+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-14704 | |||||||||
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タイトル | Structure of human Apoferritin obtained from ssDNA coated grid | |||||||||
マップデータ | Final map | |||||||||
試料 |
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キーワード | human 80S ribosome / ssDNA covered grid / ssDNA / cryoEM / RIBOSOME | |||||||||
生物種 | Homo sapiens (ヒト) | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.8 Å | |||||||||
データ登録者 | Hrebik D / Plevka P | |||||||||
資金援助 | チェコ, 1件
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引用 | ジャーナル: Acta Crystallogr D Struct Biol / 年: 2022 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 | emd_14704.map.gz | 94.8 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-14704-v30.xml emd-14704.xml | 21.8 KB 21.8 KB | 表示 表示 | EMDBヘッダ |
FSC (解像度算出) | emd_14704_fsc.xml | 18.1 KB | 表示 | FSCデータファイル |
画像 | emd_14704.png | 81.6 KB | ||
Filedesc metadata | emd-14704.cif.gz | 6.9 KB | ||
その他 | emd_14704_half_map_1.map.gz emd_14704_half_map_2.map.gz | 94.6 MB 94.6 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-14704 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14704 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_14704_validation.pdf.gz | 1 MB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_14704_full_validation.pdf.gz | 1 MB | 表示 | |
XML形式データ | emd_14704_validation.xml.gz | 21.3 KB | 表示 | |
CIF形式データ | emd_14704_validation.cif.gz | 28.6 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14704 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14704 | 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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-マップ
ファイル | ダウンロード / ファイル: emd_14704.map.gz / 形式: CCP4 / 大きさ: 103 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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注釈 | Final map | ||||||||||||||||||||
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 |
-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 / ID: 1 / 親要素: 0 / 含まれる分子: all |
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由来(天然) | 生物種: Homo sapiens (ヒト) |
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: Homo sapiens (ヒト) |
分子量 | 理論値: 1.222873375 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)GGA AAGUUGAAAA GAACUUUGAA G(A2M)GAGAGUU CAAGAGGGCG UGAAACCGUU AAGAGGUAAA CGGGUGGGGU CCGCGCAGUC CGCCCGGAGG AUUCAAC CC GGCGGCGGGU CCGGCCGUGU CGGCGGCCCG GCGGAUCUUU CCCGCGCGGG GGACCGUCCC CCGACCGGCG ACCGGCCG C CGCCGGGCGC AUUUCCACCG CGGCGGUGCG CCGCGACCGG CUCCGGGACG GCU(2MG)GGGAAG GCCCGGCGGG GAAGG UGGC UCGGGGGCCC CCGAGUGUUA CAGCCCCCCC GGCAGCAGCA CUCGCCGAAU CCCGGGGCCG AGGGAGCGAG ACCCGU CGC CGCGCUCUCC CCCCUCCCGG CGCGCCGGGG GGGGCCGGGC CACCCCUCCC ACGGCGCGAC CGCUCGGGGC GGACUGU CC CCAGUGCGCC CCGGGCGGGU CGCGCCGUCG GGCCCGGGGG AGGCCACGCG CGCGUCCCCC GAAGAGGGGG ACGGCGGA G CGAGCGCACG GGGUCGGCGG CGACGUCGGC UACCCACCCG ACCCGUCUUG AAACACGGAC CAAGGAGUCU AACACGUGC GCGAGUCGGG GGCUCGCACG AAAGCCGCCG UGGCGCAAUG AAGGUGAAGG CCGGCGCGCU CGCCGGCCGA GGUGGGAUCC CGAGGCCUC UCCAGUCCGC CGAGGGCGCA CCACCGGCCC GUCUCGCCCG CCGCGCCGGG GAGGUGGAGC ACGAGCGCAC G UGUUAGGA CCCGAAAGAU GGUGAACUAU GCCUGGGCAG GGCGAAGCCA GAGGAAACUC UGGUGGAGGU CCGUAGCGGU CC UGACGUG CAAAUCGGUC GUCCGACCUG GGUAUAGGGG CGAAAGACUA AUCGAACCAU CUAGUAGCUG GUUCCCUCCG AAG UUUCCC UCAGGAUAGC UGGCGCUCUC GCAGACCCGA CGCCCGCCAC GCAGUUUUAU CCGGUAAAGC GAAUGAUUAG AGGU CUUGG GGCCGAAACG AUCUCAACCU AUUCUCAAAC UUUAAAUGGG UAAGAAGCCC GGCUCGCUGG CGUGGAGCCG GGCGU GGAA UGCGAGUGCC UAGUGGGCCA CUUUUGGUAA GCAGAACUGG CGCUGCGGGA UGAACCGAAC GCCGGGUUAA GGCGCC CGA UGCCGACGCU CAUCAGACCC CAGAAAAGGU GUUGGUUGAU AUAGACAGCA GGACGGUGGC CAUGGAAGUC GGAAUCC GC UAAGGAGUGU GUAACAACUC ACCUGCCGAA UCAACUAGCC CUGAAAAUGG AUGGCGCUGG AGCGUCGGGC CCAUACCC G GCCGUCGCCG GCAGUCGAGA GUGGACGGGA GCGGCGGGCC GGAGCCCCGC GGACGCUACG CCGCGACGAG UAGGAGGGC CGCUGCGGUG AGCCUUGAAG CCUAGGGCGC GGGCCCGGGU GGAGCCGCCG CAGGUGCAGA UCUUGGUGGU AGUAGCAAAU AUUCAAACG AGAACUUUGA AGGCCGAAGU GGAGAAGGGU UCCAUGUGAA CAGCAGUUGA ACAUGGGUCA GUCGGUCCUG A GAGAUGGG CGAGCGCCGU UCCGAAGGGA CGGGCGAUGG CCUCCGUUGC CCUCGGCCGA UCGAAAGGGA GUCGGGUUCA GA UCCCCGA AUCCGGAGUG GCGGAGAUGG GCGCCGCGAG GCGUCCAGUG CGGUAACGCG ACCGAUCCCG GAGAAGCCGG CGG GAGCCC CGGGGAGAGU UCUCUUUUCU UUGUGAAGGG CAGGGCGCCC UGGAAUGGGU UCGCCCCGAG AGAGGGGCCC GUGC CUUGG AAAGCGUCGC GGUUCCGGCG GCGUCCGGUG AGCUCUCGCU GGCCCUUGAA AAUCCGGGGG AGAGGGUGUA AAUCU CGCG CCGGGCCGUA CCCAUAUCCG CAGCAGGUCU CCAAGGUGAA CAGCCUCUGG CAUGUUGGAA CAAUGUAGGU AAGGGA AGU CGGCAAGCCG GAUCCGUAAC UUCGGGAUAA GGAUUGGCUC UAAGGGCUGG GUCGGUCGCG GCCGGCGCCU AGCAGCC GA CUUAGAACUG GUGCGGACCA GGGGAAUCCG ACUGUUUAAU UAAAACAAAG CAUCGCGAAG GCCCGCGGCG GGUGUUGA C GCGAUGUGAU UUCUGCCCAG UGCUCUGAAU GUCAAAGUGA AGAAAUUCAA UGAAGCGCGG GUAAACGGCG GGAGUAACU AUGACUCUCU UAAGGUAGCC AAAUGCCUCG UCAUCUAAUU AGUGACGCGC AUGAAUGGAU GAACGAGAUU CCCACUGUCC CUACCUACU AUCCAGCGAA ACCACAGCCA AGGGAACGGG CUUGGCGGAA UCAGCGGGGA AAGAAGACCC UGUUGAGCUU G ACUCUAGU CUGGCACGGU GAAGAGACAU GAGAGGUGUA GAAUAAGUGG GAGGCCCCCG GCGCCCCCCC GGUGUCCCCG CG AGGGGCC CGGGGCGGGG UCCGCCGGCC CUGCGGGCCG CCGGUGAAAU ACCACUACUC UGAUCGUUUU UUCACUGACC CGG UGAGGC GGGGGGGCGA GCCCCGAGGG GCUCUCGCUU CUGGCGCCAA GCGCCCGGCC GCGCGCCGGC CGGGCGCGAC CCGC UCCGG GGACAGUGCC AGGUGGGGAG UUUGACUGGG GCGGUACACC UGUCAAACGG UAACGCAGGU GUCCUAAGGC GAGCU CAGG GAGGACAGAA ACCUCCCGUG GAGCAGAAGG GCAAAAGCUC GCUUGAUCUU GAUUUUCAGU ACGAAUACAG ACCGUG AAA GCGGGGCCUC ACGAUCCUUC UGACCUUUUG GGUUUUAAGC AGGAGGUGUC AGAAAAGUUA CCACAGGGAU AACUGGC UU GUGGCGGCCA AGCGUUCAUA GCGACGUCGC UUUUUGAUCC UUCGAUGUCG GCUCUUCCUA UCAUUGUGAA GCAGAAUU C ACCAAGCGUU GGAUUGUUCA CCCACUAAUA GGGAACGUGA GCUGGGUUUA GACCGUCGUG AGACAGGUUA GUUUUACCC UACUGAUGAU GUGUUGUUGC CAUGGUAAUC CUGCUCAGUA CGAGAGGAAC CGCAGGUUCA GACAUUUGGU GUAUGUGCUU GGCUGAGGA GCCAAUGGGG CGAAGCUACC AUCUGUGGGA UUAUGACUGA ACGCCUCUAA GUCAGAAUCC CGCCCAGGCG G AACGAUAC GGCAGCGCCG CGGAGCCUCG GUUGGCCUCG GAUAGCCGGU CCCCCGCCGG GGUCCGGUGC GGAGUGCCCU UC GUCCUGG GAAACGGGGC GCGGCCGGAG AGGCGGCCGC CCCCUCGCCC GUCACGCACC GCACGUUCGU GGGGAACCUG GCG CUAAAC CAUUCGUAGA CGACCUGCUU CUGGGUCGGG GUUUCGUACG UAGCAGAGCA GCUCCCUCGC UGCGAUCUAU UGAA AGUCA GCCCUCGACA CAAGGGUUUG U |
-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
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 / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 20 sec. / 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. |
-電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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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 / 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 |
電子線 | 加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN |
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 / Cs: 2.7 mm / 最大 デフォーカス(公称値): 1.7 µm / 最小 デフォーカス(公称値): 0.3 µm / 倍率(公称値): 75000 |
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 |
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |