+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-2605 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
タイトル | Cryo-EM structures of the 50S ribosome subunit bound with ObgE | |||||||||
マップデータ | Reconstruction of 50S-ObgE complex | |||||||||
試料 |
| |||||||||
キーワード | (p)ppGpp / Obg / ribosome assembly / stringent response / GTPase | |||||||||
機能・相同性 | 機能・相同性情報 guanyl ribonucleotide binding / dormancy process / negative regulation of ribosome biogenesis / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis ...guanyl ribonucleotide binding / dormancy process / negative regulation of ribosome biogenesis / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / ribosomal large subunit assembly / chromosome segregation / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / 加水分解酵素; 酸無水物に作用; GTPに作用・細胞または細胞小器官の運動に関与 / response to radiation / mRNA 5'-UTR binding / GDP binding / large ribosomal subunit / ribosome binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / rRNA binding / negative regulation of translation / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / GTPase activity / mRNA binding / GTP binding / magnesium ion binding / DNA binding / RNA binding / zinc ion binding / cytoplasm / cytosol 類似検索 - 分子機能 | |||||||||
生物種 | Escherichia coli K-12 (大腸菌) | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 5.5 Å | |||||||||
データ登録者 | Feng B / Mandava CS / Guo Q / Wang J / Cao W / Li N / Zhang Y / Wang Z / Wu J / Sanyal S ...Feng B / Mandava CS / Guo Q / Wang J / Cao W / Li N / Zhang Y / Wang Z / Wu J / Sanyal S / Lei J / Gao N | |||||||||
引用 | ジャーナル: PLoS Biol / 年: 2014 タイトル: Structural and functional insights into the mode of action of a universally conserved Obg GTPase. 著者: Boya Feng / Chandra Sekhar Mandava / Qiang Guo / Jie Wang / Wei Cao / Ningning Li / Yixiao Zhang / Yanqing Zhang / Zhixin Wang / Jiawei Wu / Suparna Sanyal / Jianlin Lei / Ning Gao / 要旨: Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed ...Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p)ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p)ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues. | |||||||||
履歴 |
|
-構造の表示
ムービー |
ムービービューア |
---|---|
構造ビューア | EMマップ: SurfViewMolmilJmol/JSmol |
添付画像 |
-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_2605.map.gz | 59 MB | EMDBマップデータ形式 | |
---|---|---|---|---|
ヘッダ (付随情報) | emd-2605-v30.xml emd-2605.xml | 11.7 KB 11.7 KB | 表示 表示 | EMDBヘッダ |
画像 | EMD-2605.png | 119.6 KB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-2605 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2605 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_2605_validation.pdf.gz | 253.1 KB | 表示 | EMDB検証レポート |
---|---|---|---|---|
文書・詳細版 | emd_2605_full_validation.pdf.gz | 252.2 KB | 表示 | |
XML形式データ | emd_2605_validation.xml.gz | 5.6 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2605 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2605 | HTTPS FTP |
-関連構造データ
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
---|---|
「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_2605.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 50S-ObgE complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.5 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
|
-添付データ
-試料の構成要素
-全体 : 50S-ObgE complex
全体 | 名称: 50S-ObgE complex |
---|---|
要素 |
|
-超分子 #1000: 50S-ObgE complex
超分子 | 名称: 50S-ObgE complex / タイプ: sample / ID: 1000 / Number unique components: 2 |
---|---|
分子量 | 実験値: 1.5 MDa / 理論値: 1.5 MDa |
-超分子 #1: prokaryotic 50S ribosome subunit
超分子 | 名称: prokaryotic 50S ribosome subunit / タイプ: complex / ID: 1 / Name.synonym: 50S subunit / 組換発現: No / Ribosome-details: ribosome-prokaryote: LSU 50S |
---|---|
由来(天然) | 生物種: Escherichia coli K-12 (大腸菌) / 株: K12 / 細胞中の位置: cytoplasm |
分子量 | 実験値: 1.5 MDa / 理論値: 1.5 MDa |
-分子 #1: ObgE
分子 | 名称: ObgE / タイプ: protein_or_peptide / ID: 1 / Name.synonym: CgtAE / コピー数: 1 / 集合状態: monomer / 組換発現: Yes |
---|---|
由来(天然) | 生物種: Escherichia coli K-12 (大腸菌) / 株: K12 |
分子量 | 実験値: 50 KDa / 理論値: 43 KDa |
組換発現 | 生物種: Escherichia coli BL21 (大腸菌) / 組換株: BL21 / 組換プラスミド: pET28a |
-実験情報
-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 |
-試料調製
緩衝液 | pH: 7.5 / 詳細: 20mM Tris-HCl, 100mM NH4Cl, 10mM MgCl2 |
---|---|
グリッド | 詳細: 200 mesh copper grid with thin carbon support, glow discharged |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / 装置: FEI VITROBOT MARK IV / 手法: Blot for 1 seconds before plunging |
-電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
---|---|
日付 | 2011年7月27日 |
撮影 | カテゴリ: CCD / フィルム・検出器のモデル: FEI EAGLE (4k x 4k) / 平均電子線量: 20 e/Å2 |
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: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 最大 デフォーカス(公称値): 4.0 µm / 最小 デフォーカス(公称値): 1.0 µm / 倍率(公称値): 59000 |
試料ステージ | 試料ホルダー: Liquid Nitrogen cooled 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 |
-画像解析
詳細 | Particles were first picked using a method based on a locally normalized cross-correlation function, subjected to correspondence analysis and then manually verified. Then all particles were first classified in two groups, according to the presence or absence of ObgE on the 50S subunit using a modified supervised classification method. The resulting ObgE-containing particles were further applied to another round of 3D classification using RELION. The particles were finally split into four groups in 30 iterations using a final angle sampling of 1.8 degree. One of the four groups was used for final refinement. The refinement was performed using RELION. Amplitude correction using the B-factor sharpening approach was applied to the final volume. |
---|---|
CTF補正 | 詳細: Each particle |
最終 再構成 | 想定した対称性 - 点群: C1 (非対称) / アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 5.5 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: 102814 |
-原子モデル構築 1
初期モデル | PDB ID: 3ofc |
---|---|
ソフトウェア | 名称: MDFF |
精密化 | 空間: REAL / プロトコル: FLEXIBLE FIT |
得られたモデル | PDB-4csu: |