EMDB-5800: Structure of the Ribosome with Elongation Factor G Trapped in the...

基本情報

• 登録情報: データベース: EMDB / ID: EMD-5800
• タイトル: Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State
• マップデータ: Reconstruction of a pre-translocation ribosome with EF-G bound

試料

• 試料: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and P/E hybrid state tRNA
• 複合体: 70S ribosome
• タンパク質・ペプチド: Elongation Factor G
• RNA: Transfer RNA
• RNA: Messenger RNA
• リガンド: Viomycin

• キーワード: protein structure / translation / EF-G / electron cryo-microscopy / single particle analysis

機能・相同性

分子機能:

intracellular anatomical structure / ribosome disassembly / guanosine tetraphosphate binding / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / translational elongation / translation elongation factor activity / transcriptional attenuation / positive regulation of ribosome biogenesis / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / translational termination / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / regulation of mRNA stability / negative regulation of DNA-templated DNA replication initiation / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / ribosome assembly / transcription elongation factor complex / regulation of DNA-templated transcription elongation / cytosolic ribosome assembly / response to reactive oxygen species / DNA endonuclease activity / transcription antitermination / regulation of cell growth / DNA-templated transcription termination / response to radiation / maintenance of translational fidelity / mRNA 5'-UTR binding / regulation of translation / ribosome biogenesis / large ribosomal subunit / transferase activity / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / small ribosomal subunit rRNA binding / 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 / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / GTPase activity / mRNA binding / GTP binding / protein homodimerization activity / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm

ドメイン・相同性:

Translation elongation factor EFG/EF2 / Translation elongation factor EFG/EF2 / : / Elongation factor G, domain III / EFG, domain V / Ribosomal protein L7/L12, oligomerisation / Ribosomal protein L7/L12, oligomerisation domain superfamily / Ribosomal protein L7/L12 dimerisation domain / Ribosomal protein L7/L12 / Ribosomal protein L7/L12, C-terminal / Ribosomal protein L7/L12 C-terminal domain / Ribosomal protein L7/L12, C-terminal/adaptor protein ClpS-like / Ribosomal protein L1, bacterial-type / Elongation Factor G, domain II / Elongation Factor G, domain III / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / Ribosomal protein L10 / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / EF-G domain III/V-like / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / : / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Translation elongation factor EFTu-like, domain 2 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L11, bacterial-type / Elongation factor Tu domain 2 / Ribosomal protein L25, short-form / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ribosomal protein S21 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / : / Ribosomal protein L16 signature 1. / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 2. / Ribosomal protein L16, conserved site / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / : / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L17 signature. / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L28 / Ribosomal protein L35, non-mitochondrial / : / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / : / Ribosomal protein S19, bacterial-type

構成要素:

Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Elongation factor G / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL12 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25

• 生物種: Escherichia coli (大腸菌) / synthetic construct (人工物) / unidentified (未定義)
• 手法: 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 7.6 Å
• データ登録者: Brilot AF / Korostelev AA / Ermolenko DN / Grigorieff N
• 引用: ジャーナル: Proc Natl Acad Sci U S A / 年: 2013; タイトル: Structure of the ribosome with elongation factor G trapped in the pretranslocation state.; 著者: Axel F Brilot / Andrei A Korostelev / Dmitri N Ermolenko / Nikolaus Grigorieff / ; 要旨: During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by a universally conserved ribosome-dependent GTPase [elongation factor G (EF-G) in prokaryotes and elongation factor 2 (EF-2) in eukaryotes]. Although the high-resolution structure of EF-G bound to the posttranslocation ribosome has been determined, the pretranslocation conformation of the ribosome bound with EF-G and A-site tRNA has evaded visualization owing to the transient nature of this state. Here we use electron cryomicroscopy to determine the structure of the 70S ribosome with EF-G, which is trapped in the pretranslocation state using antibiotic viomycin. Comparison with the posttranslocation ribosome shows that the small subunit of the pretranslocation ribosome is rotated by ∼12° relative to the large subunit. Domain IV of EF-G is positioned in the cleft between the body and head of the small subunit outwardly of the A site and contacts the A-site tRNA. Our findings suggest a model in which domain IV of EF-G promotes the translocation of tRNA from the A to the P site as the small ribosome subunit spontaneously rotates back from the hybrid, rotated state into the nonrotated posttranslocation state.

履歴

登録	2013年11月19日	-
ヘッダ(付随情報) 公開	2013年12月18日	-
マップ公開	2013年12月18日	-
更新	2014年7月23日	-
現状	2014年7月23日	処理サイト: RCSB / 状態: 公開

マップ

• ファイル: ダウンロード / ファイル: emd_5800.map.gz / 形式: CCP4 / 大きさ: 122.1 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• 注釈: Reconstruction of a pre-translocation ribosome with EF-G bound
• ボクセルのサイズ: X=Y=Z: 1.04 Å

密度

表面レベル	登録者による: 0.32 / ムービー #1: 0.32
最小 - 最大	-0.45880854 - 1.44600463
平均 (標準偏差)	-0.02902501 (±0.17701891)

• 対称性: 空間群: 1

詳細

EMDB XML:

マップ形状	Axis order X Y Z Origin 0 0 0 サイズ 320 320 320 Spacing 320 320 320
セル	A=B=C: 332.8 Å α=β=γ: 90.0 °

CCP4マップ ヘッダ情報:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.04	1.04	1.04
M x/y/z	320	320	320
origin x/y/z	0.000	0.000	0.000
length x/y/z	332.800	332.800	332.800
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-95	-75	153
NX/NY/NZ	200	200	200
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	320	320	320
D min/max/mean	-0.459	1.446	-0.029

添付データ

試料の構成要素

全体 : Pre-translocation 70S ribosome with bound EF-G and bound A/P* and...

• 全体: 名称: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and P/E hybrid state tRNA

要素

• 試料: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and P/E hybrid state tRNA
• 複合体: 70S ribosome
• タンパク質・ペプチド: Elongation Factor G
• RNA: Transfer RNA
• RNA: Messenger RNA
• リガンド: Viomycin

超分子 #1000: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and...

• 超分子: 名称: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and P/E hybrid state tRNA; タイプ: sample / ID: 1000 / 詳細: Sample was monodisperse / Number unique components: 5
• 分子量: 理論値: 3 MDa

超分子 #1: 70S ribosome

• 超分子: 名称: 70S ribosome / タイプ: complex / ID: 1 / 組換発現: No / データベース: NCBI / Ribosome-details: ribosome-prokaryote: ALL
• 由来(天然): 生物種: Escherichia coli (大腸菌) / 株: MRE600
• 分子量: 理論値: 3 MDa

分子 #1: Elongation Factor G

• 分子: 名称: Elongation Factor G / タイプ: protein_or_peptide / ID: 1 / Name.synonym: EF-G / コピー数: 1 / 集合状態: monomer / 組換発現: Yes
• 由来(天然): 生物種: Escherichia coli (大腸菌) / 株: K-12
• 分子量: 理論値: 78 KDa
• 組換発現: 生物種: Escherichia coli (大腸菌) / 組換株: BL21 / 組換プラスミド: pET24b
• 配列: UniProtKB: Elongation factor G; GO: translational elongation, GTP binding, translation elongation factor activity, intracellular anatomical structure; InterPro: Translation elongation factor EFG/EF2

分子 #2: Transfer RNA

• 分子: 名称: Transfer RNA / タイプ: rna / ID: 2 / Name.synonym: tRNA / 詳細: Two tRNA (P and E site) / 分類: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
• 由来(天然): 生物種: Escherichia coli (大腸菌) / 株: K-12
• 分子量: 実験値: 25 KDa

分子 #3: Messenger RNA

• 分子: 名称: Messenger RNA / タイプ: rna / ID: 3 / Name.synonym: mRNA / 分類: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
• 由来(天然): 生物種: synthetic construct (人工物)
• 分子量: 理論値: 12 KDa

配列

文字列:

GGCAAGGAGG UAAAAAUGUU UAAACGUAAA UCUACU

分子 #4: Viomycin

• 分子: 名称: Viomycin / タイプ: ligand / ID: 4 / Name.synonym: Vio / コピー数: 1 / 組換発現: No / データベース: NCBI
• 由来(天然): 生物種: unidentified (未定義)
• 分子量: 理論値: 1 KDa

Chemical component information

化合物 画像なし

ChemComp-PRD_000226:
Unknown entry

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

• 濃度: 0.4 mg/mL
• 緩衝液: pH: 7.6 ; 詳細: 10 mM HEPES-KOH, 5 mM MgCl2, 90 mM NH4Cl, 2 mM spermidine, 0.1 mM spermine, 6 mM BME, 0.5 mM viomycin, 0.5 mM GTP, 0.5 mM fusidic acid
• グリッド: 詳細: C-flat 1.2/1.3 holey carbon 400 mesh copper grid, glow discharged with a current of -20 mA for 45 seconds in an EMITECH K100X glow discharge unit
• 凍結: 凍結剤: ETHANE / チャンバー内湿度: 95 % / 装置: FEI VITROBOT MARK II; 手法: Freshly glow-disharged grids were loaded into an FEI Mark II Vitrobot and equilibrated to 95% relative humidity at 22 degrees Celsius. 2 microliters of sample was applied through the side port, blotted for 7 seconds with a positional offset of 2, and plunged into liquid ethane.

電子顕微鏡法

• 顕微鏡: FEI TITAN KRIOS
• アライメント法: Legacy - 非点収差: Automatically corrected using FEI software
• 日付: 2012年11月2日
• 撮影: カテゴリ: CCD; フィルム・検出器のモデル: FEI FALCON I (4k x 4k); デジタル化 - サンプリング間隔: 14.0 µm / 実像数: 13341 / 平均電子線量: 30 e/Ų / ビット/ピクセル: 16
• 電子線: 加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
• 電子光学系: 倍率（補正後）: 134615 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 0.01 mm / 最大 デフォーカス（公称値）: 6.95 µm / 最小 デフォーカス（公称値）: 1.15 µm / 倍率（公称値）: 133333
• 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

画像解析

• 詳細: Refinement and 3D classification performed by Frealign. See primary citation Supplementary Information for details.
• CTF補正: 詳細: CTFFIND3, FREALIGN per micrograph
• 最終 再構成: アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 7.6 Å / 解像度の算出法: OTHER; ソフトウェア - 名称: EMAN2, IMAGIC, FREALIGN, RSAMPLE, CTFFIND3; 詳細: Refinement included data to 12 Angstrom resolution to limit FSC bias. See primary citation Supplementary Information for details.; 使用した粒子像数: 85115

原子モデル構築 1

初期モデル

PDB ID:

4gd1
PDB 未公開エントリ

• ソフトウェア: 名称: Chimera, CNS
• 詳細: Chain Y not used in refinement. See primary citation Supplementary Information for details.
• 精密化: 空間: REAL / プロトコル: RIGID BODY FIT / 当てはまり具合の基準: Cross-correlation

得られたモデル

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

原子モデル構築 2

初期モデル

PDB ID:

3r8s
PDB 未公開エントリ

• ソフトウェア: 名称: Chimera, CNS
• 詳細: See primary citation Supplementary Information for details.
• 精密化: 空間: REAL / プロトコル: RIGID BODY FIT / 当てはまり具合の基準: Cross-correlation

得られたモデル

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

原子モデル構築 3

初期モデル

PDB ID:

2bm0

• ソフトウェア: 名称: Chimera, CNS
• 詳細: A homology model of E. coli EF-G was created using the SWISS-MODEL from T. thermophilus EF-G. See primary citation Supplementary Information for details.
• 精密化: 空間: REAL / プロトコル: RIGID BODY FIT / 当てはまり具合の基準: Cross-correlation

得られたモデル

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

原子モデル構築 4

初期モデル

PDB ID:

1mms

• ソフトウェア: 名称: Chimera, CNS
• 詳細: Used to rebuild L11 stalk.
• 精密化: 空間: REAL / プロトコル: RIGID BODY FIT / 当てはまり具合の基準: Cross-correlation

得られたモデル

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

原子モデル構築 5

初期モデル

PDB ID:

1dd3

• ソフトウェア: 名称: Chimera, CNS
• 詳細: Used to rebuild L12.
• 精密化: 空間: REAL / プロトコル: RIGID BODY FIT / 当てはまり具合の基準: Cross-correlation

得られたモデル

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

原子モデル構築 6

初期モデル

PDB ID:

3u4m

• ソフトウェア: 名称: Chimera, CNS
• 詳細: Used to build homology model for L1.
• 精密化: 空間: REAL / プロトコル: RIGID BODY FIT / 当てはまり具合の基準: Cross-correlation

得られたモデル

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

原子モデル構築 7

初期モデル

PDB ID:

1zav

• ソフトウェア: 名称: Chimera, CNS
• 詳細: Used to build homology model for L10.
• 精密化: 空間: REAL / プロトコル: RIGID BODY FIT / 当てはまり具合の基準: Cross-correlation

得られたモデル

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)