+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-13978 | |||||||||||||||||||||||||||||||||
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タイトル | S. cerevisiae CMGE nucleating origin DNA melting | |||||||||||||||||||||||||||||||||
マップデータ | PEHNIX Resolve cryoEM density modified map generated from RELION half maps | |||||||||||||||||||||||||||||||||
試料 |
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機能・相同性 | 機能・相同性情報 DNA-templated DNA replication maintenance of fidelity / 遺伝子変換 / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / GINS complex ...DNA-templated DNA replication maintenance of fidelity / 遺伝子変換 / Unwinding of DNA / DNA replication initiation / epsilon DNA polymerase complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / GINS complex / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / SUMO binding / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / Termination of translesion DNA synthesis / single-stranded DNA 3'-5' DNA exonuclease activity / nuclear pre-replicative complex / Activation of ATR in response to replication stress / MCM complex / DNA replication preinitiation complex / double-strand break repair via break-induced replication / replication fork protection complex / mitotic DNA replication initiation / mitotic DNA replication checkpoint signaling / mitotic intra-S DNA damage checkpoint signaling / nucleotide-excision repair, DNA gap filling / DNA replication proofreading / single-stranded DNA helicase activity / 加水分解酵素; エステル加水分解酵素; 5'-リン酸モノエステル産生エンドデオキシリボヌクレアーゼ / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / DNA strand elongation involved in DNA replication / nuclear chromosome / leading strand elongation / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / Dual incision in TC-NER / DNA replication initiation / subtelomeric heterochromatin formation / error-prone translesion synthesis / DNA helicase activity / base-excision repair, gap-filling / helicase activity / DNA複製 / デオキシリボ核酸 / 塩基除去修復 / DNA-templated DNA replication / double-strand break repair via nonhomologous end joining / double-strand break repair / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / double-stranded DNA binding / ヘリカーゼ / DNA複製 / chromosome, telomeric region / DNAポリメラーゼ / DNA-directed DNA polymerase activity / hydrolase activity / 細胞周期 / nucleotide binding / mRNA binding / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / 核質 / ATP binding / metal ion binding / 細胞核 / 細胞質 類似検索 - 分子機能 | |||||||||||||||||||||||||||||||||
生物種 | Saccharomyces cerevisiae (パン酵母) / DNA molecule (デオキシリボ核酸) | |||||||||||||||||||||||||||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.3 Å | |||||||||||||||||||||||||||||||||
データ登録者 | Lewis JS / Sousa JS / Costa A | |||||||||||||||||||||||||||||||||
資金援助 | European Union, 10件
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引用 | ジャーナル: Nature / 年: 2022 タイトル: Mechanism of replication origin melting nucleated by CMG helicase assembly. 著者: Jacob S Lewis / Marta H Gross / Joana Sousa / Sarah S Henrikus / Julia F Greiwe / Andrea Nans / John F X Diffley / Alessandro Costa / 要旨: The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex ...The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation. | |||||||||||||||||||||||||||||||||
履歴 |
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-構造の表示
添付画像 |
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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 | emd_13978.map.gz | 23.5 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-13978-v30.xml emd-13978.xml | 46.6 KB 46.6 KB | 表示 表示 | EMDBヘッダ |
画像 | emd_13978.png | 48.7 KB | ||
その他 | emd_13978_additional_1.map.gz emd_13978_half_map_1.map.gz emd_13978_half_map_2.map.gz | 478.5 MB 410.8 MB 411 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-13978 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13978 | HTTPS FTP |
-関連構造データ
-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_13978.map.gz / 形式: CCP4 / 大きさ: 25.2 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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注釈 | PEHNIX Resolve cryoEM density modified map generated from RELION half maps | ||||||||||||||||||||||||||||||||||||
投影像・断面図 | 画像のコントロール
画像は Spider により作成 これらの図は立方格子座標系で作成されたものです | ||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-追加マップ: RELION 3.1 postprocessed map generated from RELION half maps
ファイル | emd_13978_additional_1.map | ||||||||||||
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注釈 | RELION 3.1 postprocessed map generated from RELION half maps | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: RELION half map 1
ファイル | emd_13978_half_map_1.map | ||||||||||||
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注釈 | RELION half map 1 | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: RELION half map 2
ファイル | emd_13978_half_map_2.map | ||||||||||||
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注釈 | RELION half map 2 | ||||||||||||
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 : S. cerevisiae CMG helicase nucleating origin DNA melting
+超分子 #1: S. cerevisiae CMG helicase nucleating origin DNA melting
+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: DNA replication licensing factor MCM6
+分子 #5: DNA replication licensing factor MCM7
+分子 #6: DNA replication complex GINS protein PSF1
+分子 #7: DNA replication complex GINS protein PSF2
+分子 #8: DNA replication complex GINS protein PSF3
+分子 #9: DNA replication complex GINS protein SLD5
+分子 #10: Cell division control protein 45
+分子 #11: DNA polymerase epsilon subunit B
+分子 #12: DNA polymerase epsilon catalytic subunit A
+分子 #15: DNA replication licensing factor MCM5
+分子 #13: DNA (26-MER)
+分子 #14: DNA (26-MER)
+分子 #16: ADENOSINE-5'-TRIPHOSPHATE
+分子 #17: ZINC ION
+分子 #18: MAGNESIUM ION
+分子 #19: ADENOSINE-5'-DIPHOSPHATE
-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
緩衝液 | pH: 7.5 |
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凍結 | 凍結剤: ETHANE / 装置: FEI VITROBOT MARK IV |
詳細 | four microlitres of sample was applied on a grid and incubated for 2 min at room temperature before blotting with filter paper for 5.5 s and plunge-freezing in liquid ethane. |
-電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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電子線 | 加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN |
電子光学系 | C2レンズ絞り径: 50.0 µm / 照射モード: OTHER / 撮影モード: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / 最大 デフォーカス(公称値): 4.4 µm / 最小 デフォーカス(公称値): 2.0 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 |
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 / 撮影したグリッド数: 2 / 実像数: 65286 / 平均露光時間: 10.0 sec. / 平均電子線量: 1.6 e/Å2 |
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
-画像解析
粒子像選択 | 選択した数: 927109 |
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CTF補正 | ソフトウェア: (名称: RELION (ver. 3.1), Gctf (ver. 1.18)) |
初期 角度割当 | タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1) |
最終 角度割当 | タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1) |
最終 再構成 | 想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 3.3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: RELION (ver. 3.1) 詳細: Reported resolution after PHENIX resolve cryoEM density modification. Input were half maps generated in RELION 3.1 使用した粒子像数: 71348 |