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- PDB-6m78: Aromatic interactions drive the coupled folding and binding of th... -

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Basic information

Entry
Database: PDB / ID: 6m78
TitleAromatic interactions drive the coupled folding and binding of the intrinsically disordered Sesbania mosaic virus VPg protein
Components(Polyprotein) x 2
KeywordsHYDROLASE / Viral-Protein-genome-linked / complex / protease / PLANT VIRAL PROTEIN
Function / homology
Function and homology information


host cell membrane / viral translational frameshifting / serine-type endopeptidase activity / proteolysis / membrane
Similarity search - Function
Peptidase S39 / Peptidase S39B, luteovirus / Peptidase family S39 domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Biological speciesSesbania mosaic virus
MethodSOLUTION NMR / simulated annealing
AuthorsDixit, K. / Karanth, N.M. / Nair, S. / Kumari, K. / Chakraborti, K.S. / Savithri, H.S. / Sarma, S.P.
CitationJournal: Biochemistry / Year: 2020
Title: Aromatic Interactions Drive the Coupled Folding and Binding of the Intrinsically Disordered Sesbania mosaic Virus VPg Protein.
Authors: Dixit, K. / Karanth, N.M. / Nair, S. / Kumari, K. / Chakrabarti, K.S. / Savithri, H.S. / Sarma, S.P.
History
DepositionMar 17, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 27, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polyprotein
B: Polyprotein


Theoretical massNumber of molelcules
Total (without water)41,3592
Polymers41,3592
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Co-elution on an S-75 Sephacryl gelfiltration column
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1380 Å2
ΔGint-6 kcal/mol
Surface area13200 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)4 / 200structures with favorable non-bond energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Polyprotein


Mass: 32245.457 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sesbania mosaic virus / Plasmid: pRSET C / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9EB08
#2: Protein Polyprotein


Mass: 9113.224 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sesbania mosaic virus / Plasmid: pET21a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q9EB08
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
131isotropic12D 1H-15N HSQC
162isotropic12D 1H-15N HSQC
143isotropic12D 1H-13C HSQC aliphatic
151isotropic13D HNCA
171isotropic13D HN(CO)CA
181isotropic13D HN(CA)CB
191anisotropic13D HN(COCA)CB
1101isotropic13D HNCO
1113isotropic23D 1H-15N NOESY
1124isotropic12D 1H-15N HSQC
1135isotropic12D 1H-15N HSQC
1146isotropic12D 1H-15N HSQC
1157isotropic12D 1H-15N HSQC
1168isotropic12D 1H-15N HSQC
2179anisotropic12D 1H-15N HSQC
11810isotropic42D 1H-15N HSQC

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution1250 uM > 98% 2H, U-99% 13C, U-99% 15N scPVPg, 90% H2O/10% D2O20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.2H, 13C, 15N-scPVPg90% H2O/10% D2O
solution2250 uM > 98% 2H, U-99% 13C, U-99% 15N scPVPg, 100% D2O20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.2H, 13C ,15N-scPVPG100% D2O
filamentous virus9150 uM > 98% 2H,U-99% 13C,U-99% 15N scPVPg-Pf1, 90% H2O/10% D2O20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.2H,13C,15N-scPVPg90% H2O/10% D2O
solution3250 uM 99% 13CH3-ILV, > 98%2H, U-99% 13C, U-99% 15N ILV-scPVPg, 90% H2O/10% D2O20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.1H-ILV Methyl Protonated, 2H, 13C, 15N-scPVPg90% H2O/10% D2O
solution4200 uM > 98% 2H, U-98% 13C, U-98%15N SeMV Protease Domain, 90% H2O/10% D2O20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.2H,13C,15N-PRO90% H2O/10% D2O
solution5250 uM > 98% 15N scPVPg, 90% H2O/10% D2O15N - labeled in Ala, Gly, Ser, Thr, Glu, Asp, Asn, Gln and Cys. 20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.15N-scPVPg-A90% H2O/10% D2O
solution6250 uM > 98% 15N scPVPg, 90% H2O/10% D2O15N - labeled in Ala, Gly, Ser, Thr, Glu, Asp, Asn, Gln, Cys, Lys, Arg. 20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.15N-scPVPg-B90% H2O/10% D2O
solution7250 uM > 98% 15N scPVPg, 90% H2O/10% D2O15N - labeled in Ala, Gly, Ser, Thr, Glu, Asp, Asn, Gln, Ile, Leu, Val. 20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.15N-scPVPg-C90% H2O/10% D2O
solution8250 uM > 98% 15N scPVPg, 90% H2O/10% D2O15N - labeled in Ala, Gly, Ser, Thr, Glu, Asp, Asn, Gln, Phe, Tyr, Trp 20mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.15N-scPVPg-D90% H2O/10% D2O
solution10200 uM [U-99% 15N] SeMV VPg Protein (Viral Protein genome-linked), 90% H2O/10% D2O15N-labeled SeMV Viral Protein genome-linked (VPg) in complex with the SERINE PROTEASE DOMAIN OF SESBANIA MOSAIC VIRUS15N-VPg90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
250 uMscPVPg> 98% 2H, U-99% 13C, U-99% 15N1
250 uMscPVPg> 98% 2H, U-99% 13C, U-99% 15N2
150 uMscPVPg-Pf1> 98% 2H,U-99% 13C,U-99% 15N9
250 uMILV-scPVPg99% 13CH3-ILV, > 98%2H, U-99% 13C, U-99% 15N3
200 uMSeMV Protease Domain> 98% 2H, U-98% 13C, U-98%15N4
250 uMscPVPg> 98% 15N5
250 uMscPVPg> 98% 15N6
250 uMscPVPg> 98% 15N7
250 uMscPVPg> 98% 15N8
200 uMSeMV VPg Protein (Viral Protein genome-linked)[U-99% 15N]10
Sample conditions

Ionic strength: 0.38 M / pH: 7 / Pressure: 1 atm / Temperature: 303 K

Conditions-IDDetailsLabelPH errPressure errTemperature err
120mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide.2H, 13C, 15N-scPVPg0.10.010.2
220mM Potassium phosphate buffer pH 7.0, 20mM NaCl, 20mM Proline, 50mM Arginine, 50mM Glutamic acid, 1mM EDTA, 1mM DTT, 0.01% Sodium Azide with 13 mg / ml of Pf1 (filamentous phage) as co-solvent.2H,13C,15N-scPVPg-Pf1

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCEBrukerAVANCE7001Triple resonance TCI cryoprobe,probe, single z-axis gradient accessory
Bruker AVANCEBrukerAVANCE8002Triple resonance TCI cryoprobe,probe, single z-axis gradient accessory
Agilent Agilent DDS2AgilentAgilent DDS26004Varian Triple resonance Coldprobe, single z-axis gradient accessory

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Processing

NMR software
NameVersionDeveloperClassification
XwinNMR3.5Bruker Biospincollection
VNMRVnmrJ 3.2, vnmrJ4.2collection
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxpeak picking
ANSIGKraulisdata analysis
CcpNmr AnalysisCCPNdata analysis
ANSIGKraulischemical shift assignment
CcpNmr Analysis2.2CCPNchemical shift assignment
HADDOCK2.2Bonvinstructure calculation
CNSBrunger, Adams, Clore, Gros, Nilges and Readrefinement
RefinementMethod: simulated annealing / Software ordinal: 8
Details: OTHER REFINEMENT REMARKS: Authors had used the coordinates of 1zyo.pdb and the best structure from 6LXF.
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with favorable non-bond energy
Conformers calculated total number: 200 / Conformers submitted total number: 4

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