ジャーナル: Nature / 年: 2018 タイトル: Structure of the membrane-assembled retromer coat determined by cryo-electron tomography. 著者: Oleksiy Kovtun / Natalya Leneva / Yury S Bykov / Nicholas Ariotti / Rohan D Teasdale / Miroslava Schaffer / Benjamin D Engel / David J Owen / John A G Briggs / Brett M Collins / 要旨: Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular ...Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.
ダウンロード / ファイル: emd_0158.map.gz / 形式: CCP4 / 大きさ: 30.5 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
D_1200011186
ボクセルのサイズ
X=Y=Z: 1.35 Å
密度
表面レベル
登録者による: 0.15 / ムービー #1: 0.15
最小 - 最大
-0.20608203 - 0.5279172
平均 (標準偏差)
0.007617583 (±0.0697486)
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
200
200
200
Spacing
200
200
200
セル
A=B=C: 270.0 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
1.35
1.35
1.35
M x/y/z
200
200
200
origin x/y/z
0.000
0.000
0.000
length x/y/z
270.000
270.000
270.000
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
200
200
200
D min/max/mean
-0.206
0.528
0.008
-
添付データ
-
試料の構成要素
-
全体 : Retromer-Vps5 complex assembled on the membrane.
全体
名称: Retromer-Vps5 complex assembled on the membrane.
要素
複合体: Retromer-Vps5 complex assembled on the membrane.
タンパク質・ペプチド: Vacuolar protein sorting-associated protein 5
-
超分子 #1: Retromer-Vps5 complex assembled on the membrane.
超分子
名称: Retromer-Vps5 complex assembled on the membrane. / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all 詳細: Vps26/Vps35/Vps29 trimer recruited to the membrane via Vps5 PX-BAR protein.
由来(天然)
生物種: Chaetomium thermophilum (菌類)
組換発現
生物種: Escherichia coli (大腸菌)
-
分子 #1: Vacuolar protein sorting-associated protein 5
分子
名称: Vacuolar protein sorting-associated protein 5 / タイプ: protein_or_peptide / ID: 1 / 光学異性体: LEVO
After motion correction in "alignframes" (IMOD), each of the images in the tilt series was low-pass filtered according to the electron-dose acquired by the sample (Grant and Grigorieff, 2015).
最終 再構成
想定した対称性 - 点群: C2 (2回回転対称) / アルゴリズム: BACK PROJECTION / 解像度のタイプ: BY AUTHOR / 解像度: 9.4 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 使用したサブトモグラム数: 18680
抽出
トモグラム数: 71 / 使用した粒子像数: 194885 / 参照モデル: reference-free 手法: geometrical seeding alogn the surface of manually traced tubules
CTF補正
ソフトウェア: (名称: CTFFIND (ver. 4), IMOD) 詳細: CTF correction was performed with ctfphaseflip IMOD command using defocus values measured by CTFFIND4 on non-dose-filtered images.
最終 角度割当
タイプ: OTHER / ソフトウェア: (名称: AV3, TOM) 詳細: Image processing was performed in TOM Toolbox, AV3 and Dynamo toolboxes.