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- PDB-5fwp: Atomic cryoEM structure of Hsp90-Cdc37-Cdk4 complex -

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基本情報

登録情報
データベース: PDB / ID: 5fwp
タイトルAtomic cryoEM structure of Hsp90-Cdc37-Cdk4 complex
要素
  • CYCLIN-DEPENDENT KINASE 4
  • HEAT SHOCK PROTEIN HSP 90 BETA
  • HSP90 CO-CHAPERONE CDC37
キーワードCHAPERONE / HSP90 / CDC37 / CDK4 / KINASE / UNFOLDING
機能・相同性
機能・相同性情報


cyclin D3-CDK4 complex / cyclin D1-CDK4 complex / cyclin D2-CDK4 complex / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 / cellular response to ionomycin / regulation of transcription initiation by RNA polymerase II / Drug-mediated inhibition of CDK4/CDK6 activity / regulation of type II interferon-mediated signaling pathway / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 ...cyclin D3-CDK4 complex / cyclin D1-CDK4 complex / cyclin D2-CDK4 complex / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 / cellular response to ionomycin / regulation of transcription initiation by RNA polymerase II / Drug-mediated inhibition of CDK4/CDK6 activity / regulation of type II interferon-mediated signaling pathway / Evasion of Oncogene Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / Evasion of Oxidative Stress Induced Senescence Due to Defective p16INK4A binding to CDK4 and CDK6 / regulation of type B pancreatic cell proliferation / receptor ligand inhibitor activity / PTK6 Regulates Cell Cycle / HSP90-CDC37 chaperone complex / positive regulation of mitophagy in response to mitochondrial depolarization / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / aryl hydrocarbon receptor complex / dynein axonemal particle / histone methyltransferase binding / cellular response to phorbol 13-acetate 12-myristate / Transcriptional regulation by RUNX2 / positive regulation of protein localization to cell surface / ATP-dependent protein binding / cyclin-dependent kinase / protein kinase regulator activity / cyclin-dependent protein serine/threonine kinase activity / protein folding chaperone complex / cyclin-dependent protein serine/threonine kinase regulator activity / post-transcriptional regulation of gene expression / telomerase holoenzyme complex assembly / Respiratory syncytial virus genome replication / Uptake and function of diphtheria toxin / regulation of cyclin-dependent protein serine/threonine kinase activity / positive regulation of G2/M transition of mitotic cell cycle / Drug-mediated inhibition of ERBB2 signaling / Resistance of ERBB2 KD mutants to trastuzumab / Resistance of ERBB2 KD mutants to sapitinib / Resistance of ERBB2 KD mutants to tesevatinib / Resistance of ERBB2 KD mutants to neratinib / Resistance of ERBB2 KD mutants to osimertinib / Resistance of ERBB2 KD mutants to afatinib / Resistance of ERBB2 KD mutants to AEE788 / Resistance of ERBB2 KD mutants to lapatinib / Drug resistance in ERBB2 TMD/JMD mutants / TPR domain binding / positive regulation of transforming growth factor beta receptor signaling pathway / Assembly and release of respiratory syncytial virus (RSV) virions / cyclin-dependent protein kinase holoenzyme complex / regulation of G2/M transition of mitotic cell cycle / dendritic growth cone / regulation of type I interferon-mediated signaling pathway / : / : / Sema3A PAK dependent Axon repulsion / regulation of protein ubiquitination / The NLRP3 inflammasome / Signaling by ERBB2 / telomere maintenance via telomerase / HSF1-dependent transactivation / cyclin binding / response to unfolded protein / chaperone-mediated protein complex assembly / HSF1 activation / bicellular tight junction / Attenuation phase / protein targeting / cellular response to interleukin-4 / RHOBTB2 GTPase cycle / axonal growth cone / Purinergic signaling in leishmaniasis infection / DNA polymerase binding / supramolecular fiber organization / chaperone-mediated protein folding / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / protein folding chaperone / heat shock protein binding / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / nitric-oxide synthase regulator activity / Constitutive Signaling by Overexpressed ERBB2 / ESR-mediated signaling / Signaling by ERBB2 TMD/JMD mutants / Meiotic recombination / peptide binding / Constitutive Signaling by EGFRvIII / Signaling by ERBB2 ECD mutants / Ubiquitin-dependent degradation of Cyclin D / Signaling by ERBB2 KD Mutants / placenta development / positive regulation of cell differentiation / Transcriptional regulation of granulopoiesis / SCF(Skp2)-mediated degradation of p27/p21 / ATP-dependent protein folding chaperone / kinase binding / Downregulation of ERBB2 signaling / Hsp90 protein binding / DDX58/IFIH1-mediated induction of interferon-alpha/beta / tau protein binding
類似検索 - 分子機能
Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain ...Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain / Cdc37 N terminal kinase binding / : / Heat shock protein Hsp90, conserved site / Heat shock hsp90 proteins family signature. / HSP90, C-terminal domain / Heat shock protein Hsp90, N-terminal / Heat shock protein Hsp90 family / Hsp90 protein / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
類似検索 - ドメイン・相同性
ADENOSINE-5'-TRIPHOSPHATE / Heat shock protein HSP 90-beta / Cyclin-dependent kinase 4 / Hsp90 co-chaperone Cdc37
類似検索 - 構成要素
生物種HOMO SAPIENS (ヒト)
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: 7.2 Å
データ登録者Verba, K.A. / Wang, R.Y.R. / Arakawa, A. / Liu, Y. / Yokoyama, S. / Agard, D.A.
引用ジャーナル: Science / 年: 2016
タイトル: Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.
著者: Kliment A Verba / Ray Yu-Ruei Wang / Akihiko Arakawa / Yanxin Liu / Mikako Shirouzu / Shigeyuki Yokoyama / David A Agard /
要旨: The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" ...The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.
履歴
登録2016年2月18日登録サイト: PDBE / 処理サイト: PDBE
改定 1.02016年10月26日Provider: repository / タイプ: Initial release
改定 1.12017年8月2日Group: Data collection / カテゴリ: em_image_scans / em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
改定 2.02019年10月23日Group: Atomic model / Data collection / Derived calculations
カテゴリ: atom_site / struct_conn
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _struct_conn.pdbx_leaving_atom_flag
改定 2.12019年12月18日Group: Other / カテゴリ: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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構造の表示

ムービー
  • 登録構造単位
  • Jmolによる作画
  • ダウンロード
  • EMマップとの重ね合わせ
  • マップデータ: EMDB-3340
  • UCSF Chimeraによる作画
  • ダウンロード
ムービービューア
構造ビューア分子:
MolmilJmol/JSmol

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集合体

登録構造単位
A: HEAT SHOCK PROTEIN HSP 90 BETA
B: HEAT SHOCK PROTEIN HSP 90 BETA
E: HSP90 CO-CHAPERONE CDC37
K: CYCLIN-DEPENDENT KINASE 4
ヘテロ分子


分子量 (理論値)分子数
合計 (水以外)247,4978
ポリマ－246,4344
非ポリマー1,0634
水00
1


  • 登録構造と同一
  • 登録者が定義した集合体
タイプ名称対称操作数
identity operation1_555x,y,z1

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要素

#1: タンパク質 HEAT SHOCK PROTEIN HSP 90 BETA / HSP 90 / HEAT SHOCK 84 KDA / HSP 84 / HSP84 / HEAT SHOCK PROTEI N HSP 90 BETA


分子量: 83645.539 Da / 分子数: 2 / 由来タイプ: 組換発現 / 詳細: ATP / 由来: (組換発現) HOMO SAPIENS (ヒト) / プラスミド: PFASTBACHT
発現宿主: SPODOPTERA FRUGIPERDA (ツマジロクサヨトウ)
参照: UniProt: P08238
#2: タンパク質 HSP90 CO-CHAPERONE CDC37 / HSP90 CHAPERONE PROTEIN KINASE-TARGETING SUBUNIT / P50CDC37


分子量: 44622.363 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) HOMO SAPIENS (ヒト) / プラスミド: PFASTBACHT
発現宿主: SPODOPTERA FRUGIPERDA (ツマジロクサヨトウ)
参照: UniProt: Q16543
#3: タンパク質 CYCLIN-DEPENDENT KINASE 4 / CELL DIVISION PROTEIN KINASE 4 / PSK-J3


分子量: 34520.629 Da / 分子数: 1 / Fragment: KINASE DOMAIN / 由来タイプ: 組換発現 / 由来: (組換発現) HOMO SAPIENS (ヒト) / プラスミド: PFASTBACHT
発現宿主: SPODOPTERA FRUGIPERDA (ツマジロクサヨトウ)
参照: UniProt: P11802
#4: 化合物 ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / ATP


分子量: 507.181 Da / 分子数: 2 / 由来タイプ: 合成 / 式: C10H16N5O13P3 / コメント: ATP, エネルギー貯蔵分子*YM
#5: 化合物 ChemComp-MG / MAGNESIUM ION / マグネシウムジカチオン


分子量: 24.305 Da / 分子数: 2 / 由来タイプ: 合成 / 式: Mg

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実験情報

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実験

実験手法: 電子顕微鏡法
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
ID名称Parent-ID由来
1COMPLEX OF HUMAN HSP90BETA, HUMAN CDC37 AND HUMAN CDK40MULTIPLE SOURCES
2HUMAN HSP90BETA1RECOMBINANT
4HUMAN CDC371RECOMBINANT
3HUMAN CDC371RECOMBINANT
分子量値: 0.245 MDa / 実験値: NO
緩衝液pH: 7.5
詳細: 20mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM KCl, 10 mM MgCl2, 20 mM Na2MoO4, 2mM DTT, 0.085mM DDM
試料濃度: 0.27 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
試料支持詳細: HOLEY CARBON
急速凍結装置: FEI VITROBOT MARK III / 凍結剤: ETHANE / Temp: 95 K / 湿度: 90 % / 凍結前の試料温度: 293 K / 詳細: LIQUID ETHANE / 手法: Single blot from 4 to 6 seconds, at 20C

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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
顕微鏡モデル: FEI TITAN KRIOS / 日付: 2014年11月25日
電子銃電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: OTHER
電子レンズモード: BRIGHT FIELD / 倍率（公称値）: 22500 X / 最大 デフォーカス（公称値）: 3800 nm / 最小 デフォーカス（公称値）: 1400 nm / Cs: 2.7 mm
試料ホルダ試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER
撮影平均露光時間: 7.6 sec. / 電子線照射量: 44 e/Å2
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k)
実像数: 3718 / 詳細: 38 frames

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解析

EMソフトウェア
ID名称カテゴリ
1Rosettaモデル精密化
2UCSF Chimeraモデル精密化
3RELION３次元再構成
画像処理詳細: Image stacks were corrected for motion and summed as described previously, resulting in binned sums (1.315A/pix). For particle picking the images were binned to 5.2A/pix and Gaussian bandpass ...詳細: Image stacks were corrected for motion and summed as described previously, resulting in binned sums (1.315A/pix). For particle picking the images were binned to 5.2A/pix and Gaussian bandpass filtered between 15A and 500A using EMAN2. SamViewer template based picking was then used to pick particles from all the micrographs, followed by manual review of all the picks. After such procedure 802877 particles were picked in total and extracted from images binned to 2.6A/pix. CTFFIND4 was used to estimate defocus parameters for all the images. Relion 1.4 was used for all the following steps unless noted otherwise. Reference free 2D classification into 300 classes for 75 iterations was performed followed by manual examination of the resulting class averages. Low resolution/signal to noise/feature class averages and contributing particles were discarded, resulting in 670000 particles left. The resulting particles were 3D classified into 4 classes resulting in two classes having high-resolution features (390000 particles). At this stage particles were extracted from 1.315A/pix micrographs and all the following processing was done with these particles. Using 3D Auto-refine in Relion 1.4, a reconstruction was obtained from 390000 particles resulting from 3D classification above (using highest resolution 3D class as initial model, low pass filtered to 20A). Using the resulting parameters, the particles were further drift corrected per particle and dose weighted using the Particle Polishing feature. The B-factor weighing curve was fit by a polynomial (with a rationale that such a curve should be smooth) and used to generate new weighting parameters for Particle Polishing, with which 390000 particles were then polished. All further data processing was done using the polished particles. Re-refinement of the 390000 particles after polishing yielded the map at about 4A resolution (determined using gold standard FSC in the PostProcessing tab). The 390000 particles were then 3D classified into four different classes without particle re-alignment, using the alignment parameters from 4A reconstruction. Particles contributing to each of the four classes were grouped and a full 3D refinement with a spherical 200A mask was performed with each of the four groups of particles using the same initial model, low pass filtered to 20A. This is one of the resulting reconstructions.
粒子像の選択選択した粒子像数: 114683
対称性点対称性: C1 (非対称)
3次元再構成手法: FSC 0.143 CUT-OFF / 解像度: 7.2 Å / 粒子像の数: 114683 / ピクセルサイズ（実測値）: 1.315 Å
詳細: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3340. (DEPOSITION ID: 14281).
Refinement type: HALF-MAPS REFINED INDEPENDENTLY / 対称性のタイプ: POINT
原子モデル構築B value: 141 / プロトコル: FLEXIBLE FIT / 空間: REAL
詳細: Cdc37 crystal structure from PDB:1US7 was fit into EMD-3340 manually and then with UCSF Chimera "Fit In Map" tool. The model was truncated at residue 260, as there was no reliable density for ...詳細: Cdc37 crystal structure from PDB:1US7 was fit into EMD-3340 manually and then with UCSF Chimera "Fit In Map" tool. The model was truncated at residue 260, as there was no reliable density for the rest of the crystal structure. PDB:5FWK and the above fit crystal structure for residues 148-260 were loaded in Coot. The residues 133-147 were built in by hand into the Cdc37 Reconstruction and residues 245-260 (helix) were rotated as a rigid body. To relieve atomic clashes or bond length/angle distortions at the linker regions, the resulting model was subjected to "Cartesian space relax" protocol within EMD-3340 map using Rosetta. Final model was selected using the combined score of Rosetta all-atom physically-realistic score and electron density score.
原子モデル構築
ID 3D fitting-ID詳細
115FWK
211US7
精密化最高解像度: 7.2 Å
精密化ステップサイクル: LAST / 最高解像度: 7.2 Å
タンパク質核酸リガンド溶媒全体
原子数14123 0 64 0 14187

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