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- PDB-6sft: Solution structure of protein ARR_CleD in complex with c-di-GMP -

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Basic information

Entry
Database: PDB / ID: 6sft
TitleSolution structure of protein ARR_CleD in complex with c-di-GMP
ComponentsTwo-component receiver protein CleD
KeywordsSIGNALING PROTEIN / c-di-GMP / CleD / CheY / Response Regulator
Function / homology: / phosphorelay signal transduction system / Response regulator receiver domain / cheY-homologous receiver domain / Signal transduction response regulator, receiver domain / Response regulatory domain profile. / CheY-like superfamily / Chem-C2E / Two-component receiver protein CleD
Function and homology information
Biological speciesCaulobacter vibrioides (bacteria)
MethodSOLUTION NMR / simulated annealing
AuthorsHabazettl, J. / Hee, C.S. / Jenal, U. / Schirmer, T. / Grzesiek, S.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science FoundationSNF 31-149927 Switzerland
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2020
Title: Intercepting second-messenger signaling by rationally designed peptides sequestering c-di-GMP.
Authors: Hee, C.S. / Habazettl, J. / Schmutz, C. / Schirmer, T. / Jenal, U. / Grzesiek, S.
History
DepositionAug 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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 1.2Jun 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 1.3Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Two-component receiver protein CleD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)5,5563
Polymers4,1751
Non-polymers1,3812
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry, dissociation constant K_D - 110 nM
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2010 Å2
ΔGint9 kcal/mol
Surface area4020 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 200structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein/peptide Two-component receiver protein CleD


Mass: 4174.684 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: ARR_CleD is the arginine rich region of CleD, ranging from K140 to S174. It binds intercalated dimeric c-di-GMP
Source: (gene. exp.) Caulobacter vibrioides (strain NA1000 / CB15N) (bacteria)
Strain: NA1000 / CB15N / Gene: cleD, CCNA_03198 / Details (production host): pET28a-His-SUMO-ARR_CleD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0H3CCM2
#2: Chemical ChemComp-C2E / 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one) / c-di-GMP / Cyclic diguanosine monophosphate


Mass: 690.411 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H24N10O14P2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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
121isotropic22D 1H-15N HSQC
131isotropic13D 1H-15N NOESY
141isotropic13D 1H-15N TOCSY
151isotropic13D 1H-15N NH2 NOESY
191isotropic13D 1H-15N NH2 TOCSY
161isotropic22D 1H-1H NOESY
182isotropic33D HNCA
172isotropic33D HNCO
1132isotropic33D CBCA(CO)NH
1122isotropic33D HBHA(CO)NH
1112isotropic33D CBCANH
1102isotropic13D C(CO)NH
1152isotropic22D 1H-1H NOESY isotpe filtered
1161isotropic13D 15N t1 interleaved
1141isotropic13D 15N t2 interleaved
1171isotropic12D 1H-15N NOE without sturation
1191isotropic12D 1H-15N NOE with sturation
1242isotropic13D HN(CO)CA
2182anisotropic13D HN(CO)CA without decoupling
1252isotropic12D 1H-15N HSQC IPAP
2232anisotropic12D 1H-15N HSQC IPAP

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent systemDetails
solution11.0 mM [U-99% 15N] ARR_CleD, 3.0 mM C2E, 100 mM sodium chloride, 2 mM magnesim chloride, 20 mM sodium phosphate, 0.02 % sodium azide, 95% H2O/5% D2O15N_sample95% H2O/5% D2O
solution20.9 mM [U-99% 13C; U-99% 15N] ARR_CleD, 2.7 mM C2E, 100 mM sodium chloride, 2 mM magnesium chloride, 20 mM sodium phosphate, 0.02 % sodium azide, 95% H2O/5% D2O15N_13C_sample95% H2O/5% D2O
filamentous virus30.6 mM [U-99% 13C; U-99% 15N] ARR_CleD, 1.8 mM C2E, 66.6 mM sodium chloride, 1.33 mM magnesium chloride, 13.33 mM sodium phosphate, 9 mg/mL Pf1 phage, 90% H2O/10% D2O15N-13C-phages90% H2O/10% D2OD2O splitting of 8Hz is enough
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
1.0 mMARR_CleD[U-99% 15N]1
3.0 mMC2Enatural abundance1
100 mMsodium chloridenatural abundance1
2 mMmagnesim chloridenatural abundance1
20 mMsodium phosphatenatural abundance1
0.02 %sodium azidenatural abundance1
0.9 mMARR_CleD[U-99% 13C; U-99% 15N]2
2.7 mMC2Enatural abundance2
100 mMsodium chloridenatural abundance2
2 mMmagnesium chloridenatural abundance2
20 mMsodium phosphatenatural abundance2
0.02 %sodium azidenatural abundance2
0.6 mMARR_CleD[U-99% 13C; U-99% 15N]3
1.8 mMC2Enatural abundance3
66.6 mMsodium chloridenatural abundance3
1.33 mMmagnesium chloridenatural abundance3
13.33 mMsodium phosphatenatural abundance3
9 mg/mLPf1 phagenatural abundance3
Sample conditions

pH: 6.5 / Pressure: 1 atm / Temperature: 298 K

Conditions-IDIonic strengthLabelPH errTemperature err
10.47 Mconditions_10.040.1
20.31 Mconditions_2

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-IDDetails
Bruker DRXBrukerDRX6001TXI probe head
Bruker DRXBrukerDRX9002TCI probe head
Bruker DRXBrukerDRX8003TXI probe head

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Processing

NMR software
NameVersionDeveloperClassification
X-PLOR NIH2.34Schwieters, Kuszewski, Tjandra and Clorerefinement
X-PLOR NIH2.34Schwieters, Kuszewski, Tjandra and Clorestructure calculation
Sparky3.115Goddardchemical shift assignment
Sparky3.115Goddardpeak picking
NMRPipe8.9Delaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
ModelFree4Palmerdata analysis
PIPPGarrettpeak picking
TopSpinBruker Biospincollection
RefinementMethod: simulated annealing / Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 200 / Conformers submitted total number: 10

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