Biotechnology and Biological Sciences Research Council (BBSRC)
BB/M011151/1
英国
Wellcome Trust
222373/Z/21/Z
英国
Wellcome Trust
105220/Z/14/Z
英国
Research Foundation - Flanders (FWO)
G0G0818N
ベルギー
Royal Society
RSRP/R1/211057
英国
引用
ジャーナル: Nat Commun / 年: 2021 タイトル: The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding. 著者: Paul White / Samuel F Haysom / Matthew G Iadanza / Anna J Higgins / Jonathan M Machin / James M Whitehouse / Jim E Horne / Bob Schiffrin / Charlotte Carpenter-Platt / Antonio N Calabrese / ...著者: Paul White / Samuel F Haysom / Matthew G Iadanza / Anna J Higgins / Jonathan M Machin / James M Whitehouse / Jim E Horne / Bob Schiffrin / Charlotte Carpenter-Platt / Antonio N Calabrese / Kelly M Storek / Steven T Rutherford / David J Brockwell / Neil A Ranson / Sheena E Radford / 要旨: The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit ...The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.
エネルギーフィルター名称: GIF Bioquantum / エネルギーフィルタースリット幅: 20 eV
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解析
EMソフトウェア
ID
名称
バージョン
カテゴリ
詳細
2
EPU
画像取得
4
Gctf
1.18
CTF補正
7
UCSF Chimera
モデルフィッティング
9
RELION
3
初期オイラー角割当
10
cryoSPARC
2.2.0
最終オイラー角割当
final refinement used the non-uniform refinement in cryoSPARC
11
RELION
3
分類
12
RELION
3
3次元再構成
half maps from non-uniform refinement were postprocessed in RELION 3.0
13
cryoSPARC
2.2.0
3次元再構成
final refinement used the non-uniform refinement in cryoSPARC
14
NAMD
モデル精密化
15
MDFF
モデル精密化
16
VMD
モデル精密化
17
PHENIX
1.14
モデル精密化
18
Coot
0.9
モデル精密化
EMoptimisedversionfromccpembundle
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: 703997
3次元再構成
解像度: 5.2 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 131853 / 対称性のタイプ: POINT
原子モデル構築
B value: 183 / プロトコル: FLEXIBLE FIT / 空間: REAL 詳細: The starting BAM model was created from 5LJO (lateral-open BAM-WT cryoEM structure) with BamA residues 687-700 replaced by those from 5EKQ (crystal structure of BamACDE in a lateral-open ...詳細: The starting BAM model was created from 5LJO (lateral-open BAM-WT cryoEM structure) with BamA residues 687-700 replaced by those from 5EKQ (crystal structure of BamACDE in a lateral-open conformation). This and 7BM5 (Fab1 crystal structure) were rigid fitted into the BAM-WT Fab1 complex electron density in UCSF Chimera. Molecular dynamics based flexible fitting was then set up in VMD and run in NAMD to fit the structures into the density. The resulting structure was finally real-space refined in phenix to generate the final model.