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- PDB-5t3m: Solution structure of a triple mutant of HwTx-IV - a potent block... -

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Basic information

Entry
Database: PDB / ID: 5t3m
TitleSolution structure of a triple mutant of HwTx-IV - a potent blocker of Nav1.7
ComponentsMu-theraphotoxin-Hs2a
KeywordsTOXIN
Function / homology
Function and homology information


host cell presynaptic membrane / ion channel inhibitor activity / sodium channel regulator activity / toxin activity / extracellular region
Similarity search - Function
Huwentoxin, conserved site-1 / Huwentoxin-1 family signature. / Huwentoxin-1 family / Ion channel inhibitory toxin
Similarity search - Domain/homology
Biological speciesHaplopelma schmidti (Chinese earth tiger)
MethodSOLUTION NMR / torsion angle dynamics
AuthorsRahnama, S. / Sharma, G. / Mobli, M.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1034958 Australia
CitationJournal: PLoS ONE / Year: 2017
Title: The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant.
Authors: Rahnama, S. / Deuis, J.R. / Cardoso, F.C. / Ramanujam, V. / Lewis, R.J. / Rash, L.D. / King, G.F. / Vetter, I. / Mobli, M.
History
DepositionAug 25, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 6, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 27, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Nov 1, 2017Group: Author supporting evidence / Category: pdbx_struct_assembly_auth_evidence
Revision 1.4Jan 8, 2020Group: Author supporting evidence / Data collection
Category: pdbx_audit_support / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.5Jun 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 1.6Oct 30, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Mu-theraphotoxin-Hs2a


Theoretical massNumber of molelcules
Total (without water)4,0001
Polymers4,0001
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area2880 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)20 / 200target function
RepresentativeModel #1lowest energy
DetailsMonomer as determined by NMR and mass spectrometry

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Components

#1: Protein/peptide Mu-theraphotoxin-Hs2a / Mu-TRTX-Hs2a / Huwentoxin-4 / Huwentoxin-IV / HwTx-IV / Huwentoxin-IVa / HWTX-IVa / Huwentoxin-IVb ...Mu-TRTX-Hs2a / Huwentoxin-4 / Huwentoxin-IV / HwTx-IV / Huwentoxin-IVa / HWTX-IVa / Huwentoxin-IVb / HWTX-IVb / Huwentoxin-IVc / HWTX-IVc


Mass: 3999.777 Da / Num. of mol.: 1 / Fragment: residues 53-87 / Mutation: E1G,E4G,Y33W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Haplopelma schmidti (Chinese earth tiger)
Plasmid: PLIC-C
Details (production host): MBP fusion - periplasmic export sequence
Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: P83303
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
111isotropic12D 1H-15N HSQC
121isotropic13D HNCO
131isotropic13D HBHA(CO)NH
141isotropic13D (H)CCH-TOCSY
151isotropic13D CBCA(CO)NH
1121isotropic13D HN(CA)CB
1111isotropic14D HC(CO)NH
1101isotropic22D 1H-15N HSQC - T1 relaxation
191isotropic22D 1H-15N HSQC - T2 relaxation
181isotropic22D 1H-15N HSQC - NOE
171isotropic13D 1H-13C NOESY aliphatic
161isotropic13D 1H-13C NOESY aromatic
1131isotropic13D 1H-15N NOESY

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Sample preparation

DetailsType: solution
Contents: 400 uM [U-99% 13C; U-99% 15N] [m3]-HwTx-IV, 20 mM sodium acetate, 5 % [U-100% 2H] D2O, 95% H2O/5% D2O
Details: 300 ul sample, added into a susceptibility matched 5 mm NMR cells (Shigemi Inc. Japan), prepared by dissolving 15N/13C-labled m3-HwTx-IV to a final concentration of 400 uM in 20 mM sodium acetate solution, pH 5.
Label: 15N/13C [m3]-HwTx-IV / Solvent system: 95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
400 uM[m3]-HwTx-IV[U-99% 13C; U-99% 15N]1
20 mMsodium acetatenatural abundance1
5 %D2O[U-100% 2H]1
Sample conditionsIonic strength: 20 mM / Ionic strength err: 1 / Label: peptide sample / pH: 5 / PH err: 0.1 / Pressure: 1 atm / Pressure err: 0 / Temperature: 298 K / Temperature err: 0.2

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker AVANCE IIIBrukerAVANCE III9001Cryoprobe
Bruker AVANCE IIIBrukerAVANCE III7002Cryoprobe

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Processing

NMR software
NameVersionDeveloperClassification
AnalysisCCPNchemical shift assignment
CYANAGuntert, Mumenthaler and Wuthrichrefinement
ROWLAND NMR TOOLKIT3JC Hoch, AS Stern University of Connecticut, Health Centreprocessing
TALOSnCornilescu, Delaglio and Baxdata analysis
TopSpinBruker Biospincollection
RefinementMethod: torsion angle dynamics / Software ordinal: 2 / Details: automated NOE assignments
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 200 / Conformers submitted total number: 20

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