+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 8r4i | |||||||||
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タイトル | Cryo-EM structure of human islet amyloid polypeptide (hIAPP) | |||||||||
要素 | Islet amyloid polypeptide | |||||||||
キーワード | PROTEIN FIBRIL / Amyloid / fibril | |||||||||
機能・相同性 | 機能・相同性情報 amylin receptor signaling pathway / Calcitonin-like ligand receptors / negative regulation of amyloid fibril formation / negative regulation of bone resorption / eating behavior / positive regulation of protein kinase A signaling / negative regulation of osteoclast differentiation / Regulation of gene expression in beta cells / negative regulation of protein-containing complex assembly / bone resorption ...amylin receptor signaling pathway / Calcitonin-like ligand receptors / negative regulation of amyloid fibril formation / negative regulation of bone resorption / eating behavior / positive regulation of protein kinase A signaling / negative regulation of osteoclast differentiation / Regulation of gene expression in beta cells / negative regulation of protein-containing complex assembly / bone resorption / sensory perception of pain / positive regulation of calcium-mediated signaling / osteoclast differentiation / hormone activity / cell-cell signaling / amyloid-beta binding / G alpha (s) signalling events / positive regulation of MAPK cascade / receptor ligand activity / positive regulation of apoptotic process / Amyloid fiber formation / signaling receptor binding / lipid binding / apoptotic process / signal transduction / extracellular space / extracellular region / identical protein binding 類似検索 - 分子機能 | |||||||||
生物種 | Homo sapiens (ヒト) | |||||||||
手法 | 電子顕微鏡法 / らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 4.01 Å | |||||||||
データ登録者 | Ooi, S.A. / Valli, D. / Maj, M. | |||||||||
資金援助 | スウェーデン, 2件
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引用 | ジャーナル: Biophys J / 年: 2024 タイトル: Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins. 著者: Dylan Valli / Saik Ann Ooi / Giorgio Scattolini / Himanshu Chaudhary / Alesia A Tietze / Michał Maj / 要旨: Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the ...Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques. | |||||||||
履歴 |
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-構造の表示
構造ビューア | 分子: MolmilJmol/JSmol |
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-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 8r4i.cif.gz | 48.8 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb8r4i.ent.gz | 36.1 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 8r4i.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
文書・要旨 | 8r4i_validation.pdf.gz | 1.2 MB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 8r4i_full_validation.pdf.gz | 1.2 MB | 表示 | |
XML形式データ | 8r4i_validation.xml.gz | 24.7 KB | 表示 | |
CIF形式データ | 8r4i_validation.cif.gz | 34.3 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/r4/8r4i ftp://data.pdbj.org/pub/pdb/validation_reports/r4/8r4i | HTTPS FTP |
-関連構造データ
関連構造データ | 18887MC M: このデータのモデリングに利用したマップデータ C: 同じ文献を引用 (文献) |
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類似構造データ | 類似検索 - 機能・相同性F&H 検索 |
-リンク
-集合体
登録構造単位 |
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-要素
#1: タンパク質・ペプチド | 分子量: 3907.312 Da / 分子数: 10 / 由来タイプ: 合成 / 由来: (合成) Homo sapiens (ヒト) / 参照: UniProt: P10997 研究の焦点であるリガンドがあるか | N | |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: FILAMENT / 3次元再構成法: らせん対称体再構成法 |
-試料調製
構成要素 | 名称: Islet amyloid polypeptide / タイプ: CELL / Entity ID: all / 由来: MULTIPLE SOURCES |
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由来(天然) | 生物種: Homo sapiens (ヒト) |
由来(組換発現) | 生物種: synthetic construct (人工物) |
緩衝液 | pH: 7.4 |
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
急速凍結 | 凍結剤: ETHANE |
-電子顕微鏡撮影
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 105000 X / 最大 デフォーカス(公称値): 1700 nm / 最小 デフォーカス(公称値): 500 nm |
撮影 | 電子線照射量: 39.926 e/Å2 フィルム・検出器のモデル: GATAN K3 BIOQUANTUM (6k x 4k) |
-解析
CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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らせん対称 | 回転角度/サブユニット: 178.25 ° / 軸方向距離/サブユニット: 2.46 Å / らせん対称軸の対称性: C2 |
3次元再構成 | 解像度: 4.01 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 10566 / 対称性のタイプ: HELICAL |