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- PDB-6px4: Crystal structure of the complex between periplasmic domains of a... -

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Basic information

Entry
Database: PDB / ID: 6px4
TitleCrystal structure of the complex between periplasmic domains of antiholin RI and holin T from T4 phage, in H32
Components
  • Antiholin
  • Holin
KeywordsVIRAL PROTEIN / phage / lysis inhibition
Function / homology
Function and homology information


: / host cell periplasmic space / pore-forming activity / cytolysis / molecular function inhibitor activity / viral release from host cell by cytolysis / killing of cells of another organism / host cell plasma membrane / DNA binding / membrane
Similarity search - Function
Bacteriophage T4, GpT, holin / Bacteriophage T holin / Antiholin T4 type
Similarity search - Domain/homology
Holin / Antiholin / Holin / Antiholin
Similarity search - Component
Biological speciesEscherichia phage ECML-134 (virus)
Escherichia phage vB_EcoM_NBG2 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.65 Å
AuthorsKrieger, I.V. / Sacchettini, J.C.
Funding support United States, 1items
OrganizationGrant numberCountry
Welch FoundationA-0015 United States
CitationJournal: J Mol Biol / Year: 2020
Title: The Structural Basis of T4 Phage Lysis Control: DNA as the Signal for Lysis Inhibition.
Authors: Inna V Krieger / Vladimir Kuznetsov / Jeng-Yih Chang / Junjie Zhang / Samir H Moussa / Ryland F Young / James C Sacchettini /
Abstract: Optimal phage propagation depends on the regulation of the lysis of the infected host cell. In T4 phage infection, lysis occurs when the holin protein (T) forms lesions in the host membrane. However, ...Optimal phage propagation depends on the regulation of the lysis of the infected host cell. In T4 phage infection, lysis occurs when the holin protein (T) forms lesions in the host membrane. However, the lethal function of T can be blocked by an antiholin (RI) during lysis inhibition (LIN). LIN sets if the infected cell undergoes superinfection, then the lysis is delayed until host/phage ratio becomes more favorable for the release of progeny. It has been thought that a signal derived from the superinfection is required to activate RI. Here we report structures that suggest a radically different model in which RI binds to T irrespective of superinfection, causing it to accumulate in a membrane as heterotetrameric 2RI-2T complex. Moreover, we show the complex binds non-specifically to DNA, suggesting that the gDNA from the superinfecting phage serves as the LIN signal and that stabilization of the complex by DNA binding is what defines LIN. Finally, we show that soluble domain of free RI crystallizes in a domain-swapped homotetramer, which likely works as a sink for RI molecules released from the RI-T complex to ensure efficient lysis. These results constitute the first structural basis and a new model not only for the historic LIN phenomenon but also for the temporal regulation of phage lysis in general.
History
DepositionJul 24, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 24, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 1, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 12, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: Antiholin
T: Holin
A: Antiholin
B: Holin


Theoretical massNumber of molelcules
Total (without water)50,4754
Polymers50,4754
Non-polymers00
Water4,504250
1
R: Antiholin
T: Holin
A: Antiholin
B: Holin
x 6


Theoretical massNumber of molelcules
Total (without water)302,84924
Polymers302,84924
Non-polymers00
Water43224
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
crystal symmetry operation4_555y,x,-z1
crystal symmetry operation5_555x-y,-y,-z1
crystal symmetry operation6_555-x,-x+y,-z1
Buried area43840 Å2
ΔGint-203 kcal/mol
Surface area103790 Å2
MethodPISA
Unit cell
Length a, b, c (Å)116.546, 116.546, 223.384
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number155
Space group name H-MH32
Components on special symmetry positions
IDModelComponents
11T-304-

HOH

21A-151-

HOH

31B-353-

HOH

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Components

#1: Protein Antiholin


Mass: 8801.884 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage ECML-134 (virus) / Gene: ECML134_104 / Production host: Escherichia coli (E. coli) / References: UniProt: I7AU04, UniProt: P13304*PLUS
#2: Protein Holin


Mass: 16435.533 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage vB_EcoM_NBG2 (virus) / Gene: vBEcoMNBG2_239 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2U8QQK7, UniProt: P06808*PLUS
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 250 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.91 Å3/Da / Density % sol: 57.75 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 7 / Details: 0.8 M Na/K Tartrate

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Data collection

DiffractionMean temperature: 120 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.987 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Apr 1, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 1.65→99 Å / Num. obs: 68307 / % possible obs: 97.2 % / Redundancy: 7.29 % / Biso Wilson estimate: 22.32 Å2 / Rmerge(I) obs: 0.065 / Net I/σ(I): 16.45
Reflection shellResolution: 1.65→1.75 Å / Num. unique obs: 9569 / Rsym value: 0.3382 / % possible all: 85.1

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHENIX1.11.1_2575refinement
SAINTdata reduction
SAINTdata scaling
MOLREPphasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.65→33.644 Å / SU ML: 0.18 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 22.92 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2252 3429 5.04 %
Rwork0.1935 64659 -
obs0.1951 68088 96.92 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 65.44 Å2 / Biso mean: 25.8733 Å2 / Biso min: 12.66 Å2
Refinement stepCycle: final / Resolution: 1.65→33.644 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3499 0 0 250 3749
Biso mean---29.66 -
Num. residues----427
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0073660
X-RAY DIFFRACTIONf_angle_d0.8754961
X-RAY DIFFRACTIONf_chiral_restr0.049513
X-RAY DIFFRACTIONf_plane_restr0.006643
X-RAY DIFFRACTIONf_dihedral_angle_d7.142899
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
1.65-1.67360.26921040.2834214277
1.6736-1.69860.32451200.263225582
1.6986-1.72510.31491340.2486238387
1.7251-1.75340.32161360.2498258894
1.7534-1.78370.25431430.2346270398
1.7837-1.81610.27951380.2312272399
1.8161-1.8510.2531310.2214275099
1.851-1.88880.2771330.2216273999
1.8888-1.92990.25191700.2275273199
1.9299-1.97470.2741320.2155274599
1.9747-2.02410.211370.2093274599
2.0241-2.07880.24481440.2174275499
2.0788-2.140.23551280.20722746100
2.14-2.20910.2191330.20322784100
2.2091-2.2880.23651650.2132273799
2.288-2.37960.24771640.20512748100
2.3796-2.48790.231430.21332768100
2.4879-2.6190.27541450.20772759100
2.619-2.7830.25611660.20822776100
2.783-2.99770.23771630.19862761100
2.9977-3.29920.22491510.1939277599
3.2992-3.7760.21851440.17852820100
3.776-4.75530.16511470.1467282299
4.7553-33.60.17191580.1575290599

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