+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-0633 | |||||||||
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タイトル | RNA polymerase II elongation complex arrested at a CPD lesion | |||||||||
マップデータ | RNA polymerase II elongation complex stalled at a CPD lesion | |||||||||
試料 |
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キーワード | RNA polymerase / CPD / elongation complex / streptavidin grids / transcription / transferase-DNA-RNA complex | |||||||||
機能・相同性 | 機能・相同性情報 RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes / termination of RNA polymerase II transcription / RNA Polymerase II Promoter Escape ...RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes / termination of RNA polymerase II transcription / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA-templated transcription / termination of RNA polymerase III transcription / RNA Polymerase II Pre-transcription Events / Formation of TC-NER Pre-Incision Complex / termination of RNA polymerase I transcription / RNA Polymerase I Promoter Escape / transcription initiation at RNA polymerase III promoter / nucleolar large rRNA transcription by RNA polymerase I / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription initiation at RNA polymerase I promoter / transcription by RNA polymerase I / Estrogen-dependent gene expression / transcription by RNA polymerase III / Dual incision in TC-NER / transcription elongation by RNA polymerase I / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I complex / RNA polymerase III complex / transcription-coupled nucleotide-excision repair / RNA polymerase I activity / RNA polymerase II, core complex / translesion synthesis / transcription elongation by RNA polymerase II / transcription initiation at RNA polymerase II promoter / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / cytoplasmic stress granule / ribosome biogenesis / peroxisome / transcription by RNA polymerase II / nucleic acid binding / protein dimerization activity / mRNA binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytoplasm 類似検索 - 分子機能 | |||||||||
生物種 | Saccharomyces cerevisiae (パン酵母) | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.1 Å | |||||||||
データ登録者 | Lahiri I / Leshziner AE | |||||||||
資金援助 | 米国, 2件
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引用 | ジャーナル: J Struct Biol / 年: 2019 タイトル: 3.1 Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids. 著者: Indrajit Lahiri / Jun Xu / Bong Gyoon Han / Juntaek Oh / Dong Wang / Frank DiMaio / Andres E Leschziner / 要旨: Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact ...Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Å structure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)'s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface. | |||||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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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_0633.map.gz | 124.6 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-0633-v30.xml emd-0633.xml | 26.3 KB 26.3 KB | 表示 表示 | EMDBヘッダ |
画像 | emd_0633.png | 201.6 KB | ||
Filedesc metadata | emd-0633.cif.gz | 9.3 KB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-0633 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0633 | HTTPS FTP |
-関連構造データ
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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「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_0633.map.gz / 形式: CCP4 / 大きさ: 216 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | RNA polymerase II elongation complex stalled at a CPD lesion | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.16 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
-試料の構成要素
+全体 : RNA polymerase II elongation complex stalled at a CPD lesion
+超分子 #1: RNA polymerase II elongation complex stalled at a CPD lesion
+分子 #1: DNA-directed RNA polymerase II subunit RPB1
+分子 #2: DNA-directed RNA polymerase II subunit RPB2
+分子 #3: DNA-directed RNA polymerase II subunit RPB3
+分子 #4: DNA-directed RNA polymerases I, II, and III subunit RPABC1
+分子 #5: DNA-directed RNA polymerases I, II, and III subunit RPABC2
+分子 #6: DNA-directed RNA polymerases I, II, and III subunit RPABC3
+分子 #7: DNA-directed RNA polymerase II subunit RPB9
+分子 #8: DNA-directed RNA polymerases I, II, and III subunit RPABC5
+分子 #9: DNA-directed RNA polymerase II subunit RPB11
+分子 #10: DNA-directed RNA polymerases I, II, and III subunit RPABC4
+分子 #11: RNA (5'-R(P*AP*UP*CP*GP*AP*GP*AP*GP*G)-3')
+分子 #12: DNA (5'-D(P*GP*GP*AP*GP*AP*AP*GP*GP*AP*GP*CP*AP*GP*AP*GP*C)-3')
+分子 #13: DNA (27-MER)
+分子 #14: ZINC ION
+分子 #15: MAGNESIUM ION
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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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 |
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グリッド | モデル: Quantifoil R2/2 / 材質: GOLD / メッシュ: 300 詳細: The grids had a monolayer of streptavidin crystals on them. |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 293.15 K / 装置: FEI VITROBOT MARK IV 詳細: The sample was manually wicked from the grid. 1.2 uL sample buffer was then applied to the streptavidin side. The grid was then blotted.. |
-電子顕微鏡法
顕微鏡 | FEI TALOS ARCTICA |
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撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / 撮影したグリッド数: 1 / 平均露光時間: 6.0 sec. / 平均電子線量: 51.7 e/Å2 |
電子線 | 加速電圧: 200 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 |
実験機器 | モデル: Talos Arctica / 画像提供: FEI Company |
-画像解析
初期モデル | モデルのタイプ: PDB ENTRY PDBモデル - PDB ID: |
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最終 再構成 | 想定した対称性 - 点群: C1 (非対称) / アルゴリズム: FOURIER SPACE / 解像度のタイプ: BY AUTHOR / 解像度: 3.1 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 使用した粒子像数: 61654 |
初期 角度割当 | タイプ: PROJECTION MATCHING / ソフトウェア - 名称: RELION (ver. 3.0) |
最終 角度割当 | タイプ: PROJECTION MATCHING / ソフトウェア - 名称: RELION (ver. 3.0) |
-原子モデル構築 1
精密化 | 空間: REAL / プロトコル: FLEXIBLE FIT |
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得られたモデル | PDB-6o6c: |