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- PDB-2kho: NMR-RDC / XRAY structure of E. coli HSP70 (DNAK) chaperone (1-605... -

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Basic information

Entry
Database: PDB / ID: 2kho
TitleNMR-RDC / XRAY structure of E. coli HSP70 (DNAK) chaperone (1-605) complexed with ADP and substrate
ComponentsHeat shock protein 70Hsp70
KeywordsCHAPERONE / MOLECULAR CHAPERONE / HSP70 / PEPTIDE BINDING / PROTEIN FOLDING / ATP-binding / Cell inner membrane / Cell membrane / DNA replication / Membrane / Nucleotide-binding / Phosphoprotein / Stress response / TRANSCRIPTION
Function / homology
Function and homology information


stress response to copper ion / sigma factor antagonist activity / chaperone cofactor-dependent protein refolding / protein unfolding / cellular response to unfolded protein / inclusion body / protein folding chaperone / heat shock protein binding / ADP binding / ATP-dependent protein folding chaperone ...stress response to copper ion / sigma factor antagonist activity / chaperone cofactor-dependent protein refolding / protein unfolding / cellular response to unfolded protein / inclusion body / protein folding chaperone / heat shock protein binding / ADP binding / ATP-dependent protein folding chaperone / unfolded protein binding / response to heat / protein-folding chaperone binding / protein refolding / protein-containing complex assembly / DNA replication / ATP hydrolysis activity / protein-containing complex / zinc ion binding / ATP binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Substrate Binding Domain Of Dnak; Chain:A; Domain 2 - #10 / Substrate Binding Domain Of DNAk; Chain A, domain 1 / Chaperone DnaK / Defensin A-like - #30 / Substrate Binding Domain Of DNAk; Chain A, domain 1 / Defensin A-like / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site ...Substrate Binding Domain Of Dnak; Chain:A; Domain 2 - #10 / Substrate Binding Domain Of DNAk; Chain A, domain 1 / Chaperone DnaK / Defensin A-like - #30 / Substrate Binding Domain Of DNAk; Chain A, domain 1 / Defensin A-like / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site / Heat shock protein 70kD, peptide-binding domain superfamily / Heat shock protein 70 family / Hsp70 protein / Heat shock protein 70kD, C-terminal domain superfamily / Substrate Binding Domain Of Dnak; Chain:A; Domain 2 / ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / ATPase, nucleotide binding domain / Nucleotidyltransferase; domain 5 / Alpha-Beta Complex / Up-down Bundle / Sandwich / 2-Layer Sandwich / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Chaperone protein DnaK
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodSOLUTION NMR / RDC OPTIMIZATION
AuthorsZuiderweg, E.R.P. / Bertelsen, E.B.
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2009
Title: Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.
Authors: Bertelsen, E.B. / Chang, L. / Gestwicki, J.E. / Zuiderweg, E.R.
History
DepositionApr 10, 2009Deposition site: BMRB / Processing site: RCSB
Revision 1.0May 12, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2May 16, 2012Group: Database references
Revision 1.3Aug 18, 2021Group: Data collection / Database references ...Data collection / Database references / Experimental preparation / Structure summary
Category: database_2 / pdbx_nmr_exptl_sample ...database_2 / pdbx_nmr_exptl_sample / pdbx_nmr_representative / pdbx_nmr_sample_details / struct
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_exptl_sample.component / _pdbx_nmr_exptl_sample.isotopic_labeling / _pdbx_nmr_representative.selection_criteria / _struct.pdbx_model_details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Heat shock protein 70


Theoretical massNumber of molelcules
Total (without water)65,6511
Polymers65,6511
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)1 / 1target function
RepresentativeModel #1fewest violations

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Components

#1: Protein Heat shock protein 70 / Hsp70 / Chaperone protein dnaK / Heat shock 70 kDa protein / HSP70


Mass: 65651.023 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: dnaK, groP, grpF, seg, b0014, JW0013 / Production host: Escherichia coli (E. coli) / Strain (production host): BL2 / References: UniProt: P0A6Y8
Sequence detailsTHE SEQUENCE CONFLICT IN 1DKX, ASP530 WHICH SHOULD BE GLU530, HAS BEEN RETAINED IN THIS ENTRY.

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experimentType: KAPPA-SHIFTED 15N-1H HSQC- TROSY
NMR detailsText: NMR 1H-15N RESIDUAL DIPOLAR COUPLING (RDC) DATA IN 4% STRETCHED POLY ACRYL AMIDE. NH RDC DATA WERE OBTAINED FROM KAPPA-SHIFTED TROSY USING A VARIAN INOVA 800 MHZ NMR SPECTROMETER EQUIPPED WITH ...Text: NMR 1H-15N RESIDUAL DIPOLAR COUPLING (RDC) DATA IN 4% STRETCHED POLY ACRYL AMIDE. NH RDC DATA WERE OBTAINED FROM KAPPA-SHIFTED TROSY USING A VARIAN INOVA 800 MHZ NMR SPECTROMETER EQUIPPED WITH A HCN COLD (CRYO) PROBE. THE NUCLEOTIDE-BINDING DOMAIN (NBD) AND SUBSTRATE BINDING DOMAIN (SBD) ARE CONNECTED BY A FLEXIBLE LINKER AND MOVE WITH RESPECT TO EACHOTHER IN A CONE WITH AN ESTIMATED OPENING ANGLE OF 70 DEGREES ON THE NANO SECOND TIME SCALE.

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Sample preparation

DetailsType: solution
Contents: 0.2 MM [U-100% 13C U-100% 15N U-80% 2H] HSP70, 10 MM POTASSIUM CHLORIDE, 25 MM TRIS, 10 MM DTT, 5 MM MGCL2, 5 MM ADP, 10 MM POTASSIUM PHOSPHATE, 0.2 MM SODIUM AZIDE, 2 MM NRLLLTG, 90% H2O/ 10% D2O
Label: sample_1 / Solvent system: 90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.2 mMHsp70[U-100% 13C; U-100% 15N; U-80% 2H]1
10 mMpotassium chloridenatural abundance1
25 mMTRISnatural abundance1
10 mMDTTnatural abundance1
5 mMMgCl2natural abundance1
5 mMADPnatural abundance1
10 mMpotassium phosphatenatural abundance1
0.2 mMsodium azidenatural abundance1
2 mMNRLLLTGnatural abundance1
Sample conditionsIonic strength: 0.05 / pH: 7.2 / Pressure: AMBIENT / Temperature: 300 K

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NMR measurement

NMR spectrometerType: Varian INOVA / Manufacturer: Varian / Model: INOVA / Field strength: 800 MHz

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Processing

NMR software
NameDeveloperClassification
REDCATVALAFAR, H. AND PRESTEGARD, J.H.(2004)refinement
REDCATVALAFAR, H. AND PRESTEGARD, J.H.(2004)structure solution
own software FORTRAN 77E. ZUIDERWEG, 2008refinement
RefinementMethod: RDC OPTIMIZATION / Software ordinal: 1
Details: RDC's WERE FITTED FOR (IA, IB, IIA) (IIB) (BETA, LID) AS SEPARATE UNITS USING GRID SEARCH OVER DA, DR, AND THREE EULER ANGLES FOLLOWED BY STEEPEST DESCEND. XRAY DATA FROM PDB ENTRY 1DKG WERE ...Details: RDC's WERE FITTED FOR (IA, IB, IIA) (IIB) (BETA, LID) AS SEPARATE UNITS USING GRID SEARCH OVER DA, DR, AND THREE EULER ANGLES FOLLOWED BY STEEPEST DESCEND. XRAY DATA FROM PDB ENTRY 1DKG WERE USED FOR RESIDUES 3-378 (NBD - NUCLEOTIDE BINDING DOMAIN). XRAY DATA FROM PDB ENTRY 1DKX WERE USED FOR 397-603 (SBD - SUBSTRATE BINDING DOMAIN). MISSING LOOPS IN 1DKG WERE ANNEALED AND MINIMIZED USING SWISSPROT SERVER. ORIENTATION OF DOMAIN IIB (RESIDUES 229-307) ADAPTED TO NMR DATA. DOMAIN IIB WAS ROTATED 20 DEGREES BASED ON RDC DATA AND MINIMALLY SUPERPOSED ON IIB IN 1DKG USING TRANSLATION AND ROTATION AROUND SZZ ONLY. CONNECTING RESIDUES 225-233 AND 307-314 WERE MINIMIZED IN SWISSPROT. NBD AND SBD WERE ORIENTED WITH RESPECT TO EACHOTHER BASED ON THE NMR DIPOLAR INFORMATION TRANSLATIONAL POSITION OF SBD WITH RESPECT TO NBD WAS DETERMINED BY COMPUTING THE BEST THEORETICAL ALLIGNMENT TENSOR USING PALES FROM ZWECKSTETTER M & BAX A (2001) J BIOMOL NMR 20(4):365-377. HETATM RECORDS IDENTIFY TENSOR ORIENTAIONS FOR DOMAINS IA,IB AND IIA AND BETA-LID OBTAINED FROM SELF_VALIDATION USING 50% OF THE RDC DATA. 65 A Z SEPARATION BETWEEN COORDINATE CENTERS OF NBD AND BETA-LID. 10 A Y SEPARATION BETWEEN COORDINATE CENTERS OF NBD AND BETA-LID. LINKER RESIDUES 379-395 ARE DYNAMIC AND WERE PLACED IN ARBITRARY CONFORMATION USING MOE (MOLECULAR OPERATING ENVIRONMENT) CHEMICAL COMPUTING GROUP 1010 SHERBROOKE ST. W, SUITE 910 MONTREAL, QUEBEC, CANADA H3A 2R7. LINKER CONFORMATION WAS OPTIMIZED IN MOE AND SWISS PROT.
NMR representativeSelection criteria: fewest violations
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 1 / Conformers submitted total number: 1

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