+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-2441 | |||||||||
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タイトル | Cryo-EM structure of activated and oligomeric restriction endonuclease SgrAI | |||||||||
マップデータ | cryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI | |||||||||
試料 |
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キーワード | restriction endonuclease / helix / oligomer / allostery / DNA cleavage / parasite-host interaction | |||||||||
機能・相同性 | Restriction endonuclease, type II, Cfr10I/Bse634I / Cfr10I/Bse634I restriction endonuclease / Restriction endonuclease type II-like / identical protein binding / metal ion binding / SgraIR restriction enzyme 機能・相同性情報 | |||||||||
生物種 | Streptomyces griseus (ストレプトマイシン生産菌) | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / ネガティブ染色法 / 解像度: 8.6 Å | |||||||||
データ登録者 | Lyumkis D / Talley H / Stewart A / Shah S / Park CK / Tama F / Potter CS / Carragher B / Horton NC | |||||||||
引用 | ジャーナル: Structure / 年: 2013 タイトル: Allosteric regulation of DNA cleavage and sequence-specificity through run-on oligomerization. 著者: Dmitry Lyumkis / Heather Talley / Andrew Stewart / Santosh Shah / Chad K Park / Florence Tama / Clinton S Potter / Bridget Carragher / Nancy C Horton / 要旨: SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of ...SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200- to 500-fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating the conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI's oligomerization and allosteric behavior. | |||||||||
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_2441.map.gz | 22.4 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-2441-v30.xml emd-2441.xml | 13.9 KB 13.9 KB | 表示 表示 | EMDBヘッダ |
画像 | EMD-2441-EMDB_model_scale.png | 197.5 KB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-2441 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2441 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_2441_validation.pdf.gz | 256.9 KB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_2441_full_validation.pdf.gz | 256.1 KB | 表示 | |
XML形式データ | emd_2441_validation.xml.gz | 6 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2441 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2441 | 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_2441.map.gz / 形式: CCP4 / 大きさ: 26.4 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | cryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.42 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
-試料の構成要素
-全体 : cryo-EM reconstruction of activated and oligomeric restriction en...
全体 | 名称: cryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI |
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要素 |
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-超分子 #1000: cryo-EM reconstruction of activated and oligomeric restriction en...
超分子 | 名称: cryo-EM reconstruction of activated and oligomeric restriction endonuclease SgrAI タイプ: sample / ID: 1000 / 詳細: 1 megadalton (100 kDa) per DNA-bound dimer 集合状態: oligomeric SgrAI forms a helical array, whereby each asymmetric unit is composed of a DNA-bound SgrAI dimer Number unique components: 2 |
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分子量 | 理論値: 1 MDa |
-分子 #1: SgrAI
分子 | 名称: SgrAI / タイプ: protein_or_peptide / ID: 1 / Name.synonym: SgraIR restriction enzyme 詳細: 2 copies of pre-cleaved DNA containing sticky ends and the SgrAI recognition sequence is bound to an SgrAI dimer. The oligomeric helix is formed from multiple copies of such DNA-bound dimers 集合状態: DNA-bound dimer / 組換発現: Yes |
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由来(天然) | 生物種: Streptomyces griseus (ストレプトマイシン生産菌) |
分子量 | 理論値: 38 KDa |
組換発現 | 生物種: Escherichia coli (大腸菌) / 組換株: ER2566 / 組換プラスミド: pET21a_SgrA1R |
配列 | UniProtKB: SgraIR restriction enzyme |
-分子 #2: SgrAI recognition sequence
分子 | 名称: SgrAI recognition sequence / タイプ: dna / ID: 2 詳細: 2 copies of pre-cleaved DNA containing sticky ends and the SgrAI recognition sequence is bound to an SgrAI dimer. The oligomeric helix is formed from multiple copies of such DNA-bound dimers 分類: DNA / Structure: DOUBLE HELIX / Synthetic?: No |
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由来(天然) | 生物種: Streptomyces griseus (ストレプトマイシン生産菌) |
配列 | 文字列: GATGCGTGGG TCTTCACACC GGTGTGAAGA CCCACGCATC |
-実験情報
-構造解析
手法 | ネガティブ染色法, 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 | helical array |
-試料調製
濃度 | 0.2 mg/mL |
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緩衝液 | pH: 8 詳細: 10 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM Mg(OAc)2, 0.5 mM DTT |
染色 | タイプ: NEGATIVE 詳細: Specimens were prepared for cryo-EM by applying 3 microliters of sample in binding buffer to a holey carbon C-flat grid (CF-2/2-400) (Protochips, inc.) that had been plasma cleaned (Gatan, ...詳細: Specimens were prepared for cryo-EM by applying 3 microliters of sample in binding buffer to a holey carbon C-flat grid (CF-2/2-400) (Protochips, inc.) that had been plasma cleaned (Gatan, Solarus) for 5 sec. The sample was allowed to adsorb to the grid for 30 sec., then plunge-frozen into liquid ethane using a manual cryo-plunger at 4 C. |
グリッド | 詳細: CF-2/2-400 |
凍結 | 凍結剤: ETHANE / 装置: HOMEMADE PLUNGER 手法: Specimens were prepared for cryo-EM by applying 3 microliters of sample in binding buffer to a holey carbon C-flat grid (CF-2/2-400 (Protochips, inc.) that had been plasma cleaned (Gatan, ...手法: Specimens were prepared for cryo-EM by applying 3 microliters of sample in binding buffer to a holey carbon C-flat grid (CF-2/2-400 (Protochips, inc.) that had been plasma cleaned (Gatan, Solarus) for 5 sec. The sample was allowed to adsorb to the grid for 30 sec., then plunge-frozen into liquid ethane using a manual cryo-plunger at 4 C. |
-電子顕微鏡法
顕微鏡 | FEI TECNAI F20 |
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アライメント法 | Legacy - 非点収差: objective lens astigmatism was corrected at 120,000 times magnification |
日付 | 2012年9月19日 |
撮影 | カテゴリ: FILM フィルム・検出器のモデル: DIRECT ELECTRON DE-12 (4k x 3k) デジタル化 - スキャナー: OTHER / デジタル化 - サンプリング間隔: 6.0 µm / 実像数: 655 / 平均電子線量: 16 e/Å2 |
Tilt angle min | 0 |
電子線 | 加速電圧: 200 kV / 電子線源: FIELD EMISSION GUN |
電子光学系 | 倍率(補正後): 42134 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2 mm / 最大 デフォーカス(公称値): 3.0 µm / 最小 デフォーカス(公称値): 1.0 µm / 倍率(公称値): 62000 |
試料ステージ | 試料ホルダーモデル: GATAN LIQUID NITROGEN |
実験機器 | モデル: Tecnai F20 / 画像提供: FEI Company |
-画像解析
詳細 | The final reconstruction was obtained from 1,918 filament segments, averaged and inserted into the final reconstruction 8 times using Frealign |
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CTF補正 | 詳細: Frealign |
最終 再構成 | 想定した対称性 - らせんパラメータ - Δz: 21.6 Å 想定した対称性 - らせんパラメータ - ΔΦ: 86.2 ° 想定した対称性 - らせんパラメータ - 軸対称性: D1 (2回x1回 2面回転対称) アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 8.6 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: Xmipp, and, Frealign / 使用した粒子像数: 1918 |
-原子モデル構築 1
初期モデル | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B |
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ソフトウェア | 名称: Direx |
詳細 | A model of SgrAI bound to PC DNA generated using the x-ray crystal structure of SgrAI bound to an 18 bp DNA containing a primary site (PDB ID: 3DVO) and additional modeled flanking DNA in B form was flexibly fitted using Direx, a geometry-based conformational sampling approach under low-resolution restraints. The refinement procedure was run for 500 steps followed by the minimization procedure of 300 steps |
精密化 | 空間: REAL / プロトコル: FLEXIBLE FIT |
得られたモデル | PDB-4c3g: |