- PDB-6tup: Cryo-EM structure of Pf4 bacteriophage coat protein with single-s... -
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基本情報
登録情報
データベース: PDB / ID: 6tup
タイトル
Cryo-EM structure of Pf4 bacteriophage coat protein with single-stranded DNA
要素
Coat protein B of bacteriophage Pf1
DNA (5'-D(P*AP*AP*AP*AP*AP*A)-3')
キーワード
VIRUS / Bacteriophage / helical / filamentous
機能・相同性
Inovirus Coat protein B / Capsid protein G8P / helical viral capsid / host cell membrane / membrane / DNA / Capsid protein G8P / Coat protein B of bacteriophage Pf1
ジャーナル: Proc Natl Acad Sci U S A / 年: 2020 タイトル: Phage liquid crystalline droplets form occlusive sheaths that encapsulate and protect infectious rod-shaped bacteria. 著者: Abul K Tarafder / Andriko von Kügelgen / Adam J Mellul / Ulrike Schulze / Dirk G A L Aarts / Tanmay A M Bharat / 要旨: The opportunistic pathogen is a major cause of antibiotic-tolerant infections in humans. evades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral ...The opportunistic pathogen is a major cause of antibiotic-tolerant infections in humans. evades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral prophage, Pf4. We investigated the mechanism of phage-mediated antibiotic tolerance using biochemical reconstitution combined with structural biology and high-resolution cellular imaging. We resolved electron cryomicroscopy atomic structures of Pf4 with and without its linear single-stranded DNA genome, and studied Pf4 assembly into liquid crystalline droplets using optical microscopy and electron cryotomography. By biochemically replicating conditions necessary for antibiotic protection, we found that phage liquid crystalline droplets form phase-separated occlusive compartments around rod-shaped bacteria leading to increased bacterial survival. Encapsulation by these compartments was observed even when inanimate colloidal rods were used to mimic rod-shaped bacteria, suggesting that shape and size complementarity profoundly influences the process. Filamentous inoviruses are pervasive across prokaryotes, and in particular, several Gram-negative bacterial pathogens including , and harbor these prophages. We propose that biophysical occlusion mediated by secreted filamentous molecules such as Pf4 may be a general strategy of bacterial survival in harsh environments.
The helical filament symmetry is only applied to the Pf4 coat protein and not the DNA. The reason for this is that the DNA bases are not resolved clearly due to averaging along the ssDNA genome of the phage, meaning that refinement of the protein into the map is of a higher quality than the ssDNA, where a poly-adenine model has been built.
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要素
#1: タンパク質・ペプチド
CoatproteinBofbacteriophagePf1 / Phage coat protein B
濃度: 5 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES / 詳細: Filamentous phage purified from source
試料支持
詳細: 20 second glow discharge at 15 mA in a LeicaEM ACE200 グリッドの材料: COPPER/RHODIUM / グリッドのサイズ: 200 divisions/in. / グリッドのタイプ: Quantifoil R2/2
急速凍結
装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 283 K 詳細: Samples for cryo-EM were prepared by pipetting 2.5 ul of the sample onto freshly glow-discharged Quantifoil grids (Cu/Rh R2/2, 200 mesh). Grids were blotted for 2.5 seconds with a blot force ...詳細: Samples for cryo-EM were prepared by pipetting 2.5 ul of the sample onto freshly glow-discharged Quantifoil grids (Cu/Rh R2/2, 200 mesh). Grids were blotted for 2.5 seconds with a blot force of -15, 0.5 second drain and 0 second wait times.
B value: 93.74 / プロトコル: BACKBONE TRACE / 空間: REAL / Target criteria: Correlation coefficient 詳細: The helical filament symmetry is only applied to the Pf4 coat protein and not the DNA. The reason for this is that the DNA bases are not resolved clearly due to averaging along the ssDNA ...詳細: The helical filament symmetry is only applied to the Pf4 coat protein and not the DNA. The reason for this is that the DNA bases are not resolved clearly due to averaging along the ssDNA genome of the phage, meaning that refinement of the protein into the map is of a higher quality than the ssDNA, where a poly-adenine model has been built.