork protection complex / CIP-box / REPLICATION | |||||||||
機能・相同性 | ![]() establishment of sister chromatid cohesion / maintenance of DNA repeat elements / Unwinding of DNA / Cul8-RING ubiquitin ligase complex / replication fork arrest / meiotic chromosome segregation / DNA strand elongation involved in mitotic DNA replication / GINS complex / MCM core complex / Assembly of the pre-replicative complex ...establishment of sister chromatid cohesion / maintenance of DNA repeat elements / Unwinding of DNA / Cul8-RING ubiquitin ligase complex / replication fork arrest / meiotic chromosome segregation / DNA strand elongation involved in mitotic DNA replication / GINS complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / mitotic DNA replication preinitiation complex assembly / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / mitotic DNA replication / DNA replication checkpoint signaling / CMG complex / establishment of mitotic sister chromatid cohesion / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / mitotic DNA replication initiation / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / mitotic sister chromatid cohesion / nuclear chromosome / DNA strand elongation involved in DNA replication / replication fork processing / nuclear replication fork / : / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / meiotic cell cycle / helicase activity / transcription elongation by RNA polymerase II / DNA-templated DNA replication / heterochromatin formation / nucleosome assembly / mitotic cell cycle / single-stranded DNA binding / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / DNA helicase / cohesin loader activity / DNA clamp loader activity / DNA replication / chromosome, telomeric region / DNA repair / DNA damage response / chromatin binding / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytosol / 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: 3.7 Å | |||||||||
![]() | Baretic D / Jenkyn-Bedford M | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork. 著者: Domagoj Baretić / Michael Jenkyn-Bedford / Valentina Aria / Giuseppe Cannone / Mark Skehel / Joseph T P Yeeles / ![]() 要旨: The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric ...The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 "grip" duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability. | |||||||||
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構造の表示
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構造ビューア | EMマップ: ![]() ![]() ![]() |
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リンク
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「今月の分子」の関連する項目 |
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マップ
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注釈 | Conformation1 CMG-Csm3-Tof1-Mrc1-Ctf4 and a DNA fork | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
投影像・断面図 | 画像のコントロール
画像は Spider により作成 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.049 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 : Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
+超分子 #1: Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA
+超分子 #2: CMG-Csm3-Tof1-Mrc1-Ctf4
+超分子 #3: DNA
+分子 #1: DNA replication licensing factor MCM2
+分子 #2: DNA replication licensing factor MCM3
+分子 #3: DNA replication licensing factor MCM4
+分子 #4: Minichromosome maintenance protein 5
+分子 #5: DNA replication licensing factor MCM6
+分子 #6: DNA replication licensing factor MCM7
+分子 #7: DNA replication complex GINS protein PSF1
+分子 #8: DNA replication complex GINS protein PSF2
+分子 #9: DNA replication complex GINS protein PSF3
+分子 #10: DNA replication complex GINS protein SLD5
+分子 #11: Cell division control protein 45
+分子 #12: DNA polymerase alpha-binding protein
+分子 #15: Topoisomerase 1-associated factor 1
+分子 #16: Chromosome segregation in meiosis protein 3
+分子 #13: DNA fork, leading-strand template
+分子 #14: DNA fork, lagging-strand template
+分子 #17: ZINC ION
+分子 #18: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
+分子 #19: MAGNESIUM 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 | 0.5 mg/mL | |||||||||||||||||||||
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緩衝液 | pH: 7.5 構成要素:
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グリッド | モデル: Quantifoil R2/2 / 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: CONTINUOUS / 支持フィルム - Film thickness: 5 / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 5 sec. / 前処理 - 雰囲気: AIR / 詳細: 15 mA | |||||||||||||||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 80 % / チャンバー内温度: 277.15 K / 装置: HOMEMADE PLUNGER 詳細: Three microlitres of sample was applied on a grid and incubated for 15-30 s at 4 degC before manually blotting with filter paper for 10 s and plunge-freezing in liquid ethane.. | |||||||||||||||||||||
詳細 | In vitro reconstitution from individual components: CMG, Csm3-Tof1, Mrc1, Ctf4 and a DNA fork |
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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 Bioquantum / エネルギーフィルター - スリット幅: 20 eV |
撮影 | フィルム・検出器のモデル: GATAN K2 QUANTUM (4k x 4k) 検出モード: COUNTING / デジタル化 - 画像ごとのフレーム数: 1-20 / 撮影したグリッド数: 2 / 実像数: 6682 / 平均露光時間: 7.0 sec. / 平均電子線量: 37.0 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | C2レンズ絞り径: 50.0 µm / 最大 デフォーカス(補正後): 3.0 µm / 最小 デフォーカス(補正後): 0.5 µm / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 最大 デフォーカス(公称値): 2.6 µm 最小 デフォーカス(公称値): 1.4000000000000001 µm 倍率(公称値): 130000 |
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
初期モデル |
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精密化 | 空間: REAL / プロトコル: OTHER / 温度因子: 20 / 当てはまり具合の基準: FSC 0.5 | ||||||||
得られたモデル | ![]() PDB-6skl: |