+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-16888 | ||||||||||||
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タイトル | Cryo-EM structure of ADP-bound, filamentous beta-actin harboring the R183W mutation | ||||||||||||
マップデータ | Sharpened, local-resolution filtered cryo-EM density map of filamentous beta-actin harboring the R183W mutation. | ||||||||||||
試料 |
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キーワード | Actin filament / cytoskeletal protein / ATPase / STRUCTURAL PROTEIN | ||||||||||||
機能・相同性 | 機能・相同性情報 positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / morphogenesis of a polarized epithelium / bBAF complex / postsynaptic actin cytoskeleton organization / protein localization to adherens junction / postsynaptic actin cytoskeleton / npBAF complex / Tat protein binding ...positive regulation of norepinephrine uptake / cellular response to cytochalasin B / regulation of transepithelial transport / morphogenesis of a polarized epithelium / bBAF complex / postsynaptic actin cytoskeleton organization / protein localization to adherens junction / postsynaptic actin cytoskeleton / npBAF complex / Tat protein binding / brahma complex / structural constituent of postsynaptic actin cytoskeleton / nBAF complex / GBAF complex / regulation of G0 to G1 transition / dense body / Formation of annular gap junctions / Gap junction degradation / Cell-extracellular matrix interactions / Folding of actin by CCT/TriC / apical protein localization / regulation of double-strand break repair / regulation of nucleotide-excision repair / adherens junction assembly / Prefoldin mediated transfer of substrate to CCT/TriC / RSC-type complex / RHOF GTPase cycle / Adherens junctions interactions / tight junction / regulation of norepinephrine uptake / Sensory processing of sound by outer hair cells of the cochlea / regulation of mitotic metaphase/anaphase transition / Interaction between L1 and Ankyrins / Sensory processing of sound by inner hair cells of the cochlea / SWI/SNF complex / positive regulation of double-strand break repair / regulation of synaptic vesicle endocytosis / positive regulation of T cell differentiation / apical junction complex / establishment or maintenance of cell polarity / regulation of cyclin-dependent protein serine/threonine kinase activity / maintenance of blood-brain barrier / cortical cytoskeleton / positive regulation of stem cell population maintenance / NuA4 histone acetyltransferase complex / nitric-oxide synthase binding / Recycling pathway of L1 / regulation of G1/S transition of mitotic cell cycle / kinesin binding / brush border / calyx of Held / negative regulation of cell differentiation / positive regulation of double-strand break repair via homologous recombination / EPH-ephrin mediated repulsion of cells / RHO GTPases Activate WASPs and WAVEs / RHO GTPases activate IQGAPs / regulation of protein localization to plasma membrane / positive regulation of myoblast differentiation / EPHB-mediated forward signaling / substantia nigra development / axonogenesis / negative regulation of protein binding / Translocation of SLC2A4 (GLUT4) to the plasma membrane / RHO GTPases Activate Formins / cell motility / actin filament / positive regulation of cell differentiation / regulation of transmembrane transporter activity / FCGR3A-mediated phagocytosis / adherens junction / DNA Damage Recognition in GG-NER / 加水分解酵素; 酸無水物に作用; 酸無水物に作用・細胞または細胞小器官の運動に関与 / Signaling by high-kinase activity BRAF mutants / Schaffer collateral - CA1 synapse / MAP2K and MAPK activation / tau protein binding / B-WICH complex positively regulates rRNA expression / structural constituent of cytoskeleton / Regulation of actin dynamics for phagocytic cup formation / kinetochore / cytoplasmic ribonucleoprotein granule / nuclear matrix / platelet aggregation / VEGFA-VEGFR2 Pathway / Signaling by RAF1 mutants / UCH proteinases / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / nucleosome / Signaling by BRAF and RAF1 fusions / cell-cell junction / actin cytoskeleton / presynapse / lamellipodium / Clathrin-mediated endocytosis / Factors involved in megakaryocyte development and platelet production / HATs acetylate histones / regulation of apoptotic process / vesicle 類似検索 - 分子機能 | ||||||||||||
生物種 | Homo sapiens (ヒト) | ||||||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 2.28 Å | ||||||||||||
データ登録者 | Oosterheert W / Blanc FEC / Roy A / Belyy A / Hofnagel O / Hummer G / Bieling P / Raunser S | ||||||||||||
資金援助 | ドイツ, European Union, 3件
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引用 | ジャーナル: Nat Struct Mol Biol / 年: 2023 タイトル: Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments. 著者: Wout Oosterheert / Florian E C Blanc / Ankit Roy / Alexander Belyy / Micaela Boiero Sanders / Oliver Hofnagel / Gerhard Hummer / Peter Bieling / Stefan Raunser / 要旨: The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release ...The release of inorganic phosphate (P) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. The mechanisms underlying P release from the core and barbed end of actin filaments remain unclear. Here, using human and bovine actin isoforms, we combine cryo-EM with molecular-dynamics simulations and in vitro reconstitution to demonstrate how actin releases P through a 'molecular backdoor'. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and opens only transiently through concerted amino acid rearrangements. This explains why P escapes rapidly from the filament end but slowly from internal subunits. In a nemaline-myopathy-associated actin variant, the backdoor is predominantly open in filament-core subunits, resulting in accelerated P release and filaments with drastically shortened ADP-P caps. Our results provide the molecular basis for P release from actin and exemplify how a disease-linked mutation distorts the nucleotide-state distribution and atomic structure of the filament. | ||||||||||||
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_16888.map.gz | 129.9 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-16888-v30.xml emd-16888.xml | 24.2 KB 24.2 KB | 表示 表示 | EMDBヘッダ |
FSC (解像度算出) | emd_16888_fsc.xml | 13.6 KB | 表示 | FSCデータファイル |
画像 | emd_16888.png | 89.9 KB | ||
マスクデータ | emd_16888_msk_1.map | 216 MB | マスクマップ | |
Filedesc metadata | emd-16888.cif.gz | 7.2 KB | ||
その他 | emd_16888_additional_1.map.gz emd_16888_half_map_1.map.gz emd_16888_half_map_2.map.gz | 169.1 MB 170.8 MB 170.8 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-16888 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-16888 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_16888_validation.pdf.gz | 810.4 KB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_16888_full_validation.pdf.gz | 809.9 KB | 表示 | |
XML形式データ | emd_16888_validation.xml.gz | 20.7 KB | 表示 | |
CIF形式データ | emd_16888_validation.cif.gz | 26.7 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16888 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16888 | 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_16888.map.gz / 形式: CCP4 / 大きさ: 216 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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注釈 | Sharpened, local-resolution filtered cryo-EM density map of filamentous beta-actin harboring the R183W mutation. | ||||||||||||||||||||||||||||||||||||
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: 0.695 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
-マスク #1
ファイル | emd_16888_msk_1.map | ||||||||||||
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投影像・断面図 |
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密度ヒストグラム |
-追加マップ: 3D refined, unsharpened cryo-EM density map of filamentous...
ファイル | emd_16888_additional_1.map | ||||||||||||
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注釈 | 3D refined, unsharpened cryo-EM density map of filamentous beta-actin harboring the R183W mutation. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Half map 1 of the refinement of filamentous...
ファイル | emd_16888_half_map_1.map | ||||||||||||
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注釈 | Half map 1 of the refinement of filamentous beta-actin harboring the R183W mutation. | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Half map 2 of the refinement of filamentous...
ファイル | emd_16888_half_map_2.map | ||||||||||||
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注釈 | Half map 2 of the refinement of filamentous beta-actin harboring the R183W mutation. | ||||||||||||
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 : Actin filament harboring the R183W mutation.
全体 | 名称: Actin filament harboring the R183W mutation. |
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要素 |
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-超分子 #1: Actin filament harboring the R183W mutation.
超分子 | 名称: Actin filament harboring the R183W mutation. / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1 詳細: Actin was purified as monomer from insect cells. It was then polymerized into a filament in vitro. |
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由来(天然) | 生物種: Homo sapiens (ヒト) |
-分子 #1: Actin, cytoplasmic 1
分子 | 名称: Actin, cytoplasmic 1 / タイプ: protein_or_peptide / ID: 1 / コピー数: 5 / 光学異性体: LEVO EC番号: 加水分解酵素; 酸無水物に作用; 酸無水物に作用・細胞または細胞小器官の運動に関与 |
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由来(天然) | 生物種: Homo sapiens (ヒト) |
分子量 | 理論値: 41.792633 KDa |
組換発現 | 生物種: Trichoplusia ni (イラクサキンウワバ) |
配列 | 文字列: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG ...文字列: MDDDIAALVV DNGSGMCKAG FAGDDAPRAV FPSIVGRPRH QGVMVGMGQK DSYVGDEAQS KRGILTLKYP IE(HIC)GIV TNW DDMEKIWHHT FYNELRVAPE EHPVLLTEAP LNPKANREKM TQIMFETFNT PAMYVAIQAV LSLYASGRTT GIVMDSG DG VTHTVPIYEG YALPHAILRL DLAGWDLTDY LMKILTERGY SFTTTAEREI VRDIKEKLCY VALDFEQEMA TAASSSSL E KSYELPDGQV ITIGNERFRC PEALFQPSFL GMESAGIHET TFNSIMKCDV DIRKDLYANT VLSGGTTMYP GIADRMQKE ITALAPSTMK IKIIAPPERK YSVWIGGSIL ASLSTFQQMW ISKQEYDESG PSIVHRKCF UniProtKB: Actin, cytoplasmic 1 |
-分子 #2: ADENOSINE-5'-DIPHOSPHATE
分子 | 名称: ADENOSINE-5'-DIPHOSPHATE / タイプ: ligand / ID: 2 / コピー数: 5 / 式: ADP |
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分子量 | 理論値: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-分子 #3: MAGNESIUM ION
分子 | 名称: MAGNESIUM ION / タイプ: ligand / ID: 3 / コピー数: 5 / 式: MG |
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分子量 | 理論値: 24.305 Da |
-分子 #4: water
分子 | 名称: water / タイプ: ligand / ID: 4 / コピー数: 585 / 式: HOH |
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分子量 | 理論値: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-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 | filament |
-試料調製
濃度 | 2.3 mg/mL | ||||||||||||||||||
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緩衝液 | pH: 7.1 構成要素:
詳細: 1x KMEH (10 mM HEPES pH 7.1, 100 mM KCl, 2 mM MgCl2, 1 mM EGTA) supplemented with 0.02% Tween20 (v/v) | ||||||||||||||||||
グリッド | モデル: Quantifoil R2/1 / 材質: COPPER / メッシュ: 300 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 90 sec. | ||||||||||||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 286 K / 装置: FEI VITROBOT MARK IV | ||||||||||||||||||
詳細 | Actin filaments were reconstituted by adding salt to monomeric actin. |
-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: 15 eV |
詳細 | Titan Krios G3 microscope was aligned using Sherpa (FEI). Data collected in superresolution mode. |
撮影 | フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) 撮影したグリッド数: 1 / 実像数: 7916 / 平均電子線量: 70.0 e/Å2 |
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: SPOT SCAN / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 最大 デフォーカス(公称値): 2.0 µm / 最小 デフォーカス(公称値): 0.8 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 |