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- PDB-8dkr: Pseudomonas-phage E217 TerL nuclease domain -

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Basic information

Entry
Database: PDB / ID: 8dkr
TitlePseudomonas-phage E217 TerL nuclease domain
ComponentsLarge terminase protein
KeywordsHYDROLASE / viral genome packaging motor / large terminase / TerL / Pseudomonas phage E217 / nuclease
Function / homologyPhage terminase large subunit, N-terminal / Phage terminase large subunit / Bacteriophage terminase, large subunit / P-loop containing nucleoside triphosphate hydrolase / Large terminase protein
Function and homology information
Biological speciesPseudomonas phage vB_PaeM_E217 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.05 Å
AuthorsCingolani, G. / Lokareddy, R. / Hou, D.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM100888 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM140733 United States
National Institutes of Health/Office of the DirectorOD017987 United States
National Institutes of Health/Office of the DirectorOD023479 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA56036 United States
CitationJournal: J Mol Biol / Year: 2022
Title: Terminase Subunits from the Pseudomonas-Phage E217.
Authors: Ravi K Lokareddy / Chun-Feng David Hou / Steven G Doll / Fenglin Li / Richard E Gillilan / Francesca Forti / David S Horner / Federica Briani / Gino Cingolani /
Abstract: Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae ...Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ∼58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.
History
DepositionJul 6, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 14, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.title / _citation_author.name
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Large terminase protein
A: Large terminase protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,0485
Polymers56,9752
Non-polymers733
Water3,675204
1
B: Large terminase protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,5122
Polymers28,4871
Non-polymers241
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: Large terminase protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,5363
Polymers28,4871
Non-polymers492
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)59.601, 62.628, 149.253
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21(chain B and resid 214 through 451)

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: LYS / Beg label comp-ID: LYS / End auth comp-ID: ILE / End label comp-ID: ILE / Auth seq-ID: 214 - 451 / Label seq-ID: 9 - 246

Dom-IDSelection detailsAuth asym-IDLabel asym-ID
1chain AAB
2(chain B and resid 214 through 451)BA

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Components

#1: Protein Large terminase protein


Mass: 28487.459 Da / Num. of mol.: 2 / Fragment: C-terminal nuclease domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage vB_PaeM_E217 (virus) / Gene: vBPaeME217_00005 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A2K8HL37, Hydrolases; Acting on ester bonds
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 204 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.44 Å3/Da / Density % sol: 49.68 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.6
Details: 0.1 M sodium citrate tribasic dihydrate, pH 5.6, 0.7 M sodium citrate tribasic dihydrate, 10 mM 2-mercaptoethanol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-1 / Wavelength: 0.976 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Feb 27, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 2.05→15 Å / Num. obs: 31650 / % possible obs: 88.1 % / Redundancy: 5.8 % / Biso Wilson estimate: 30.97 Å2 / CC1/2: 0.984 / Rpim(I) all: 0.036 / Rsym value: 0.087 / Net I/σ(I): 20.3
Reflection shellResolution: 2.05→2.12 Å / Redundancy: 2 % / Num. unique obs: 2226 / CC1/2: 0.836 / Rpim(I) all: 0.332 / Rsym value: 0.44 / % possible all: 63

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Processing

Software
NameVersionClassification
PHENIX1.2refinement
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 5C12

5c12


Resolution: 2.05→14.93 Å / SU ML: 0.25 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 32.09 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2433 2751 5.09 %
Rwork0.2172 51328 -
obs0.2185 31191 80.08 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 93.96 Å2 / Biso mean: 41.5482 Å2 / Biso min: 20.37 Å2
Refinement stepCycle: final / Resolution: 2.05→14.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3810 0 3 204 4017
Biso mean--26.37 43.24 -
Num. residues----486
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2275X-RAY DIFFRACTION7.495TORSIONAL
12B2275X-RAY DIFFRACTION7.495TORSIONAL
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.05-2.090.3796790.3718140444
2.09-2.120.4043840.3448153248
2.12-2.160.3953830.3153174253
2.16-2.210.29381040.2887179456
2.21-2.260.30541230.2851191260
2.26-2.310.27251200.2694204164
2.31-2.370.28981060.2761222869
2.37-2.430.27661200.2716231272
2.43-2.50.29621480.2652233873
2.5-2.580.24161430.2308267484
2.58-2.670.27051540.2538288091
2.67-2.780.29841630.2555313797
2.78-2.90.27911600.2253313598
2.9-3.060.33421620.2294317699
3.06-3.250.27561820.2171316999
3.25-3.490.21691540.2001318798
3.49-3.840.2041630.1943313098
3.84-4.380.17861710.1691317599
4.38-5.480.18491660.1767318499
5.48-14.90.24781660.2106317899

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