- PDB-6w6m: Single particle cryoEM structure of V. cholerae Type IV competenc... -
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基本情報
登録情報
データベース: PDB / ID: 6w6m
タイトル
Single particle cryoEM structure of V. cholerae Type IV competence pilus secretin PilQ
要素
Type IV pilus secretin PilQ family protein
キーワード
TRANSPORT PROTEIN / secretion system / outer membrane protein
機能・相同性
機能・相同性情報
pilus assembly / protein secretion by the type II secretion system / protein secretion / cell outer membrane 類似検索 - 分子機能
Type IV pilus secretin PilQ / Secretin/TonB, short N-terminal domain / Secretin and TonB N terminus short domain / GspD/PilQ family / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / NolW-like / Bacterial type II/III secretion system short domain / NolW-like superfamily / Type II/III secretion system / Bacterial type II and III secretion system protein 類似検索 - ドメイン・相同性
Type IV pilus secretin PilQ family protein / Fimbrial assembly protein 類似検索 - 構成要素
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM128674
米国
引用
ジャーナル: Nat Commun / 年: 2020 タイトル: CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae. 著者: Sara J Weaver / Davi R Ortega / Matthew H Sazinsky / Triana N Dalia / Ankur B Dalia / Grant J Jensen / 要旨: Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of ...Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.
A: Type IV pilus secretin PilQ family protein B: Type IV pilus secretin PilQ family protein C: Type IV pilus secretin PilQ family protein D: Type IV pilus secretin PilQ family protein E: Type IV pilus secretin PilQ family protein F: Type IV pilus secretin PilQ family protein G: Type IV pilus secretin PilQ family protein H: Type IV pilus secretin PilQ family protein I: Type IV pilus secretin PilQ family protein J: Type IV pilus secretin PilQ family protein K: Type IV pilus secretin PilQ family protein L: Type IV pilus secretin PilQ family protein M: Type IV pilus secretin PilQ family protein N: Type IV pilus secretin PilQ family protein
名称: Vibrio cholerae Type IV competence pilus secretin PilQ タイプ: COMPLEX / Entity ID: all / 由来: NATURAL
分子量
値: 0.826 MDa / 実験値: NO
由来(天然)
生物種: Vibrio cholerae (コレラ菌) / 株: TND1751
緩衝液
pH: 8
緩衝液成分
ID
濃度
名称
式
Buffer-ID
1
50mM
TrisHCl
1
2
300mM
sodiumchloride
NaCl
1
試料
濃度: 0.8 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: The protein was purified using Ni NTA agarose beads (Anatrace, SUPER-NINTA25) and concentrated to ~1 mg/mL. PilQ was then exchanged into Amphipol A8-35 (0.585 mg for a 3:1 ratio, Anatrace, ...詳細: The protein was purified using Ni NTA agarose beads (Anatrace, SUPER-NINTA25) and concentrated to ~1 mg/mL. PilQ was then exchanged into Amphipol A8-35 (0.585 mg for a 3:1 ratio, Anatrace, A835). BioBeads were used to remove excess DDM and the sample was concentrated to ~0.8 mg/mL
エネルギーフィルター名称: GIF Quantum LS / エネルギーフィルタースリット幅: 20 eV
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解析
ソフトウェア
名称: PHENIX / バージョン: 1.16_3549: / 分類: 精密化
EMソフトウェア
ID
名称
バージョン
カテゴリ
詳細
1
cryoSPARC
粒子像選択
2
SerialEM
画像取得
4
CTFFIND
CTF補正
wholemicrographs
5
RELION
CTF補正
PerparticleCtfRefine
10
RELION
初期オイラー角割当
11
RELION
最終オイラー角割当
13
RELION
3次元再構成
20
PHENIX
1.16-dev3549
モデル精密化
画像処理
詳細: MotionCor2 was used for motion correction and dose weighting of 3,808 movies. CTF correction was used to evaluate micrograph quality. 2,510 micrographs were selected for particle picking.
CTF補正
詳細: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion. タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION
粒子像の選択
選択した粒子像数: 252319 詳細: CryoSPARC blob picking on 2,510 micrographs yielded 3,100,353 potential particles. After inspection in the cryoSPARC GUI, the 252,319 particles were extracted.
対称性
点対称性: C14 (14回回転対称)
3次元再構成
解像度: 2.7 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 100543 / 対称性のタイプ: POINT