EMDB-6647: Ensemble cryo-EM uncovers inchworm-like translocation of a viral ...

基本情報

• 登録情報: データベース: EMDB / ID: EMD-6647
• タイトル: Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome
• マップデータ: Saccharomyces cerevisiae 80S ribosome bound to eEF2-GDP-sordarin and TSV IRES, Structure V. For classification, 2D masking around the A site was used, including PKI of the IRES and domain IV of eEF2.

試料

• 試料: Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure V (least rotated 40S subunit)
• 複合体: Ribosome
• RNA: Internal Ribosome Entry Site
• タンパク質・ペプチド: eukaryotic elongation factor 2

• キーワード: cryo-EM / translocation / IRES / ribosome / eEF2

機能・相同性

分子機能:

Peptide chain elongation / Synthesis of diphthamide-EEF2 / positive regulation of translational elongation / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, LSU-rRNA,5S) / negative regulation of glucose mediated signaling pathway / negative regulation of translational frameshifting / Negative regulators of DDX58/IFIH1 signaling / RMTs methylate histone arginines / positive regulation of translational fidelity / Protein methylation / mTORC1-mediated signalling / Protein hydroxylation / ribosome-associated ubiquitin-dependent protein catabolic process / GDP-dissociation inhibitor activity / hexon binding / positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / nonfunctional rRNA decay / pre-mRNA 5'-splice site binding / preribosome, small subunit precursor / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Ribosomal scanning and start codon recognition / response to cycloheximide / Major pathway of rRNA processing in the nucleolus and cytosol / mRNA destabilization / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / negative regulation of mRNA splicing, via spliceosome / preribosome, large subunit precursor / regulation of amino acid metabolic process / L13a-mediated translational silencing of Ceruloplasmin expression / translational elongation / ribosomal large subunit export from nucleus / 90S preribosome / G-protein alpha-subunit binding / positive regulation of protein kinase activity / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of translational fidelity / Ub-specific processing proteases / protein-RNA complex assembly / translation elongation factor activity / ribosomal subunit export from nucleus / ribosomal small subunit export from nucleus / translation regulator activity / translational termination / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / DNA-(apurinic or apyrimidinic site) endonuclease activity / maturation of LSU-rRNA / Neutrophil degranulation / cellular response to amino acid starvation / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ribosome assembly / rescue of stalled ribosome / ribosomal large subunit biogenesis / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / small-subunit processome / translational initiation / protein kinase C binding / macroautophagy / maintenance of translational fidelity / modification-dependent protein catabolic process / cytoplasmic stress granule / protein tag activity / rRNA processing / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / ribosome biogenesis / viral capsid / ribosome binding / ribosomal small subunit assembly / protein-folding chaperone binding / small ribosomal subunit / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / ribosomal large subunit assembly / 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 / cytoplasmic translation / cytosolic large ribosomal subunit / negative regulation of translation / rRNA binding / ribosome / protein ubiquitination / structural constituent of ribosome / ribonucleoprotein complex / positive regulation of protein phosphorylation / G protein-coupled receptor signaling pathway / translation / negative regulation of gene expression / response to antibiotic / GTPase activity / mRNA binding / ubiquitin protein ligase binding / host cell nucleus / GTP binding

ドメイン・相同性:

60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / Pre-hexon-linking protein VIII / Adenovirus hexon associated protein, protein VIII / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / 50S ribosomal protein L10, insertion domain superfamily / : / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / EF-G domain III/V-like / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / : / Ribosomal protein S26e signature. / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S26e / Ribosomal protein S21e, conserved site / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein S21e signature. / : / Ribosomal protein S12e signature. / Ribosomal protein S12e / Ribosomal protein L1, conserved site / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Ribosomal protein L1 signature. / Ribosomal protein L29e / Ribosomal L29e protein family / Ribosomal protein L1 / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal protein S2, eukaryotic / Small (40S) ribosomal subunit Asc1/RACK1 / Ribosomal protein L22e / Ribosomal protein L22e superfamily / S27a-like superfamily / Ribosomal L22e protein family / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal L38e protein family / 40S Ribosomal protein S10 / Ribosomal protein S10, eukaryotic/archaeal / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein L44e signature. / Plectin/S10, N-terminal / Ribosomal protein L10e, conserved site / Plectin/S10 domain / Ribosomal protein L10e signature. / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein L10e / Ribosomal protein L13e / Ribosomal protein L13e / Ribosomal protein L19, eukaryotic / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S2, eukaryotic/archaeal / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein S17e, conserved site / Ribosomal protein S17e signature. / 60S ribosomal protein L18a/ L20, eukaryotes / Ribosomal protein S30 / : / Ribosomal protein S30 / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / : / Ribosomal protein S7e signature. / Ribosomal protein L24e, conserved site / Ribosomal protein L19/L19e conserved site / Ribosomal protein L19e signature. / Ribosomal protein L24e signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L44e / Ribosomal protein L44 / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / : / Ribosomal protein L30e signature 1. / Ribosomal protein L6e signature. / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / Ribosomal protein S19e

構成要素:

Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS1A / Small ribosomal subunit protein uS4A / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein eS17A / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL29 / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL22A / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS15 / Ubiquitin-ribosomal protein eS31 fusion protein / Small ribosomal subunit protein uS11A / Small ribosomal subunit protein eS19A / Small ribosomal subunit protein eS21A / Small ribosomal subunit protein uS8A / Large ribosomal subunit protein uL5A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Ubiquitin-ribosomal protein eL40A fusion protein / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein eL42A / Small ribosomal subunit protein uS12A / Small ribosomal subunit protein eS24A / Small ribosomal subunit protein eS30A / Small ribosomal subunit protein eS4A / Small ribosomal subunit protein eS6A / Small ribosomal subunit protein eS8A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL1A / Large ribosomal subunit protein uL2A / Small ribosomal subunit protein uS17A / Pre-hexon-linking protein VIII / Small ribosomal subunit protein uS9A / Large ribosomal subunit protein uL11A / Small ribosomal subunit protein uS13A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Small ribosomal subunit protein eS32A / Large ribosomal subunit protein uL4A / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein uL18 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL13A / Small ribosomal subunit protein eS7A / Elongation factor 2 / Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS1A / Small ribosomal subunit protein eS27A / Large ribosomal subunit protein eL14A / Small ribosomal subunit protein RACK1 / Large ribosomal subunit protein eL32 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein eS26A / Small ribosomal subunit protein uS14A / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein eS12 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Large ribosomal subunit protein eL34A / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Small ribosomal subunit protein eS10A / Large ribosomal subunit protein eL13A / Small ribosomal subunit protein eS25A / Small ribosomal subunit protein eS28A

• 生物種: Saccharomyces cerevisiae (パン酵母) / Taura syndrome virus (ウイルス)
• 手法: 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 4.0 Å
• データ登録者: Abeyrathne PD / Koh CS / Grant T / Grigorieff N / Korostelev AA
• 引用: ジャーナル: Elife / 年: 2016; タイトル: Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome.; 著者: Priyanka D Abeyrathne / Cha San Koh / Timothy Grant / Nikolaus Grigorieff / Andrei A Korostelev / ; 要旨: Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation.

履歴

登録	2016年5月10日	-
ヘッダ(付随情報) 公開	2016年6月1日	-
マップ公開	2016年6月1日	-
更新	2016年6月1日	-
現状	2016年6月1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_6647.map.gz / 形式: CCP4 / 大きさ: 500 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• 注釈: Saccharomyces cerevisiae 80S ribosome bound to eEF2-GDP-sordarin and TSV IRES, Structure V. For classification, 2D masking around the A site was used, including PKI of the IRES and domain IV of eEF2.
• ボクセルのサイズ: X=Y=Z: 0.82 Å

密度

表面レベル	By EMDB: 0.017 / ムービー #1: 0.014
最小 - 最大	-0.0251419 - 0.06681734
平均 (標準偏差)	-0.00168754 (±0.0050078)

• 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 512 512 512 Spacing 512 512 512
セル	A=B=C: 419.84 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	0.82	0.82	0.82
M x/y/z	512	512	512
origin x/y/z	0.000	0.000	0.000
length x/y/z	419.840	419.840	419.840
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	512	512	512
D min/max/mean	-0.025	0.067	-0.002

添付データ

試料の構成要素

全体 : Saccharomyces cerevisiae 80S ribosome bound to elongation factor ...

• 全体: 名称: Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure V (least rotated 40S subunit)

要素

• 試料: Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure V (least rotated 40S subunit)
• 複合体: Ribosome
• RNA: Internal Ribosome Entry Site
• タンパク質・ペプチド: eukaryotic elongation factor 2

超分子 #1000: Saccharomyces cerevisiae 80S ribosome bound to elongation factor ...

• 超分子: 名称: Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure V (least rotated 40S subunit); タイプ: sample / ID: 1000 / Number unique components: 3
• 分子量: 理論値: 3.2 MDa

超分子 #1: Ribosome

• 超分子: 名称: Ribosome / タイプ: complex / ID: 1 / 組換発現: No / データベース: NCBI / Ribosome-details: ribosome-eukaryote: ALL
• 由来(天然): 生物種: Saccharomyces cerevisiae (パン酵母) / 別称: Yeast
• 分子量: 理論値: 3.2 MDa

分子 #1: Internal Ribosome Entry Site

• 分子: 名称: Internal Ribosome Entry Site / タイプ: rna / ID: 1 / Name.synonym: IRES / 分類: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
• 由来(天然): 生物種: Taura syndrome virus (ウイルス)

分子 #2: eukaryotic elongation factor 2

• 分子: 名称: eukaryotic elongation factor 2 / タイプ: protein_or_peptide / ID: 2 / Name.synonym: eEF2 / 組換発現: No / データベース: NCBI
• 由来(天然): 生物種: Saccharomyces cerevisiae (パン酵母) / 別称: Yeast
• 分子量: 理論値: 94 KDa

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: 0.3 mg/mL
• グリッド: 詳細: Quantifoil Cu 200 mesh
• 凍結: 凍結剤: ETHANE / チャンバー内湿度: 85 % / チャンバー内温度: 103 K / 装置: FEI VITROBOT MARK II

電子顕微鏡法

• 顕微鏡: FEI TITAN KRIOS
• アライメント法: Legacy - 非点収差: Objective lens astigmatism was corrected at 120,000 times magnification.; Legacy - Electron beam tilt params: 0
• 詳細: 10 electrons/physical pixel/second
• 日付: 2015年11月11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); デジタル化 - サンプリング間隔: 5 µm / 実像数: 2662 / 平均電子線量: 70 e/Ų / 詳細: Images recorded as movies in super-resolution mode. / 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: 倍率（補正後）: 30487 / 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）: 2.5 µm / 最小 デフォーカス（公称値）: 0.7 µm / 倍率（公称値）: 18000
• 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: FREALIGN
• CTF補正: 詳細: each particle
• 最終 再構成: アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 4.0 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: Unblur, CTFFIND4, FREALIGN; 詳細: The highest resolution included in the refinement was 7 Angstrom.; 使用した粒子像数: 1105737

原子モデル構築 1

初期モデル

PDB ID:

3j6y

• ソフトウェア: 名称: Chimera
• 精密化: 空間: REAL / プロトコル: FLEXIBLE FIT

得られたモデル

PDB-5juu:
Saccharomyces cerevisiae 80S ribosome bound with elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure V (least rotated 40S subunit)