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- PDB-2l5h: Solution Structure of the H189Q mutant of the Enzyme I dimer Usin... -

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Basic information

Entry
Database: PDB / ID: 2l5h
TitleSolution Structure of the H189Q mutant of the Enzyme I dimer Using Residual Dipolar Couplings and Small Angle X-Ray Scattering
ComponentsPhosphoenolpyruvate-protein phosphotransferase
KeywordsTRANSFERASE / protein / dimer
Function / homology
Function and homology information


phosphoenolpyruvate-protein phosphotransferase / phosphoenolpyruvate-protein phosphotransferase activity / phosphoenolpyruvate-dependent sugar phosphotransferase system / kinase activity / identical protein binding / metal ion binding / cytosol
Similarity search - Function
Phosphotransferase system, enzyme I / Phosphotransferase system, enzyme I-like / Phosphotransferase system, enzyme I N-terminal / PtsI, HPr-binding domain superfamily / : / PEP-utilising enzyme, N-terminal / PEP-utilising enzyme, active site / PEP-utilizing enzymes phosphorylation site signature. / PEP-utilising enzyme, conserved site / PEP-utilizing enzymes signature 2. ...Phosphotransferase system, enzyme I / Phosphotransferase system, enzyme I-like / Phosphotransferase system, enzyme I N-terminal / PtsI, HPr-binding domain superfamily / : / PEP-utilising enzyme, N-terminal / PEP-utilising enzyme, active site / PEP-utilizing enzymes phosphorylation site signature. / PEP-utilising enzyme, conserved site / PEP-utilizing enzymes signature 2. / PEP-utilising enzyme, C-terminal / PEP-utilising enzyme, PEP-binding domain / PEP-utilising enzyme, mobile domain / Phosphohistidine domain superfamily / PEP-utilising enzyme, mobile domain / Phosphoenolpyruvate-binding domains / Pyruvate kinase-like domain superfamily / Pyruvate/Phosphoenolpyruvate kinase-like domain superfamily / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
Phosphoenolpyruvate-protein phosphotransferase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodSOLUTION NMR / SOLUTION SCATTERING / simulated annealing
Model detailslowest energy, model 1
AuthorsTakayama, Y.D. / Schwieters, C.D. / Grishaev, A. / Guirlando, R. / Clore, G.
CitationJournal: J.Am.Chem.Soc. / Year: 2011
Title: Combined Use of Residual Dipolar Couplings and Solution X-ray Scattering To Rapidly Probe Rigid-Body Conformational Transitions in a Non-phosphorylatable Active-Site Mutant of the 128 kDa Enzyme I Dimer.
Authors: Takayama, Y. / Schwieters, C.D. / Grishaev, A. / Ghirlando, R. / Clore, G.M.
History
DepositionNov 1, 2010Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jan 12, 2011Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Apr 25, 2012Group: Database references
Revision 1.3May 1, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phosphoenolpyruvate-protein phosphotransferase
B: Phosphoenolpyruvate-protein phosphotransferase


Theoretical massNumber of molelcules
Total (without water)126,8192
Polymers126,8192
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)2 / 120target function
RepresentativeModel #1lowest energy

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Components

#1: Protein Phosphoenolpyruvate-protein phosphotransferase / Phosphotransferase system / enzyme I


Mass: 63409.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ptsI, b2416, JW2409 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 StarTM (DE3)
References: UniProt: P08839, phosphoenolpyruvate-protein phosphotransferase

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Experimental details

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Experiment

Experiment
Method
SOLUTION NMR
SOLUTION SCATTERING
NMR experimentType: TROSY-based 1H-15N correlation spectroscopy

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Sample preparation

DetailsContents: 20 mM TRIS-1, 100 mM sodium chloride-2, 10 mM DTT-3, 4 mM MgCl2-4, 1 mM EDTA-5, 10 % D2O-6, 0.15 mM EI dimer-8, 90% H2O/10% D2O
Solvent system: 90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentSolution-ID
20 mMTRIS-11
100 mMsodium chloride-21
10 mMDTT-31
4 mMMgCl2-41
1 mMEDTA-51
10 %D2O-61
0.15 mMEI dimer-81
Sample conditionspH: 7.4 / Pressure: ambient / Temperature: 310 K

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Data collection

NMR spectrometerType: Bruker DRX / Manufacturer: Bruker / Model: DRX / Field strength: 800 MHz
Soln scatterType: x-ray
Buffer name: 20 mM Tris, 100 mM NaCl, 10 mM DTT, 4 mM MgCl2, 1 mM EDTA, 1 tablet protease inhibitor cocktail (SigmaFAST S8830)
Conc. range: 5 mg/ml
Data reduction software list: Mar_Detector, Igor, Primus, Gnom
Detector specific: ECT division, Argonne National Laboratory
Detector type: Gold CCD / Mean guiner radius: 4.1 nm / Mean guiner radius esd: 0.1 nm / Num. of time frames: 20 / Protein length: 15.5 / Sample pH: 7.4 / Source beamline: 12-IDC / Source class: Y / Source type: APS / Temperature: 298 K

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Processing

NMR software
NameVersionDeveloperClassification
Xplor-NIH2.25Schwieters, Kuszewski, Tjandra and Clorestructure solution
Xplor-NIH2.25Schwieters, Kuszewski, Tjandra and Clorerefinement
RefinementMethod: simulated annealing / Software ordinal: 1
NMR constraintsProtein chi angle constraints total count: 72 / Protein other angle constraints total count: 46 / Protein phi angle constraints total count: 32 / Protein psi angle constraints total count: 30
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 120 / Conformers submitted total number: 2
Soln scatter modelMethod: simulated annealing / Conformer selection criteria: lowest energy
Details: The initial structure of the H189Q mutant of the EI dimer was taken from the calculated structures of the wildtype EI dimer summarized in PDB entry 2KX9. Throughout the structure ...Details: The initial structure of the H189Q mutant of the EI dimer was taken from the calculated structures of the wildtype EI dimer summarized in PDB entry 2KX9. Throughout the structure determination, the backbone atomic coordinates of the c-terminus of each EI subunit (residues 262-573) were held fixed in space, while the two subdomains of each n-terminus (residues 25-142 in the alpha subdomain, and residues 1-21 and 147-254 in the alpha-beta subdomain) were treated as rigid bodies. Coordinates in the linker regions (residues 22-24, 143-146, and 255-261) and interfactial sidechains were allowed varying degrees of freedom during the calculation through the use of the internal variable module (IVM) of Xplor-NIH. Model 1 corresponds to the regularized mean of the 100 structures for which data was reported in the primary publication, with the B-factor column representing the per-atom spread (in B-factor units). Model 2 is the lowest energy structure. The calculated structural statistics for the original 96 structures and for the two reported structures are: MODEL 1: SAXS CHI2 Q->0.44: 0.63 SAXS CHI2 FULL RANGE: 0.80 RDC R-FACTOR: 18.76 RDC DA: 11.16 RDC RH: 0.59 MODEL 2: SAXS CHI2 Q->0.44: 0.50 SAXS CHI2 FULL RANGE: 0.72 RDC R-FACTOR: 19.18 RDC DA: 11.15 RDC RH: 0.60 AVERAGE OVER THE FULL 99-MEMBER ENSEMBLE: SAXS CHI2 Q->0.44: 0.58 +/- 0.10 SAXS CHI2 FULL RANGE: 0.76 +/- 0.16 RDC R-FACTOR: 19.02 +/- 0.37 RDC DA: 11.13+/- 0.19 RDC RH: 0.59 +/- 0.02
Entry fitting list: free wildtype EI structures, PDB ID 2KX9
Num. of conformers calculated: 120 / Num. of conformers submitted: 2 / Representative conformer: 1
Software author list: C.D. Schwieters, J.J. Kuszewski, N. Tjandra,G.M. Clore
Software list: XPLOR-NIH

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