EMDB-2595: Deep classification of a large cryo-EM dataset defines the confor...

基本情報

• 登録情報: データベース: EMDB / ID: EMD-2595
• タイトル: Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome
• マップデータ: Reconstruction of Intermediate state (s2).

試料

• 試料: 26S Proteasome from Saccharomyces cerevisiaeプロテアソーム
• タンパク質・ペプチド: 26S Proteasomeプロテアソーム

• キーワード: Proteasome (プロテアソーム) / AAA-ATPase / ATP-analog / classification

機能・相同性

分子機能:

SAGA complex localization to transcription regulatory region / peroxisome fission / proteasome storage granule assembly / transcription export complex 2 / proteasome regulatory particle assembly / protein deneddylation / nonfunctional rRNA decay / maintenance of DNA trinucleotide repeats / filamentous growth / COP9 signalosome / proteasome regulatory particle / cytosolic proteasome complex / proteasome regulatory particle, lid subcomplex / proteasome-activating activity / protein-containing complex localization / mitochondrial fission / proteasome regulatory particle, base subcomplex / K48-linked polyubiquitin modification-dependent protein binding / proteasome core complex assembly / nuclear outer membrane-endoplasmic reticulum membrane network / Cross-presentation of soluble exogenous antigens (endosomes) / TNFR2 non-canonical NF-kB pathway / proteasomal ubiquitin-independent protein catabolic process / Ub-specific processing proteases / peptide catabolic process / proteasome binding / regulation of protein catabolic process / protein deubiquitination / proteasome storage granule / polyubiquitin modification-dependent protein binding / endopeptidase activator activity / proteasome assembly / positive regulation of RNA polymerase II transcription preinitiation complex assembly / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / enzyme regulator activity / mRNA export from nucleus / : / protein folding chaperone / Neutrophil degranulation / proteasome complex / ubiquitin binding / proteasomal protein catabolic process / nucleotide-excision repair / positive regulation of transcription elongation by RNA polymerase II / double-strand break repair via homologous recombination / positive regulation of protein catabolic process / metallopeptidase activity / protein-macromolecule adaptor activity / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / endopeptidase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / molecular adaptor activity / regulation of cell cycle / クロマチンリモデリング / protein domain specific binding / mRNA binding / ubiquitin protein ligase binding / endoplasmic reticulum membrane / structural molecule activity / 小胞体 / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / ミトコンドリア / ATP binding / identical protein binding / metal ion binding / 細胞核 / 細胞質基質 / 細胞質

ドメイン・相同性:

Rpn9, C-terminal helix / Rpn9 C-terminal helix / Proteasomal ubiquitin receptor Rpn13/ADRM1 / Proteasomal ubiquitin receptor Rpn13/ADRM1, Pru domain superfamily / Rpn13/ADRM1, Pru domain / Proteasome complex subunit Rpn13, Pru domain / Pru (pleckstrin-like receptor for ubiquitin) domain profile. / : / 26S proteasome regulatory subunit RPN7/PSMD6 C-terminal helix / 26S proteasome non-ATPase regulatory subunit Rpn12 / 26S proteasome regulatory subunit, C-terminal / Proteasome regulatory subunit C-terminal / DSS1/SEM1 / 26S proteasome regulatory subunit RPN5, C-terminal domain / : / DSS1/SEM1 family / 26S proteasome regulatory subunit RPN5 C-terminal domain / 26S proteasome subunit RPN2, N-terminal domain / DSS1_SEM1 / 26S proteasome regulatory subunit Rpn6, N-terminal / 6S proteasome subunit Rpn6, C-terminal helix domain / 26S proteasome regulatory subunit RPN6 N-terminal domain / 26S proteasome subunit RPN6 C-terminal helix domain / 26S proteasome regulatory complex, non-ATPase subcomplex, Rpn2/Psmd1 subunit / 26S Proteasome non-ATPase regulatory subunit 13 / 26S proteasome regulatory subunit RPN2, C-terminal / 26S proteasome regulatory subunit RPN2 C-terminal domain / 26S Proteasome non-ATPase regulatory subunit 7/8 / : / 26S proteasome regulatory subunit 7, OB domain / 26S proteasome regulatory complex, non-ATPase subcomplex, Rpn1 subunit / RPN1, N-terminal / 26S proteasome non-ATPase regulatory subunit RPN1, C-terminal / RPN1 N-terminal domain / 26S proteasome non-ATPase regulatory subunit RPN1 C-terminal / 26S proteasome regulatory subunit Rpn7, N-terminal / 26S proteasome regulatory subunit Rpn7/COP9 signalosome complex subunit 1 / 26S proteasome subunit RPN7 / 26S Proteasome non-ATPase regulatory subunit 12/COP9 signalosome complex subunit 4 / Proteasome/cyclosome repeat / Proteasome/cyclosome repeat / PCI/PINT associated module / von Willebrand factor type A domain / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / HEAT repeats / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / PCI domain / Proteasome component (PCI) domain / PCI domain profile. / Ubiquitin interacting motif / Ubiquitin-interacting motif (UIM) domain profile. / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Proteasome beta subunit, C-terminal / Proteasome beta subunits C terminal / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / VWFA domain profile. / Proteasome subunit / Proteasome, subunit alpha/beta / von Willebrand factor, type A / AAA ATPase, AAA+ lid domain / AAA+ lid domain / TPR repeat region circular profile. / ATPase, AAA-type, conserved site / AAA-protein family signature. / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / TPR repeat profile. / MPN domain / MPN domain profile. / Nucleophile aminohydrolases, N-terminal / von Willebrand factor A-like domain superfamily / Tetratricopeptide repeat / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / ATPases associated with a variety of cellular activities

構成要素:

26S proteasome regulatory subunit RPN13 / 26S proteasome complex subunit SEM1 / Probable proteasome subunit alpha type-7 / Proteasome subunit alpha type-1 / Proteasome subunit beta type-4 / Proteasome subunit alpha type-3 / Proteasome subunit alpha type-2 / Proteasome subunit beta type-6 / Proteasome subunit beta type-2 / Proteasome subunit beta type-3 / Proteasome subunit beta type-5 / Proteasome subunit beta type-7 / Proteasome subunit alpha type-5 / 26S proteasome regulatory subunit RPN12 / 26S proteasome regulatory subunit RPN2 / 26S proteasome regulatory subunit 6A / 26S proteasome regulatory subunit 6B homolog / 26S proteasome regulatory subunit 7 homolog / Proteasome subunit beta type-1 / 26S proteasome regulatory subunit RPN1 / 26S proteasome regulatory subunit RPN10 / 26S proteasome regulatory subunit RPN3 / Proteasome subunit alpha type-6 / Proteasome subunit alpha type-4 / 26S proteasome regulatory subunit 4 homolog / Ubiquitin carboxyl-terminal hydrolase RPN11 / 26S proteasome subunit RPT4 / 26S proteasome regulatory subunit 8 homolog / 26S proteasome regulatory subunit RPN9 / 26S proteasome regulatory subunit RPN7 / 26S proteasome regulatory subunit RPN8 / 26S proteasome regulatory subunit RPN5 / 26S proteasome regulatory subunit RPN6

• 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: 9.3 Å
• データ登録者: Unverdorben P / Beck F / Sledz P / Schweitzer A / Pfeifer G / Plitzko JM / Baumeister W / Foerster F
• 引用: ジャーナル: Proc Natl Acad Sci U S A / 年: 2014; タイトル: Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome.; 著者: Pia Unverdorben / Florian Beck / Paweł Śledź / Andreas Schweitzer / Günter Pfeifer / Jürgen M Plitzko / Wolfgang Baumeister / Friedrich Förster / ; 要旨: The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle.

履歴

登録	2014年2月25日	-
ヘッダ(付随情報) 公開	2014年3月26日	-
マップ公開	2014年4月2日	-
更新	2016年2月17日	-
現状	2016年2月17日	処理サイト: PDBe / 状態: 公開

マップ

• ファイル: ダウンロード / ファイル: emd_2595.map.gz / 形式: CCP4 / 大きさ: 81.8 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• 注釈: Reconstruction of Intermediate state (s2).
• ボクセルのサイズ: X=Y=Z: 1.99 Å

密度

表面レベル	登録者による: 0.63 / ムービー #1: 0.63
最小 - 最大	-3.27841187 - 4.67604017
平均 (標準偏差)	0.01016377 (±0.1450758)

• 対称性: 空間群: 1

詳細

EMDB XML:

マップ形状	Axis order X Y Z Origin 0 0 0 サイズ 280 280 280 Spacing 280 280 280
セル	A=B=C: 557.2 Å α=β=γ: 90.0 °

CCP4マップ ヘッダ情報:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.99	1.99	1.99
M x/y/z	280	280	280
origin x/y/z	0.000	0.000	0.000
length x/y/z	557.200	557.200	557.200
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-207	-207	-206
NX/NY/NZ	414	414	414
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	280	280	280
D min/max/mean	-3.278	4.676	0.010

添付データ

試料の構成要素

全体 : 26S Proteasome from Saccharomyces cerevisiae

• 全体: 名称: 26S Proteasome from Saccharomyces cerevisiaeプロテアソーム

要素

• 試料: 26S Proteasome from Saccharomyces cerevisiaeプロテアソーム
• タンパク質・ペプチド: 26S Proteasomeプロテアソーム

超分子 #1000: 26S Proteasome from Saccharomyces cerevisiae

• 超分子: 名称: 26S Proteasome from Saccharomyces cerevisiae / タイプ: sample / ID: 1000 / Number unique components: 1
• 分子量: 実験値: 2.5 MDa / 理論値: 2.5 MDa

分子 #1: 26S Proteasome

• 分子: 名称: 26S Proteasome / タイプ: protein_or_peptide / ID: 1 / 組換発現: No
• 由来(天然): 生物種: Saccharomyces cerevisiae (パン酵母) / 別称: Baker's yeast
• 分子量: 実験値: 2.5 MDa / 理論値: 2.5 MDa

実験情報

構造解析

• 手法: クライオ電子顕微鏡法
• 解析: 単粒子再構成法
• 試料の集合状態: particle

試料調製

• 濃度: 0.3 mg/mL
• 凍結: 凍結剤: ETHANE / 装置: OTHER

電子顕微鏡法

• 顕微鏡: FEI TITAN KRIOS
• 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: FLOOD BEAM / 撮影モード: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / 最大 デフォーカス（公称値）: 3.5 µm / 最小 デフォーカス（公称値）: 1.0 µm
• 試料ステージ: 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER
• アライメント法: Legacy - Electron beam tilt params: 0
• 日付: 2013年12月24日
• 撮影: カテゴリ: CCD; フィルム・検出器のモデル: TVIPS TEMCAM-F816 (8k x 8k); 実像数: 30000 / 平均電子線量: 25 e/Ų / ビット/ピクセル: 14
• Tilt angle min: 0
• Tilt angle max: 0
• 実験機器: モデル: Titan Krios / 画像提供: FEI Company

画像解析

• CTF補正: 詳細: micrograph
• 最終 再構成: 想定した対称性 - 点群: C₁ (非対称) / アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 9.3 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: xmipp / 使用した粒子像数: 300000
• 詳細: The particles were selected using an automatic selection program. Each physical 26S particles was considered as two particles for processing according to pseudo-C2 symmetry.