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基本情報
登録情報 | データベース: PDB / ID: 6r70 | |||||||||
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タイトル | Endogeneous native human 20S proteasome | |||||||||
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![]() | HYDROLASE / native / human / 20S proteasome / proteasome / cryo-EM / microfluidic / CryoWriter / protein isolation / endogeneous | |||||||||
機能・相同性 | ![]() purine ribonucleoside triphosphate binding / regulation of endopeptidase activity / Regulation of ornithine decarboxylase (ODC) / proteasome core complex / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / immune system process / myofibril / NF-kappaB binding / proteasome endopeptidase complex ...purine ribonucleoside triphosphate binding / regulation of endopeptidase activity / Regulation of ornithine decarboxylase (ODC) / proteasome core complex / Cross-presentation of soluble exogenous antigens (endosomes) / Somitogenesis / immune system process / myofibril / NF-kappaB binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / negative regulation of inflammatory response to antigenic stimulus / : / sarcomere / proteasome complex / Regulation of activated PAK-2p34 by proteasome mediated degradation / ciliary basal body / proteolysis involved in protein catabolic process / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / SCF-beta-TrCP mediated degradation of Emi1 / NIK-->noncanonical NF-kB signaling / Ubiquitin-dependent degradation of Cyclin D / AUF1 (hnRNP D0) binds and destabilizes mRNA / TNFR2 non-canonical NF-kB pathway / Vpu mediated degradation of CD4 / Assembly of the pre-replicative complex / Degradation of DVL / Ubiquitin Mediated Degradation of Phosphorylated Cdc25A / Dectin-1 mediated noncanonical NF-kB signaling / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / Hh mutants are degraded by ERAD / lipopolysaccharide binding / Degradation of AXIN / Degradation of GLI1 by the proteasome / Activation of NF-kappaB in B cells / Hedgehog ligand biogenesis / Defective CFTR causes cystic fibrosis / Negative regulation of NOTCH4 signaling / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / G2/M Checkpoints / Vif-mediated degradation of APOBEC3G / Autodegradation of the E3 ubiquitin ligase COP1 / Hedgehog 'on' state / Regulation of RUNX3 expression and activity / P-body / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / MAPK6/MAPK4 signaling / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / response to virus / Degradation of beta-catenin by the destruction complex / ABC-family proteins mediated transport / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / response to organic cyclic compound / CDK-mediated phosphorylation and removal of Cdc6 / CLEC7A (Dectin-1) signaling / SCF(Skp2)-mediated degradation of p27/p21 / Regulation of expression of SLITs and ROBOs / nuclear matrix / FCERI mediated NF-kB activation / Regulation of PTEN stability and activity / Interleukin-1 signaling / Orc1 removal from chromatin / Regulation of RAS by GAPs / Separation of Sister Chromatids / Regulation of RUNX2 expression and activity / The role of GTSE1 in G2/M progression after G2 checkpoint / UCH proteinases / KEAP1-NFE2L2 pathway / Antigen processing: Ubiquitination & Proteasome degradation / Downstream TCR signaling / Neddylation / RUNX1 regulates transcription of genes involved in differentiation of HSCs / peptidase activity / positive regulation of NF-kappaB transcription factor activity / ER-Phagosome pathway / regulation of inflammatory response / proteasome-mediated ubiquitin-dependent protein catabolic process / secretory granule lumen / endopeptidase activity / response to oxidative stress / postsynapse / ficolin-1-rich granule lumen / nuclear body / ribosome / Ub-specific processing proteases / cadherin binding / intracellular membrane-bounded organelle / centrosome / ubiquitin protein ligase binding / synapse / Neutrophil degranulation / mitochondrion / proteolysis / RNA binding 類似検索 - 分子機能 | |||||||||
生物種 | ![]() | |||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.5 Å | |||||||||
![]() | Schmidli, C. / Albiez, S. / Rima, L. / Righetto, R. / Mohammed, I. / Oliva, P. / Kovacik, L. / Stahlberg, H. / Braun, T. | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Microfluidic protein isolation and sample preparation for high-resolution cryo-EM. 著者: Claudio Schmidli / Stefan Albiez / Luca Rima / Ricardo Righetto / Inayatulla Mohammed / Paolo Oliva / Lubomir Kovacik / Henning Stahlberg / Thomas Braun / ![]() 要旨: High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often ...High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes. | |||||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | 分子: ![]() ![]() |
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ダウンロードとリンク
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ダウンロード
PDBx/mmCIF形式 | ![]() | 1 MB | 表示 | ![]() |
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PDB形式 | ![]() | 874 KB | 表示 | ![]() |
PDBx/mmJSON形式 | ![]() | ツリー表示 | ![]() | |
その他 | ![]() |
-検証レポート
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
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-関連構造データ
関連構造データ | ![]() 4738MC ![]() 4628C ![]() 6r7mC C: 同じ文献を引用 ( M: このデータのモデリングに利用したマップデータ |
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類似構造データ | |
電子顕微鏡画像生データ | ![]() Data size: 136.5 Data #1: Unaligned multi-frame micrographs of human 20S proteasome and TMV [micrographs - multiframe]) |
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リンク
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集合体
登録構造単位 | ![]()
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要素
-Proteasome subunit beta type- ... , 7種, 14分子 MaHVIWJXKYLZNb
#1: タンパク質 | 分子量: 24081.402 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #9: タンパク質 | 分子量: 23745.256 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #10: タンパク質 | 分子量: 22841.701 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #11: タンパク質 | 分子量: 22365.721 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #12: タンパク質 | 分子量: 22199.072 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #13: タンパク質 | 分子量: 23578.986 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #14: タンパク質 | 分子量: 21753.643 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() |
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-Proteasome subunit alpha type- ... , 7種, 14分子 AOBPCQDRESFTGU
#2: タンパク質 | 分子量: 25525.068 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #3: タンパク質 | 分子量: 27859.896 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #4: タンパク質 | 分子量: 26623.291 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #5: タンパク質 | 分子量: 25406.785 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #6: タンパク質 | 分子量: 26807.551 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #7: タンパク質 | 分子量: 26813.619 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() #8: タンパク質 | 分子量: 27186.174 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) ![]() |
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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: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: Native human 20S proteasome with bound Fabs to the two alpha-4 subunits タイプ: COMPLEX 詳細: Fab fragment generated by proteolytic cleavage of monoclonal IgG1 antibody. Entity ID: all / 由来: NATURAL | |||||||||||||||||||||||||
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分子量 | 単位: MEGADALTONS / 実験値: NO | |||||||||||||||||||||||||
由来(天然) | 生物種: ![]() | |||||||||||||||||||||||||
緩衝液 | pH: 7.4 / 詳細: PBS was purchased from Sigma # D8537, Switzerland. | |||||||||||||||||||||||||
緩衝液成分 |
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試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: 20S proteasome was purified using Fabs and eluted in a buffer containing 1mg/ml tobacco mosaic virus (TMV). The sample contains also unbound Fabs. | |||||||||||||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||
急速凍結 | 凍結剤: ETHANE 詳細: CryoWriter. (see https://doi.org/10.1016/j.jsb.2016.11.002 for more information) |
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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: ![]() |
電子レンズ | モード: BRIGHT FIELD / Cs: 2.7 mm / C2レンズ絞り径: 70 µm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 平均露光時間: 6 sec. / 電子線照射量: 72 e/Å2 / 検出モード: SUPER-RESOLUTION フィルム・検出器のモデル: GATAN K2 QUANTUM (4k x 4k) 撮影したグリッド数: 1 / 実像数: 523 |
画像スキャン | 横: 7676 / 縦: 7420 / 動画フレーム数/画像: 30 / 利用したフレーム数/画像: 0-30 |
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解析
ソフトウェア | 名称: PHENIX / バージョン: (1.14_3260: phenix.real_space_refine) / 分類: 精密化 | ||||||||||||||||||||||||||||||||||||||||
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EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 523 | ||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C2 (2回回転対称) | ||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.5 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 16015 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 5LE5 Accession code: 5LE5 / Source name: PDB / タイプ: experimental model |