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- PDB-8v3w: CryoEM Structure of Diffocin - precontracted - Baseplate - focuse... -

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Basic information

Entry
Database: PDB / ID: 8v3w
TitleCryoEM Structure of Diffocin - precontracted - Baseplate - focused refinement on triplex region
Components
  • Hub-Hydrolase (CD1368)
  • Sheath (CD1363)
  • Sheath initiator (CD1370)
  • Spike (CD1369)
  • TRI-1 (CD1372)
  • TRI-2 (CD1371)
  • Tape measure protein (CD1366)
  • Tube (CD1364)
  • Tube tail (CD1367)
KeywordsVIRUS LIKE PARTICLE / Phage tail-like / bacteriocin / baseplate / pre-contraction
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases) / hydrolase activity
Similarity search - Function
Protein of unknown function, DUF2577 / Protein of unknown function (DUF2577) / Bacteriophage Mu-like, Gp48 / Protein of unknown function DUF2634 / : / Bacteriophage Mu-like, Gp48 / Protein of unknown function (DUF2634) / : / Phage tail tube protein / XkdM-like superfamily ...Protein of unknown function, DUF2577 / Protein of unknown function (DUF2577) / Bacteriophage Mu-like, Gp48 / Protein of unknown function DUF2634 / : / Bacteriophage Mu-like, Gp48 / Protein of unknown function (DUF2634) / : / Phage tail tube protein / XkdM-like superfamily / Phage tail tube protein / Baseplate protein J-like / Baseplate J-like protein / NlpC/P60 domain profile. / Endopeptidase, NLPC/P60 domain / NlpC/P60 family / 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 / Papain-like cysteine peptidase superfamily
Similarity search - Domain/homology
DUF2577 family protein / Phage portal protein / XkdP-like protein / DUF2634 domain-containing protein / Base plate protein / Base plate protein / C40 family peptidase / Phage tail sheath protein / Phage-related protein
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 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

Downloads & links

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Assembly

Deposited unit
E: TRI-2 (CD1371)
A: TRI-2 (CD1371)
N: TRI-1 (CD1372)
C: Spike (CD1369)
B: Tape measure protein (CD1366)
G: Tube tail (CD1367)
L: Sheath initiator (CD1370)
c: Sheath (CD1363)
a: Tube (CD1364)
I: Tube (CD1364)
D: Sheath (CD1363)
H: Hub-Hydrolase (CD1368)
S: Sheath (CD1363)
R: Tube (CD1364)
P: Tube (CD1364)
K: Sheath (CD1363)
J: TRI-2 (CD1371)
M: TRI-2 (CD1371)
Q: TRI-1 (CD1372)
v: TRI-2 (CD1371)
r: TRI-2 (CD1371)
4: TRI-1 (CD1372)
t: Spike (CD1369)
s: Tape measure protein (CD1366)
w: Tube tail (CD1367)
2: Sheath initiator (CD1370)
AA: Sheath (CD1363)
9: Tube (CD1364)
z: Tube (CD1364)
u: Sheath (CD1363)
x: Hub-Hydrolase (CD1368)
8: Sheath (CD1363)
7: Tube (CD1364)
5: Tube (CD1364)
1: Sheath (CD1363)
O: Sheath initiator (CD1370)
T: Tube tail (CD1367)
0: TRI-2 (CD1371)
3: TRI-2 (CD1371)
6: TRI-1 (CD1372)
Y: TRI-2 (CD1371)
U: TRI-2 (CD1371)
i: TRI-1 (CD1372)
W: Spike (CD1369)
V: Tape measure protein (CD1366)
y: Tube tail (CD1367)
Z: Tube tail (CD1367)
g: Sheath initiator (CD1370)
F: Sheath initiator (CD1370)
q: Sheath (CD1363)
p: Tube (CD1364)
d: Tube (CD1364)
X: Sheath (CD1363)
b: Hub-Hydrolase (CD1368)
n: Sheath (CD1363)
m: Tube (CD1364)
k: Tube (CD1364)
f: Sheath (CD1363)
j: Sheath initiator (CD1370)
o: Tube tail (CD1367)
e: TRI-2 (CD1371)
h: TRI-2 (CD1371)
l: TRI-1 (CD1372)


Theoretical massNumber of molelcules
Total (without water)1,995,97863
Polymers1,995,97863
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

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Protein , 9 types, 63 molecules EAJMvr03YUehNQ46ilCtWBsVGwTyZo...

#1: Protein
TRI-2 (CD1371)


Mass: 39603.281 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: rtbJ / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A1X9JZB1
#2: Protein
TRI-1 (CD1372)


Mass: 26473.488 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: rtbK / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A1X9JZH3
#3: Protein Spike (CD1369)


Mass: 12626.413 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: BN1095_340096 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A031WFB8
#4: Protein Tape measure protein (CD1366)


Mass: 89157.289 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: SAMEA3375112_00268 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A9Q9TVH0
#5: Protein
Tube tail (CD1367)


Mass: 16234.616 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: BN1095_340093 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A031WFY8
#6: Protein
Sheath initiator (CD1370)


Mass: 16549.959 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: BN1095_340097 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A069AE46
#7: Protein
Sheath (CD1363)


Mass: 39268.430 Da / Num. of mol.: 12
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
#8: Protein
Tube (CD1364)


Mass: 16028.353 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: xkdM / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A031WFC4
#9: Protein Hub-Hydrolase (CD1368)


Mass: 65426.082 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridioides difficile (bacteria) / Gene: rtbG / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A1X9K255

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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
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

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 116539 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008118659
ELECTRON MICROSCOPYf_angle_d0.687160068
ELECTRON MICROSCOPYf_dihedral_angle_d4.29915648
ELECTRON MICROSCOPYf_chiral_restr0.04518387
ELECTRON MICROSCOPYf_plane_restr0.00520349

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