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データを開く
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基本情報
登録情報 | ![]() | |||||||||
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タイトル | Structure of human 80S ribosome obtained from ssDNA coated grid | |||||||||
![]() | Final map | |||||||||
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生物種 | ![]() ![]() | |||||||||
手法 | ![]() ![]() | |||||||||
![]() | Hrebik D / Plevka P | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules. 著者: Dominik Hrebík / Mária Gondová / Lucie Valentová / Tibor Füzik / Antonín Přidal / Jiří Nováček / Pavel Plevka / ![]() 要旨: Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, ...Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, the preparation of high-quality samples has become a bottleneck in the cryo-EM structure-determination pipeline. Macromolecules can be damaged during the purification or preparation of vitrified samples for cryo-EM, making them prone to binding to the grid support, to aggregation or to the adoption of preferential orientations at the air-water interface. Here, it is shown that coating cryo-EM grids with a negatively charged polyelectrolyte, such as single-stranded DNA, before applying the sample reduces the aggregation of macromolecules and improves their distribution. The single-stranded DNA-coated grids enabled the determination of high-resolution structures from samples that aggregated on conventional grids. The polyelectrolyte coating reduces the diffusion of macromolecules and thus may limit the negative effects of the contact of macromolecules with the grid support and blotting paper, as well as of the shear forces on macromolecules during grid blotting. Coating grids with polyelectrolytes can readily be employed in any laboratory dealing with cryo-EM sample preparation, since it is fast, simple, inexpensive and does not require specialized equipment. | |||||||||
履歴 |
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構造の表示
添付画像 |
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ダウンロードとリンク
-EMDBアーカイブ
マップデータ | ![]() | 94.8 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 22.2 KB 22.2 KB | 表示 表示 | ![]() |
FSC (解像度算出) | ![]() | 18.1 KB | 表示 | ![]() |
画像 | ![]() | 81.6 KB | ||
その他 | ![]() ![]() | 94.6 MB 94.6 MB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
EMDBのページ | ![]() ![]() |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||
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注釈 | Final map | ||||||||||||||||||||||||||||||||||||
投影像・断面図 | 画像のコントロール
画像は Spider により作成 | ||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-ハーフマップ: Half Map 1
ファイル | emd_14704_half_map_1.map | ||||||||||||
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注釈 | Half Map 1 | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Half Map 2
ファイル | emd_14704_half_map_2.map | ||||||||||||
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注釈 | Half Map 2 | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
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試料の構成要素
-全体 : human 80S ribosome
全体 | 名称: human 80S ribosome![]() |
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要素 |
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-超分子 #1: human 80S ribosome
超分子 | 名称: human 80S ribosome / タイプ: complex / キメラ: Yes / ID: 1 / 親要素: 0 / 含まれる分子: all |
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由来(天然) | 生物種: ![]() ![]() |
分子量 | 理論値: 4.3 MDa |
-分子 #1: RNA (615-MER)
分子 | 名称: RNA (615-MER) / タイプ: rna / ID: 1 / コピー数: 1 |
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由来(天然) | 生物種: ![]() ![]() |
分子量 | 理論値: 1.22354775 MDa |
配列 | 文字列: CGCGACCUCA GAUCAGACGU GGCGACCCGC UGAAUUUAAG CAUAUUAGUC AGCGGAGGAG AAGAAACUAA CCAGGAUUCC CUCAGUAAC GGCGAGUGAA CAGGGAAGAG CCCAGCGCCG AAUCCCCGCC CCGCGGCGGG GCGCGGGACA UGUGGCGUAC G GAAGACCC ...文字列: CGCGACCUCA GAUCAGACGU GGCGACCCGC UGAAUUUAAG CAUAUUAGUC AGCGGAGGAG AAGAAACUAA CCAGGAUUCC CUCAGUAAC GGCGAGUGAA CAGGGAAGAG CCCAGCGCCG AAUCCCCGCC CCGCGGCGGG GCGCGGGACA UGUGGCGUAC G GAAGACCC GCUCCCCGGC GCCGCUCGUG GGGGGCCCAA GUCCUUCUGA UCGAGGCCCA GCCCGUGGAC GGUGUGAGGC CG GUAGCGG CCCCCGGCGC GCCGGGCCCG GGUCUUCCCG GAGUCGGGUU GCUUGGGAAU GCAGCCCAAA GCGGGUGGUA AAC UCCAUC UAAGGCUAAA UACCGGCACG AGACCGAUAG UCAACAAGUA CCGUAA(OMG)GGG AAAGUUGAAA AGAACUUUGA AG(A2M)AGAGAG UUCAAGAGGG CGUGAAACCG UUAAGAGGUA AACGGGUGGG GUCCGCGCAG UCCGCCCGGA GGAUUCA AC CCGGCGGCGG GUCCGGCCGU GUCGGCGGCC CGGCGGAUCU UUCCCGCGCG GGGGACCGUC CCCCGACCGG CGACCGGC C GCCGCCGGGC GCAUUUCCAC CGCGGCGGUG CGCCGCGACC GGCUCCGGGA CGGCU(2MG)GGGA AGGCCCGGCG GGGAA GGUG GCUCGGGGGC CCCCGAGUGU UACAGCCCCC CCGGCAGCAG CACUCGCCGA AUCCCGGGGC CGAGGGAGCG AGACCC GUC GCCGCGCUCU CCCCCCUCCC GGCGCGCCGG GGGGGGCCGG GCCACCCCUC CCACGGCGCG ACCGCUCGGG GCGGACU GU CCCCAGUGCG CCCCGGGCGG GUCGCGCCGU CGGGCCCGGG GGAGGCCACG CGCGCGUCCC CCGAAGAGGG GGACGGCG G AGCGAGCGCA CGGGGUCGGC GGCGACGUCG GCUACCCACC CGACCCGUCU UGAAACACGG ACCAAGGAGU CUAACACGU GCGCGAGUCG GGGGCUCGCA CGAAAGCCGC CGUGGCGCAA UGAAGGUGAA GGCCGGCGCG CUCGCCGGCC GAGGUGGGAU CCCGAGGCC UCUCCAGUCC GCCGAGGGCG CACCACCGGC CCGUCUCGCC CGCCGCGCCG GGGAGGUGGA GCACGAGCGC A CGUGUUAG GACCCGAAAG AUGGUGAACU AUGCCUGGGC AGGGCGAAGC CAGAGGAAAC UCUGGUGGAG GUCCGUAGCG GU CCUGACG UGCAAAUCGG UCGUCCGACC UGGGUAUAGG GGCGAAAGAC UAAUCGAACC AUCUAGUAGC UGGUUCCCUC CGA AGUUUC CCUCAGGAUA GCUGGCGCUC UCGCAGACCC GACGCCCGCC ACGCAGUUUU AUCCGGUAAA GCGAAUGAUU AGAG GUCUU GGGGCCGAAA CGAUCUCAAC CUAUUCUCAA ACUUUAAAUG GGUAAGAAGC CCGGCUCGCU GGCGUGGAGC CGGGC GUGG AAUGCGAGUG CCUAGUGGGC CACUUUUGGU AAGCAGAACU GGCGCUGCGG GAUGAACCGA ACGCCGGGUU AAGGCG CCC GAUGCCGACG CUCAUCAGAC CCCAGAAAAG GUGUUGGUUG AUAUAGACAG CAGGACGGUG GCCAUGGAAG UCGGAAU CC GCUAAGGAGU GUGUAACAAC UCACCUGCCG AAUCAACUAG CCCUGAAAAU GGAUGGCGCU GGAGCGUCGG GCCCAUAC C CGGCCGUCGC CGGCAGUCGA GAGUGGACGG GAGCGGCGGG CCGGAGCCCC GCGGACGCUA CGCCGCGACG AGUAGGAGG GCCGCUGCGG UGAGCCUUGA AGCCUAGGGC GCGGGCCCGG GUGGAGCCGC CGCAGGUGCA GAUCUUGGUG GUAGUAGCAA AUAUUCAAA CGAGAACUUU GAAGGCCGAA GUGGAGAAGG GUUCCAUGUG AACAGCAGUU GAACAUGGGU CAGUCGGUCC U GAGAGAUG GGCGAGCGCC GUUCCGAAGG GACGGGCGAU GGCCUCCGUU GCCCUCGGCC GAUCGAAAGG GAGUCGGGUU CA GAUCCCC GAAUCCGGAG UGGCGGAGAU GGGCGCCGCG AGGCGUCCAG UGCGGUAACG CGACCGAUCC CGGAGAAGCC GGC GGGAGC CCCGGGGAGA GUUCUCUUUU CUUUGUGAAG GGCAGGGCGC CCUGGAAUGG GUUCGCCCCG AGAGAGGGGC CCGU GCCUU GGAAAGCGUC GCGGUUCCGG CGGCGUCCGG UGAGCUCUCG CUGGCCCUUG AAAAUCCGGG GGAGAGGGUG UAAAU CUCG CGCCGGGCCG UACCCAUAUC CGCAGCAGGU CUCCAAGGUG AACAGCCUCU GGCAUGUUGG AACAAUGUAG GUAAGG GAA GUCGGCAAGC CGGAUCCGUA ACUUCGGGAU AAGGAUUGGC UCUAAGGGCU GGGUCGGUCG CGGCCGGCGC CUAGCAG CC GACUUAGAAC UGGUGCGGAC CAGGGGAAUC CGACUGUUUA AUUAAAACAA AGCAUCGCGA AGGCCCGCGG CGGGUGUU G ACGCGAUGUG AUUUCUGCCC AGUGCUCUGA AUGUCAAAGU GAAGAAAUUC AAUGAAGCGC GGGUAAACGG CGGGAGUAA CUAUGACUCU CUUAAGGUAG CCAAAUGCCU CGUCAUCUAA UUAGUGACGC GCAUGAAUGG AUGAACGAGA UUCCCACUGU CCCUACCUA CUAUCCAGCG AAACCACAGC CAAGGGAACG GGCUUGGCGG AAUCAGCGGG GAAAGAAGAC CCUGUUGAGC U UGACUCUA GUCUGGCACG GUGAAGAGAC AUGAGAGGUG UAGAAUAAGU GGGAGGCCCC CGGCGCCCCC CCGGUGUCCC CG CGAGGGG CCCGGGGCGG GGUCCGCCGG CCCUGCGGGC CGCCGGUGAA AUACCACUAC UCUGAUCGUU UUUUCACUGA CCC GGUGAG GCGGGGGGGC GAGCCCCGAG GGGCUCUCGC UUCUGGCGCC AAGCGCCCGG CCGCGCGCCG GCCGGGCGCG ACCC GCUCC GGGGACAGUG CCAGGUGGGG AGUUUGACUG GGGCGGUACA CCUGUCAAAC GGUAACGCAG GUGUCCUAAG GCGAG CUCA GGGAGGACAG AAACCUCCCG UGGAGCAGAA GGGCAAAAGC UCGCUUGAUC UUGAUUUUCA GUACGAAUAC AGACCG UGA AAGCGGGGCC UCACGAUCCU UCUGACCUUU UGGGUUUUAA GCAGGAGGUG UCAGAAAAGU UACCACAGGG AUAACUG GC UUGUGGCGGC CAAGCGUUCA UAGCGACGUC GCUUUUUGAU CCUUCGAUGU CGGCUCUUCC UAUCAUUGUG AAGCAGAA U UCACCAAGCG UUGGAUUGUU CACCCACUAA UAGGGAACGU GAGCUGGGUU UAGACCGUCG UGAGACAGGU UAGUUUUAC CCUACUGAUG AUGUGUUGUU GCCAUGGUAA UCCUGCUCAG UACGAGAGGA ACCGCAGGUU CAGACAUUUG GUGUAUGUGC UUGGCUGAG GAGCCAAUGG GGCGAAGCUA CCAUCUGUGG GAUUAUGACU GAACGCCUCU AAGUCAGAAU CCCGCCCAGG C GGAACGAU ACGGCAGCGC CGCGGAGCCU CGGUUGGCCU CGGAUAGCCG GUCCCCCGCC GGGGUCCGGU GCGGAGUGCC CU UCGUCCU GGGAAACGGG GCGCGGCCGG AGAGGCGGCC GCCCCCUCGC CCGUCACGCA CCGCACGUUC GUGGGGAACC UGG CGCUAA ACCAUUCGUA GACGACCUGC UUCUGGGUCG GGGUUUCGUA CGUAGCAGAG CAGCUCCCUC GCUGCGAUCU AUUG AAAGU CAGCCCUCGA CACAAGGGUU UGU |
-実験情報
-構造解析
手法 | ![]() |
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試料の集合状態 | particle |
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試料調製
濃度 | 1.2 mg/mL | ||||||||||||||||||
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緩衝液 | pH: 7.5 構成要素:
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グリッド | モデル: Quantifoil R2/2 / 材質: COPPER / メッシュ: 300 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY / 支持フィルム - Film thickness: 5.0 nm / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 雰囲気: NITROGEN | ||||||||||||||||||
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277.15 K / 装置: FEI VITROBOT MARK IV 詳細: 3 s blot time,30 s waiting time, ssDNA covered grid. |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | C2レンズ絞り径: 50.0 µm / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD![]() |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
撮影 | フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 検出モード: INTEGRATING / デジタル化 - サイズ - 横: 4096 pixel / デジタル化 - サイズ - 縦: 4096 pixel / デジタル化 - サンプリング間隔: 14.0 µm / 撮影したグリッド数: 1 / 実像数: 4527 / 平均露光時間: 1.0 sec. / 平均電子線量: 64.0 e/Å2 |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |