+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 6mwq | ||||||
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タイトル | Single particle cryoEM structure of a DARPin-aldolase platform in complex with GFP | ||||||
要素 |
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キーワード | LYASE/FLUORESCENT PROTEIN / synthetic construct / platform / single particle cryoEM / small protein display / LYASE-FLUORESCENT PROTEIN complex | ||||||
機能・相同性 | 機能・相同性情報 negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / M band / I band / fructose 1,6-bisphosphate metabolic process / bioluminescence / generation of precursor metabolites and energy / glycolytic process / protein homotetramerization ...negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / M band / I band / fructose 1,6-bisphosphate metabolic process / bioluminescence / generation of precursor metabolites and energy / glycolytic process / protein homotetramerization / positive regulation of cell migration / cytosol 類似検索 - 分子機能 | ||||||
生物種 | synthetic construct (人工物) Oryctolagus cuniculus (ウサギ) Aequorea victoria (オワンクラゲ) | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3 Å | ||||||
データ登録者 | Weaver, S.J. / Yao, Q. | ||||||
資金援助 | 米国, 1件
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引用 | ジャーナル: Structure / 年: 2019 タイトル: Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM. 著者: Qing Yao / Sara J Weaver / Jee-Young Mock / Grant J Jensen / 要旨: Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of ...Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 Å resolution overall, with 5-8 Å resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element. #1: ジャーナル: Biorxiv / 年: 2018 タイトル: Fusion of DARPin to aldolase enables visualization of small protein by cryoEM 著者: Yao, Q. / Weaver, S.J. / Mock, J.Y. / Jensen, G.J. | ||||||
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 | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 6mwq.cif.gz | 474.5 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb6mwq.ent.gz | 372 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 6mwq.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
文書・要旨 | 6mwq_validation.pdf.gz | 1.2 MB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 6mwq_full_validation.pdf.gz | 1.2 MB | 表示 | |
XML形式データ | 6mwq_validation.xml.gz | 85.8 KB | 表示 | |
CIF形式データ | 6mwq_validation.cif.gz | 128.6 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/mw/6mwq ftp://data.pdbj.org/pub/pdb/validation_reports/mw/6mwq | HTTPS FTP |
-関連構造データ
-リンク
-集合体
登録構造単位 |
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-要素
#1: タンパク質 | 分子量: 53113.434 Da / 分子数: 4 / 由来タイプ: 組換発現 詳細: Rabbit (Oryctolagus cuniculus) muscle aldolase and a synthetic DARPin were fused to create this synthetic construct 由来: (組換発現) synthetic construct (人工物), (組換発現) Oryctolagus cuniculus (ウサギ) プラスミド: pET21b / 遺伝子: ALDOA 詳細 (発現宿主): C-terminal His-tag of DARPin-aldolase chimeric fusion 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / 株 (発現宿主): BL21(DE3) / 参照: UniProt: P00883, fructose-bisphosphate aldolase #2: タンパク質 | 分子量: 25473.697 Da / 分子数: 4 / 由来タイプ: 組換発現 由来: (組換発現) Aequorea victoria (オワンクラゲ) 遺伝子: GFP / プラスミド: pACYCDuet / 詳細 (発現宿主): no tag / 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / 株 (発現宿主): BL21(DE3) / 参照: UniProt: P42212 |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
-試料調製
構成要素 |
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分子量 | 値: 0.314 MDa / 実験値: NO | ||||||||||||||||||||||||
由来(天然) |
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由来(組換発現) |
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緩衝液 | pH: 8 | ||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 2.5 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: The protein complex was then purified with Ni-NTA affinity chromatography (Qiagen), and Superdex 200 chromatography (GE healthcare). The purified GFP-DARPin-aldolase complex was concentrated ...詳細: The protein complex was then purified with Ni-NTA affinity chromatography (Qiagen), and Superdex 200 chromatography (GE healthcare). The purified GFP-DARPin-aldolase complex was concentrated to 2.5mg/ml in a buffer containing 25 mM Tris-HCl pH 8.0 and 150 mM NaCl. | ||||||||||||||||||||||||
試料支持 | グリッドの材料: GOLD / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: UltrAuFoil | ||||||||||||||||||||||||
急速凍結 | 装置: HOMEMADE PLUNGER / 凍結剤: ETHANE / 湿度: 95 % / 凍結前の試料温度: 277.15 K 詳細: Grids were frozen on a manual plunger at the Scripps Research Institute Core Microscopy Facility in a 4 degrees C cold room humidified to >95%. |
-電子顕微鏡撮影
実験機器 | モデル: Titan Krios / 画像提供: FEI Company | ||||||||||||||||
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顕微鏡 | モデル: FEI TITAN KRIOS | ||||||||||||||||
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM | ||||||||||||||||
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 165000 X / 最大 デフォーカス(公称値): 30000 nm / 最小 デフォーカス(公称値): 10000 nm / Cs: 2.7 mm / C2レンズ絞り径: 70 µm | ||||||||||||||||
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER | ||||||||||||||||
撮影 | Imaging-ID: 1 / 平均露光時間: 4 sec. / 検出モード: SUPER-RESOLUTION / フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) / 撮影したグリッド数: 1
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電子光学装置 | エネルギーフィルター名称: GIF Quantum LS / エネルギーフィルタースリット幅: 20 eV | ||||||||||||||||
画像スキャン | 動画フレーム数/画像: 40 / 利用したフレーム数/画像: 1-40 |
-解析
EMソフトウェア |
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画像処理 | 詳細: Particles from all three microscopy sessions were processed together. | |||||||||||||||||||||||||||||||||||||||||||||
CTF補正 | 詳細: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion. タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 851776 詳細: Manually picked particles were used to generate references for autopicking. Particles from all three microscopy sessions were processed together. Microscope session 1 - 358,381 particles ...詳細: Manually picked particles were used to generate references for autopicking. Particles from all three microscopy sessions were processed together. Microscope session 1 - 358,381 particles Microscope session 2 - 64,368 particles Microscope session 3 - 402,427 particles | |||||||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | |||||||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 236339 / 対称性のタイプ: POINT | |||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: RIGID BODY FIT 詳細: PDB models 5vy5 and 5ma6 were used as starting points. These models were mutated to match the sequence of the DARPin-aldolase platform in complex with GFP that were used. The PDB models were ...詳細: PDB models 5vy5 and 5ma6 were used as starting points. These models were mutated to match the sequence of the DARPin-aldolase platform in complex with GFP that were used. The PDB models were docked into the cryoEM density using Chimera fit into map function. No model refinement was used. | |||||||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | 3D fitting-ID: 1 / Source name: PDB / タイプ: experimental model
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