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- PDB-8v3t: CryoEM Structure of Diffocin - precontracted - Collar -

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Basic information

Entry
Database: PDB / ID: 8v3t
TitleCryoEM Structure of Diffocin - precontracted - Collar
Components
  • Collar (CD1362)
  • Sheath (CD1363)
  • Tube (CD1364)
KeywordsVIRUS LIKE PARTICLE / Phage tail-like / bacteriocin / collar / pre-contraction
Function / homology
Function and homology information


: / Domain of unknown function (DUF6838) / Phage tail tube protein / XkdM-like superfamily / Phage tail tube protein / Tail sheath protein, subtilisin-like domain / Phage tail sheath protein subtilisin-like domain / Tail sheath protein, C-terminal domain / Phage tail sheath C-terminal domain
Similarity search - Domain/homology
Phage portal protein / RtbA / Phage tail sheath protein
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsCai, X.Y. / He, Y. / Zhou, Z.H.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI085318 United States
CitationJournal: Nat Commun / Year: 2024
Title: Atomic structures of a bacteriocin targeting Gram-positive bacteria.
Authors: Xiaoying Cai / Yao He / Iris Yu / Anthony Imani / Dean Scholl / Jeff F Miller / Z Hong Zhou /
Abstract: Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; ...Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics.
History
DepositionNov 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 28, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: Sheath (CD1363)
A: Sheath (CD1363)
C: Sheath (CD1363)
a: Tube (CD1364)
B: Tube (CD1364)
E: Tube (CD1364)
b: Collar (CD1362)
V: Sheath (CD1363)
M: Sheath (CD1363)
S: Sheath (CD1363)
k: Tube (CD1364)
P: Tube (CD1364)
Y: Tube (CD1364)
n: Collar (CD1362)
W: Sheath (CD1363)
N: Sheath (CD1363)
T: Sheath (CD1363)
l: Tube (CD1364)
Q: Tube (CD1364)
Z: Tube (CD1364)
o: Collar (CD1362)
I: Sheath (CD1363)
F: Sheath (CD1363)
H: Sheath (CD1363)
K: Tube (CD1364)
G: Tube (CD1364)
J: Tube (CD1364)
L: Collar (CD1362)
X: Sheath (CD1363)
O: Sheath (CD1363)
U: Sheath (CD1363)
m: Tube (CD1364)
R: Tube (CD1364)
c: Tube (CD1364)
p: Collar (CD1362)
g: Sheath (CD1363)
d: Sheath (CD1363)
f: Sheath (CD1363)
i: Tube (CD1364)
e: Tube (CD1364)
h: Tube (CD1364)
j: Collar (CD1362)


Theoretical massNumber of molelcules
Total (without water)1,098,53542
Polymers1,098,53542
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Sheath (CD1363)


Mass: 39268.430 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: SAMEA3375112_00264 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A9Q7ZU73
#2: Protein
Tube (CD1364)


Mass: 16028.353 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: xkdM / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A031WFC4
#3: Protein
Collar (CD1362)


Mass: 17198.816 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: rtbA / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A1X9JZ99

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Diffocin / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Clostridioides difficile (bacteria)
Source (recombinant)Organism: Bacillus subtilis (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144368 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00877358
ELECTRON MICROSCOPYf_angle_d0.716104184
ELECTRON MICROSCOPYf_dihedral_angle_d4.35110242
ELECTRON MICROSCOPYf_chiral_restr0.04412330
ELECTRON MICROSCOPYf_plane_restr0.00413044
NMR softwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement

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