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- EMDB-3337: 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: EMDB / ID: EMD-3337
タイトルAtomic cryoEM structure of Hsp90/Cdc37/Cdk4 complex
マップデータReconstruction of Hsp90:Cdc37:Cdk4. Part of series of maps, the highest resolution one out of the series.
試料
  • 試料: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4
  • タンパク質・ペプチド: Heat Shock Protein HSP 90 betaHeat shock response
  • タンパク質・ペプチド: Hsp90 co-chaperone Cdc37
  • タンパク質・ペプチド: Cyclin-dependent kinase 4サイクリン依存性キナーゼ4
キーワードHsp90 (Hsp90) / Cdc37 / Cdk4 (サイクリン依存性キナーゼ4) / chaperone (シャペロン) / kinase (キナーゼ) / 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 / citrulline metabolic process / regulation of transcription initiation by RNA polymerase II / regulation of type II interferon-mediated signaling pathway / Drug-mediated inhibition of CDK4/CDK6 activity ...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 / citrulline metabolic process / regulation of transcription initiation by RNA polymerase II / regulation of type II interferon-mediated signaling pathway / Drug-mediated inhibition of CDK4/CDK6 activity / 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 / : / very long-chain fatty acid metabolic process / HSP90-CDC37 chaperone complex / positive regulation of cyclin-dependent protein kinase activity / positive regulation of mitophagy in response to mitochondrial depolarization / Aryl hydrocarbon receptor signalling / negative regulation of proteasomal protein catabolic process / dynein axonemal particle / aryl hydrocarbon receptor complex / histone methyltransferase binding / Transcriptional regulation by RUNX2 / cellular response to phorbol 13-acetate 12-myristate / mitochondrial genome maintenance / protein kinase regulator activity / positive regulation of protein localization to cell surface / ATP-dependent protein binding / protein folding chaperone complex / negative regulation of protein metabolic process / cyclin-dependent protein serine/threonine kinase regulator activity / positive regulation of tau-protein kinase activity / post-transcriptional regulation of gene expression / telomerase holoenzyme complex assembly / Uptake and function of diphtheria toxin / 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 / PTK6 Regulates Cell Cycle / positive regulation of transforming growth factor beta receptor signaling pathway / regulation of cyclin-dependent protein serine/threonine kinase activity / dendritic growth cone / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / regulation of type I interferon-mediated signaling pathway / positive regulation of phosphoprotein phosphatase activity / Sema3A PAK dependent Axon repulsion / The NLRP3 inflammasome / regulation of protein ubiquitination / HSF1-dependent transactivation / telomere maintenance via telomerase / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / response to unfolded protein / bicellular tight junction / cyclin-dependent protein kinase holoenzyme complex / HSF1 activation / protein targeting / chaperone-mediated protein complex assembly / Attenuation phase / サイクリン依存性キナーゼ / RHOBTB2 GTPase cycle / cyclin-dependent protein serine/threonine kinase activity / Purinergic signaling in leishmaniasis infection / DNA polymerase binding / supramolecular fiber organization / axonal growth cone / Signaling by ERBB2 / positive regulation of telomerase activity / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / heat shock protein binding / positive regulation of G2/M transition of mitotic cell cycle / regulation of G2/M transition of mitotic cell cycle / cellular response to interleukin-4 / nitric-oxide synthase regulator activity / cyclin binding / Constitutive Signaling by Overexpressed ERBB2 / ESR-mediated signaling / placenta development / response to organic substance / Ubiquitin-dependent degradation of Cyclin D / positive regulation of cell differentiation / peptide binding / ATP-dependent protein folding chaperone / Signaling by ERBB2 TMD/JMD mutants / Hsp90 protein binding / G1/S transition of mitotic cell cycle / tau protein binding / DDX58/IFIH1-mediated induction of interferon-alpha/beta / Constitutive Signaling by EGFRvIII / Signaling by ERBB2 ECD mutants / Oncogene Induced Senescence / Signaling by ERBB2 KD Mutants
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 / Heat shock protein Hsp90 family / Hsp90 protein / 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. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase domain / Protein kinase-like domain superfamily
類似検索 - ドメイン・相同性
Heat shock protein HSP 90-beta / サイクリン依存性キナーゼ4 / Hsp90 co-chaperone Cdc37
類似検索 - 構成要素
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: 3.9 Å
データ登録者Verba KA / Wang RYR / Arakawa A / Liu Y / Shirouzu M / Yokoyama S / Agard DA
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17日-
ヘッダ(付随情報) 公開2016年3月9日-
マップ公開2016年6月29日-
更新2016年7月20日-
現状2016年7月20日処理サイト: PDBe / 状態: 公開

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構造の表示

ムービー
  • 表面図（断面を密度値に従い着色）
  • 表面レベル: 0.02
  • UCSF Chimeraによる作画
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  • 表面図（半径に従い着色）
  • 表面レベル: 0.02
  • UCSF Chimeraによる作画
  • ダウンロード
  • あてはめたモデルとの重ね合わせ
  • 原子モデル: PDB-5fwk
  • 表面レベル: 0.02
  • 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EMマップ:
SurfViewMolmilJmol/JSmol
添付画像

emd_3337.tif

ダウンロードとリンク

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マップ

ファイルダウンロード / ファイル: emd_3337.map.gz / 形式: CCP4 / 大きさ: 62.5 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈Reconstruction of Hsp90:Cdc37:Cdk4. Part of series of maps, the highest resolution one out of the series.
ボクセルのサイズX=Y=Z: 1.315 Å
密度
表面レベル登録者による: 0.02 / ムービー #1: 0.02
最小 - 最大-0.05036226 - 0.10603088
平均 (標準偏差)0.00003407 (±0.00230758)
対称性空間群: 1
詳細

EMDB XML:

マップ形状
Axis orderXYZ
Origin000
サイズ256256256
Spacing256256256
セルA=B=C: 336.64 Å
α=β=γ: 90.0 °

CCP4マップ ヘッダ情報:

modeImage stored as Reals
Å/pix. X/Y/Z1.3151.3151.315
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z336.640336.640336.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0500.1060.000

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添付データ

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試料の構成要素

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全体 : Complex of Human Hsp90 beta, human Cdc37 and human Cdk4

全体名称: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4
要素
  • 試料: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4
  • タンパク質・ペプチド: Heat Shock Protein HSP 90 betaHeat shock response
  • タンパク質・ペプチド: Hsp90 co-chaperone Cdc37
  • タンパク質・ペプチド: Cyclin-dependent kinase 4サイクリン依存性キナーゼ4

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超分子 #1000: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4

超分子名称: Complex of Human Hsp90 beta, human Cdc37 and human Cdk4
タイプ: sample / ID: 1000 / 詳細: All three proteins were co-expressed in Sf9 cells.
集合状態: One Hsp90 homodimer binds to one Cdc37 and one Cdk4
Number unique components: 3
分子量実験値: 245 KDa / 理論値: 245 KDa / 手法: As cloned, verified by SDS-PAGE

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分子 #1: Heat Shock Protein HSP 90 beta

分子名称: Heat Shock Protein HSP 90 beta / タイプ: protein_or_peptide / ID: 1 / Name.synonym: Hsp90 / コピー数: 2 / 集合状態: Dimer / 組換発現: Yes
由来(天然)生物種: Homo sapiens (ヒト) / 別称: Human / 細胞中の位置: cytoplasm
分子量理論値: 83 KDa
組換発現生物種: Spodoptera frugiperda (ツマジロクサヨトウ)
組換プラスミド: pFastBacHT
配列UniProtKB: Heat shock protein HSP 90-beta / GO: citrulline metabolic process / InterPro: Heat shock protein Hsp90 family

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分子 #2: Hsp90 co-chaperone Cdc37

分子名称: Hsp90 co-chaperone Cdc37 / タイプ: protein_or_peptide / ID: 2 / Name.synonym: Cdc37 / コピー数: 1 / 組換発現: Yes
由来(天然)生物種: Homo sapiens (ヒト) / 別称: Human / 細胞中の位置: throughout
分子量理論値: 44.5 KDa
組換発現生物種: Spodoptera frugiperda (ツマジロクサヨトウ)
組換プラスミド: pFastBacHT
配列UniProtKB: Hsp90 co-chaperone Cdc37 / GO: mitochondrial genome maintenance

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分子 #3: Cyclin-dependent kinase 4

分子名称: Cyclin-dependent kinase 4 / タイプ: protein_or_peptide / ID: 3 / Name.synonym: Cdk4 / コピー数: 1 / 組換発現: Yes
由来(天然)生物種: Homo sapiens (ヒト) / 別称: Human / 細胞中の位置: throughout
分子量理論値: 33.7 KDa
組換発現生物種: Spodoptera frugiperda (ツマジロクサヨトウ)
組換プラスミド: pFastBacHT
配列UniProtKB: サイクリン依存性キナーゼ4 / GO: very long-chain fatty acid metabolic process / InterPro: Protein kinase domain

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実験情報

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構造解析

手法クライオ電子顕微鏡法
解析単粒子再構成法
試料の集合状態particle

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試料調製

濃度0.27 mg/mL
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
グリッド詳細: Glow discharged for 30 sec, C-flat 400 mesh 1.2/1.3 thick carbon grids (Protochips)
凍結凍結剤: ETHANE / チャンバー内湿度: 90 % / チャンバー内温度: 95 K / 装置: FEI VITROBOT MARK III / 手法: Single blot from 4 to 6 seconds, at 20C

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電子顕微鏡法

顕微鏡FEI TITAN KRIOS
電子線加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系照射モード: OTHER / 撮影モード: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / 最大 デフォーカス（公称値）: 3.8 µm / 最小 デフォーカス（公称値）: 1.4 µm / 倍率（公称値）: 22500
試料ステージ試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER
アライメント法Legacy - 非点収差: At high mag via FT.
日付2014年11月25日
撮影カテゴリ: CCD
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k)
実像数: 3718 / 平均電子線量: 44 e/Å2 / 詳細: 38 frames, 7.6 seconds total exposure / ビット/ピクセル: 8
実験機器
モデル: Titan Krios / 画像提供: FEI Company

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画像解析

最終 2次元分類クラス数: 1
最終 再構成想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 3.9 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: Relion / 使用した粒子像数: 388688
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). Raw particles were sharpened with a B-factor of -50, low pass filtered with Gaussian filter to 3A and the refinement was continued for 10 more iterations (until convergence) with these particles (the rationale was that due to extremely low noise levels of K2 direct detector, this would yield more accurate alignments due to presence of more high resolution data in the images). This resulted in similar resolution but one with visually sharper features. Lastly, this reconstruction was tightly masked around Hsp90 region and refinement was further continued for 4 iterations (until convergence), with rationale that Hsp90 region is more coherent. This yielded a 3.9A reconstruction with the best density for Hsp90 core region.
FSC曲線 (解像度の算出)

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原子モデル構築 1

初期モデルPDB ID:

2cg9


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
ソフトウェア名称: Rosetta, MDFF, Chimera, COLORES (Situs)
詳細Atomic model building and refinement the Hsp90/Cdc37/Cdk4 complex was performed incrementally in five stages: 1) de novo model-building for Cdc37, 2) structure refinement of the Hsp90/Cdc37 complex, 3) de novo model extension for Cdk4 in the presence of the refined Hsp90/Cdc37 complex, and 4) structure refinement of the Hsp90/Cdc37/Cdk4 complex. The atomic structure of Hsp90/Cdc37/Cdk4 complex was used in the modelling of other low-resolution maps.
精密化空間: REAL / プロトコル: FLEXIBLE FIT / 温度因子: 95
当てはまり具合の基準: compound of Rosetta energy function and electron density fitting function
得られたモデル

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

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原子モデル構築 2

初期モデルPDB ID:

3g33


Chain - Chain ID: A
ソフトウェア名称: Rosetta, MDFF, Chimera, COLORES (Situs)
精密化空間: REAL / プロトコル: FLEXIBLE FIT / 温度因子: 95
当てはまり具合の基準: compound of Rosetta energy function and electron density fitting function
得られたモデル

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

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