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データを開く
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基本情報
登録情報 | データベース: PDB / ID: 6xkl | ||||||||||||
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タイトル | SARS-CoV-2 HexaPro S One RBD up | ||||||||||||
![]() | Spike glycoprotein | ||||||||||||
![]() | VIRAL PROTEIN / SARS-CoV-2 / glycoprotein / fusion protein | ||||||||||||
機能・相同性 | ![]() Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / identical protein binding / membrane / plasma membrane 類似検索 - 分子機能 | ||||||||||||
生物種 | ![]() ![]() | ||||||||||||
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: 3.21 Å | ||||||||||||
![]() | Wrapp, D. / Hsieh, C.-L. / Goldsmith, J.A. / McLellan, J.S. | ||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. 著者: Ching-Lin Hsieh / Jory A Goldsmith / Jeffrey M Schaub / Andrea M DiVenere / Hung-Che Kuo / Kamyab Javanmardi / Kevin C Le / Daniel Wrapp / Alison G Lee / Yutong Liu / Chia-Wei Chou / Patrick ...著者: Ching-Lin Hsieh / Jory A Goldsmith / Jeffrey M Schaub / Andrea M DiVenere / Hung-Che Kuo / Kamyab Javanmardi / Kevin C Le / Daniel Wrapp / Alison G Lee / Yutong Liu / Chia-Wei Chou / Patrick O Byrne / Christy K Hjorth / Nicole V Johnson / John Ludes-Meyers / Annalee W Nguyen / Juyeon Park / Nianshuang Wang / Dzifa Amengor / Jason J Lavinder / Gregory C Ippolito / Jennifer A Maynard / Ilya J Finkelstein / Jason S McLellan / ![]() 要旨: The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and ...The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). #1: ジャーナル: bioRxiv / 年: 2020 タイトル: Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes. 著者: Ching-Lin Hsieh / Jory A Goldsmith / Jeffrey M Schaub / Andrea M DiVenere / Hung-Che Kuo / Kamyab Javanmardi / Kevin C Le / Daniel Wrapp / Alison Gene-Wei Lee / Yutong Liu / Chia-Wei Chou / ...著者: Ching-Lin Hsieh / Jory A Goldsmith / Jeffrey M Schaub / Andrea M DiVenere / Hung-Che Kuo / Kamyab Javanmardi / Kevin C Le / Daniel Wrapp / Alison Gene-Wei Lee / Yutong Liu / Chia-Wei Chou / Patrick O Byrne / Christy K Hjorth / Nicole V Johnson / John Ludes-Meyers / Annalee W Nguyen / Juyeon Park / Nianshuang Wang / Dzifa Amengor / Jennifer A Maynard / Ilya J Finkelstein / Jason S McLellan / ![]() 要旨: The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key ...The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to ~10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2. #2: ジャーナル: bioRxiv / 年: 2020 タイトル: Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes. 著者: Ching-Lin Hsieh / Jory A Goldsmith / Jeffrey M Schaub / Andrea M DiVenere / Hung-Che Kuo / Kamyab Javanmardi / Kevin C Le / Daniel Wrapp / Alison Gene-Wei Lee / Yutong Liu / Chia-Wei Chou / ...著者: Ching-Lin Hsieh / Jory A Goldsmith / Jeffrey M Schaub / Andrea M DiVenere / Hung-Che Kuo / Kamyab Javanmardi / Kevin C Le / Daniel Wrapp / Alison Gene-Wei Lee / Yutong Liu / Chia-Wei Chou / Patrick O Byrne / Christy K Hjorth / Nicole V Johnson / John Ludes-Meyers / Annalee W Nguyen / Juyeon Park / Nianshuang Wang / Dzifa Amengor / Jennifer A Maynard / Ilya J Finkelstein / Jason S McLellan / ![]() 要旨: The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key ...The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to ~10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2. | ||||||||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | 分子: ![]() ![]() |
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ダウンロードとリンク
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ダウンロード
PDBx/mmCIF形式 | ![]() | 524.9 KB | 表示 | ![]() |
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PDB形式 | ![]() | 431.9 KB | 表示 | ![]() |
PDBx/mmJSON形式 | ![]() | ツリー表示 | ![]() | |
その他 | ![]() |
-検証レポート
文書・要旨 | ![]() | 1.9 MB | 表示 | ![]() |
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文書・詳細版 | ![]() | 1.9 MB | 表示 | |
XML形式データ | ![]() | 96.5 KB | 表示 | |
CIF形式データ | ![]() | 144.3 KB | 表示 | |
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 142427.438 Da / 分子数: 3 / 変異: F817P, A892P, A899P, A942P, K986P, V987P / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: S, 2 / 発現宿主: ![]() #2: 多糖 | 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose #3: 糖 | ChemComp-NAG / 研究の焦点であるリガンドがあるか | N | |
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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 |
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試料調製
構成要素 | 名称: Prefusion-stabilized SARS-CoV-2 spike trimer with a single RBD up タイプ: COMPLEX / Entity ID: #1 / 由来: RECOMBINANT | ||||||||||||||||||||
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分子量 | 値: 0.6 MDa / 実験値: YES | ||||||||||||||||||||
由来(天然) | 生物種: ![]() ![]() | ||||||||||||||||||||
由来(組換発現) | 生物種: ![]() | ||||||||||||||||||||
緩衝液 | pH: 8 | ||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.35 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES | ||||||||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: C-flat-1.2/1.3 | ||||||||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 277.15 K |
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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: ![]() |
電子レンズ | モード: BRIGHT FIELD |
撮影 | 電子線照射量: 45 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k) |
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解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 695490 | ||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||||||||
3次元再構成 | 解像度: 3.21 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 85675 / 詳細: Non-uniform refinement / 対称性のタイプ: POINT |