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- PDB-6psh: Crystal structure of periplasmic domain of antiholin RI from T4 phage -

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Basic information

Entry
Database: PDB / ID: 6psh
TitleCrystal structure of periplasmic domain of antiholin RI from T4 phage
ComponentsAntiholin
KeywordsVIRAL PROTEIN / phage / lysis inhibition
Function / homologyAntiholin T4 type / host cell periplasmic space / molecular function inhibitor activity / killing of cells of another organism / host cell plasma membrane / DNA binding / Antiholin
Function and homology information
Biological speciesEnterobacteria phage T4 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.21 Å
AuthorsKuznetsov, V.B. / Krieger, 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 12, 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

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Assembly

Deposited unit
A: Antiholin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)10,3912
Polymers10,3291
Non-polymers621
Water18010
1
A: Antiholin
hetero molecules

A: Antiholin
hetero molecules

A: Antiholin
hetero molecules

A: Antiholin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,5638
Polymers41,3144
Non-polymers2484
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_655-x+1,-y,z1
crystal symmetry operation8_556x-y,-y,-z+11
crystal symmetry operation11_656-x+y+1,y,-z+11
MethodPISA
Unit cell
Length a, b, c (Å)49.975, 49.975, 118.677
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number180
Space group name H-MP6222
Components on special symmetry positions
IDModelComponents
11A-304-

HOH

21A-308-

HOH

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Components

#1: Protein Antiholin / Protein rI


Mass: 10328.606 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T4 (virus) / Gene: rI, 58.6, rIA, tk.-2 / Production host: Escherichia coli (E. coli) / References: UniProt: P13304
#2: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C2H6O2
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.07 Å3/Da / Density % sol: 40.61 %
Crystal growTemperature: 289 K / Method: vapor diffusion / pH: 4.5
Details: 100 mM Na citrate (pH 4.5), 1M Na acetate, 0.5% Anapoe X114

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 0.9795 Å
DetectorType: MAR scanner 300 mm plate / Detector: IMAGE PLATE / Date: Apr 5, 2010
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.21→50 Å / Num. obs: 4888 / % possible obs: 99.9 % / Redundancy: 25.1 % / Biso Wilson estimate: 45.99 Å2 / Rmerge(I) obs: 0.106 / Χ2: 1.831 / Net I/σ(I): 6.3 / Num. measured all: 122852
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsΧ2Diffraction-ID% possible all
2.21-2.2519.20.72400.5171100
2.25-2.2920.50.6082290.442199.6
2.29-2.3324.50.6962300.5591100
2.33-2.3825.10.6162390.481100
2.38-2.4326.60.5022290.511100
2.43-2.4927.10.4622380.4991100
2.49-2.5526.90.3512310.561100
2.55-2.6227.90.282440.5591100
2.62-2.727.10.2232320.5871100
2.7-2.7826.90.2112410.6321100
2.78-2.8826.70.1522370.7741100
2.88-327.20.1622390.9421100
3-3.1426.10.1172471.0641100
3.14-3.326.90.1042391.3521100
3.3-3.5126.20.0952391.9471100
3.51-3.78260.0942532.3571100
3.78-4.1625.20.0872513.4631100
4.16-4.7624.60.0812543.8811100
4.76-623.50.0822655.5551100
6-5020.20.0843119.338198.7

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Processing

Software
NameVersionClassification
PHENIX1.11.1_2575refinement
HKL-2000data scaling
PDB_EXTRACT3.25data extraction
TRUNCATEdata reduction
SHARPphasing
RefinementMethod to determine structure: SAD / Resolution: 2.21→40.66 Å / SU ML: 0.29 / Cross valid method: THROUGHOUT / σ(F): 0.33 / Phase error: 38.38
RfactorNum. reflection% reflection
Rfree0.3229 476 10 %
Rwork0.2841 --
obs0.2885 4758 97.92 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 135.15 Å2 / Biso mean: 60.5893 Å2 / Biso min: 25.65 Å2
Refinement stepCycle: final / Resolution: 2.21→40.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms579 0 4 10 593
Biso mean--72.51 68.64 -
Num. residues----70
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.006616
X-RAY DIFFRACTIONf_angle_d0.731826
X-RAY DIFFRACTIONf_chiral_restr0.04581
X-RAY DIFFRACTIONf_plane_restr0.005108
X-RAY DIFFRACTIONf_dihedral_angle_d11.979517
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.2104-2.53030.39991490.3066133895
2.5303-3.18770.39191560.3162140499
3.1877-40.60.29271710.271540100

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