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- EMDB-3340: 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-3340
タイトルAtomic cryoEM structure of Hsp90/Cdc37/Cdk4 complex
マップデータReconstruction of Hsp90:Cdc37:Cdk4 complex. Part of series of maps, the highest resolution map being EMD-3337. This is a different subclass from the same particles as in EMD-3337, having well defined Cdc37MC density.
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 beta
  • 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
キーワードHsp90 / Cdc37 / Cdk4 / chaperone / 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 / citrulline metabolic process / regulation of transcription initiation by RNA polymerase II / 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 / 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 / citrulline metabolic process / regulation of transcription initiation by RNA polymerase II / 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 / Drug-mediated inhibition of CDK4/CDK6 activity / regulation of type II interferon-mediated signaling pathway / regulation of type B pancreatic cell proliferation / very long-chain fatty acid metabolic process / HSP90-CDC37 chaperone complex / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / aryl hydrocarbon receptor complex / cellular response to ionomycin / histone methyltransferase binding / Transcriptional regulation by RUNX2 / dynein axonemal particle / cellular response to phorbol 13-acetate 12-myristate / : / receptor ligand inhibitor activity / positive regulation of type 2 mitophagy / regulation of cyclin-dependent protein serine/threonine kinase activity / ATP-dependent protein binding / positive regulation of protein localization to cell surface / protein kinase regulator activity / protein folding chaperone complex / cyclin-dependent protein serine/threonine kinase regulator activity / post-transcriptional regulation of gene expression / Respiratory syncytial virus genome replication / telomerase holoenzyme complex assembly / positive regulation of transforming growth factor beta receptor signaling pathway / 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 / PTK6 Regulates Cell Cycle / regulation of type I interferon-mediated signaling pathway / dendritic growth cone / TPR domain binding / Defective binding of RB1 mutants to E2F1,(E2F2, E2F3) / Assembly and release of respiratory syncytial virus (RSV) virions / The NLRP3 inflammasome / protein phosphatase activator activity / Sema3A PAK dependent Axon repulsion / regulation of protein ubiquitination / HSF1-dependent transactivation / positive regulation of G2/M transition of mitotic cell cycle / bicellular tight junction / response to unfolded protein / HSF1 activation / cyclin-dependent kinase / cyclin-dependent protein serine/threonine kinase activity / telomere maintenance via telomerase / Attenuation phase / chaperone-mediated protein complex assembly / protein targeting / axonal growth cone / RHOBTB2 GTPase cycle / Purinergic signaling in leishmaniasis infection / cyclin-dependent protein kinase holoenzyme complex / supramolecular fiber organization / : / regulation of G2/M transition of mitotic cell cycle / DNA polymerase binding / heat shock protein binding / Signaling by ERBB2 / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / protein folding chaperone / peptide binding / cellular response to interleukin-4 / cyclin binding / ESR-mediated signaling / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Constitutive Signaling by Overexpressed ERBB2 / placenta development / nitric-oxide synthase regulator activity / Ubiquitin-dependent degradation of Cyclin D / positive regulation of cell differentiation / Hsp90 protein binding / ATP-dependent protein folding chaperone / Signaling by ERBB2 TMD/JMD mutants / G1/S transition of mitotic cell cycle / Constitutive Signaling by EGFRvIII / Signaling by ERBB2 ECD mutants / Oncogene Induced Senescence / DDX58/IFIH1-mediated induction of interferon-alpha/beta / Signaling by ERBB2 KD Mutants / Regulation of actin dynamics for phagocytic cup formation / Transcriptional regulation of white adipocyte differentiation
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 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 / 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 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18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13日-
現状2016年7月13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: 0.0068
  • UCSF Chimeraによる作画
  • ダウンロード
  • 表面図（半径に従い着色）
  • 表面レベル: 0.0068
  • 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-5fwp
  • 表面レベル: 0.0068
  • 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_3340.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_3340.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 complex. Part of series of maps, the highest resolution map being EMD-3337. This is a different subclass from the same particles as in EMD-3337, having well defined Cdc37MC density.
投影像・断面図

画像のコントロール

大きさ
明度
コントラスト
その他
軸Z (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 256 pix.
= 336.64 Å		1.32 Å/pix.
x 256 pix.
= 336.64 Å		1.32 Å/pix.
x 256 pix.
= 336.64 Å

表面

投影像

断面 (1/3)

断面 (1/2)

断面 (2/3)

画像は Spider により作成

ボクセルのサイズX=Y=Z: 1.315 Å
密度

ヒストグラム

ヒストグラム (対数)
表面レベル登録者による: 0.0068 / ムービー #1: 0.0068
最小 - 最大-0.00687688 - 0.0346454
平均 (標準偏差)0.00000484 (±0.00119831)
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-0.0070.0350.000

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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 beta
  • 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

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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: GO: 0000002

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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: Cyclin-dependent kinase 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
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
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 FIELD / 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
実験機器
モデル: Titan Krios / 画像提供: FEI Company

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画像解析

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.
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: 7.2 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: 114683
最終 2次元分類クラス数: 1
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:

5fwk


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: E / Chain - #3 - Chain ID: K
ソフトウェア名称: Chimera, Rosetta
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.
精密化空間: REAL / プロトコル: FLEXIBLE FIT / 温度因子: 141
当てはまり具合の基準: cross correlation of fit into the density with Rosetta force field score.
得られたモデル

PDB-5fwp:
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:

1us7


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: E / Chain - #3 - Chain ID: K
ソフトウェア名称: Chimera, Rosetta
精密化空間: REAL / プロトコル: FLEXIBLE FIT / 温度因子: 141
当てはまり具合の基準: cross correlation of fit into the density with Rosetta force field score.
得られたモデル

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

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