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- PDB-6e3c: NMR Solution Structure of the Monomeric Form of the Phage L Decor... -

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Basic information

Entry
Database: PDB / ID: 6e3c
TitleNMR Solution Structure of the Monomeric Form of the Phage L Decoration Protein
ComponentsDec protein
KeywordsVIRAL PROTEIN / Cementing / OB-Fold / Decoration
Function / homologyDec protein
Function and homology information
Biological speciesEnterobacteria phage L (virus)
MethodSOLUTION NMR / simulated annealing
AuthorsNewcomer, R.L. / Alexandrescu, A.T. / Teschke, C.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM076661 United States
Other privateResearch Excellence Program United States
CitationJournal: Elife / Year: 2019
Title: The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy.
Authors: Rebecca L Newcomer / Jason R Schrad / Eddie B Gilcrease / Sherwood R Casjens / Michael Feig / Carolyn M Teschke / Andrei T Alexandrescu / Kristin N Parent /
Abstract: The major coat proteins of dsDNA tailed phages (order ) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by ...The major coat proteins of dsDNA tailed phages (order ) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary 'decoration' (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.
History
DepositionJul 13, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 17, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jun 14, 2023Group: Database references / Other / Category: database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data
Revision 1.3May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: Dec protein


Theoretical massNumber of molelcules
Total (without water)14,3751
Polymers14,3751
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, We have shown that we can disrupt Dec's trimeric state by unfolding it at pH 2 for a half hour, before refolding it at pH 4, creating a monomer amenable to NMR
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area9640 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 250structures with the least restraint violations
RepresentativeModel #1closest to the average

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Components

#1: Protein Dec protein


Mass: 14374.679 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage L (virus) / Gene: dec / Plasmid: pet15b / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / References: UniProt: Q5C838

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
113isotropic13D 1H-15N NOESY
122isotropic3lrHNCO
131isotropic23D 1H-13C NOESY BB
142isotropic33D HN(CA)CB
152isotropic33D HNCO
162isotropic33D HNCA
172isotropic33D HN(CO)CA
182isotropic33D HN(CA)CO
194isotropic32D HSQC
1105isotropic33D (H)CCH-TOCSY
1115isotropic33D CCH-TOCSY
1125isotropic33D 1H-15N TOCSY

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution10.5 mM [U-13C; U-15N] Decoration Protein, 20 mM [U-99% 2H] sodium acetate, 50 mM sodium chloride, 100% D2OThe sample was lyophilized, and then unfolded at pH 2 with DCl, before being refolded at pH 4 with NaODDoubleLabeled_Structure_Sample100% D2O
solution20.5 mM [U-13C; U-15N; U-2H] Decoration Protein, 20 mM sodium acetate, 100 mM sodium chloride, 1 mM EDTA, 90% H2O/10% D2OThe sample was unfolded at pH 2 for a half hour, before being refolded at pH 4TripleLabeled_Sample90% H2O/10% D2O
solution30.5 mM [U-13C, U-15N, 50%-2H] Decoration Protein, 20 mM sodium acetate, 50 mM sodium chloride, 1 mM EDTA, 90% H2O/10% D2OThe sample was unfolded at pH 2 for a half hour, before being refolded at pH 4FracD_Sample90% H2O/10% D2O
solution40.5 mM [U-15N] Decoration Protein, 20 mM sodium acetate, 50 mM sodium chloride, 1 mM EDTA, 90% H2O/10% D2OThe sample was unfolded at pH 2 for a half hour, before being refolded at pH 415N_Sample90% H2O/10% D2O
solution50.5 mM [U-13C; U-15N] Decoration Protein, 20 mM sodium acetate, 50 mM sodiium chloride, 1 mM EDTA, 100% D2OThe sample was unfolded at pH 2 for a half hour, before being refolded at pH 4DoubleLabeled_Assign_Sample100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mMDecoration Protein[U-13C; U-15N]1
20 mMsodium acetate[U-99% 2H]1
50 mMsodium chloridenatural abundance1
0.5 mMDecoration Protein[U-13C; U-15N; U-2H]2
20 mMsodium acetatenatural abundance2
100 mMsodium chloridenatural abundance2
1 mMEDTAnatural abundance2
0.5 mMDecoration Protein[U-13C, U-15N, 50%-2H]3
20 mMsodium acetatenatural abundance3
50 mMsodium chloridenatural abundance3
1 mMEDTAnatural abundance3
0.5 mMDecoration Protein[U-15N]4
20 mMsodium acetatenatural abundance4
50 mMsodium chloridenatural abundance4
1 mMEDTAnatural abundance4
0.5 mMDecoration Protein[U-13C; U-15N]5
20 mMsodium acetatenatural abundance5
50 mMsodiium chloridenatural abundance5
1 mMEDTAnatural abundance5
Sample conditionsIonic strength: 50 (NaCl) mM / Label: Conditions_1 / pH: 4 / Pressure: 1 atm / Temperature: 306.15 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Varian INOVAVarianINOVA8003Cryogenic Probe
Varian INOVAVarianINOVA8001Room Temperature Probe
Varian INOVAVarianINOVA6002Cryogenic Probe

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Processing

NMR software
NameVersionDeveloperClassification
ARIA2.3.1Linge, O'Donoghue and Nilgesrefinement
ARIA2.3.1.Linge, O'Donoghue and Nilgesstructure calculation
Analysis2.4CCPNdata analysis
NMRBox4.1.13Maciejewski, M.W., Schuyler, A.D., Gryk, M.R., Moraru, I.I., Romero, P.R., Ulrich, E.L., Eghbalnia, H.R., Livny, M., Delaglio, F., and Hoch, J.C.data analysis
Refinement
MethodSoftware ordinal
simulated annealing1
simulated annealing2
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 250 / Conformers submitted total number: 20

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