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- PDB-2kx9: Solution Structure of the Enzyme I dimer Using Residual Dipolar C... -

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Basic information

Entry
Database: PDB / ID: 2kx9
TitleSolution Structure of the Enzyme I dimer Using Residual Dipolar Couplings and Small Angle X-Ray Scattering
ComponentsPhosphoenolpyruvate-protein phosphotransferasePhosphoenolpyruvate—protein phosphotransferase
KeywordsTRANSFERASE / SUGAR PHOSPHOTRANSFERASE SYSTEM / PTS
Function / homology
Function and homology information


phosphoenolpyruvate-protein phosphotransferase / phosphoenolpyruvate-protein phosphotransferase activity / phosphoenolpyruvate-dependent sugar phosphotransferase system / kinase activity / phosphorylation / 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. / PEP-utilising enzyme, C-terminal ...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 detailsregularized mean of the 99 structures that are reported in primary citation
AuthorsSchwieters, C.D. / Suh, J. / Grishaev, A. / Takayama, Y. / Guirlando, R. / Clore, G.
CitationJournal: J Am Chem Soc / Year: 2010
Title: Solution structure of the 128 kDa enzyme I dimer from Escherichia coli and its 146 kDa complex with HPr using residual dipolar couplings and small- and wide-angle X-ray scattering.
Authors: Charles D Schwieters / Jeong-Yong Suh / Alexander Grishaev / Rodolfo Ghirlando / Yuki Takayama / G Marius Clore /
Abstract: The solution structures of free Enzyme I (EI, ∼128 kDa, 575 × 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr (∼146 kDa) have been solved using ...The solution structures of free Enzyme I (EI, ∼128 kDa, 575 × 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr (∼146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small- (SAXS) and wide- (WAXS) angle X-ray scattering and small-angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS data that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C(2) symmetry axis of the EIC dimer, while translational, shape, and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large (∼70-90°) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.
History
DepositionApr 29, 2010Deposition site: BMRB / Processing site: RCSB
Revision 1.0Sep 15, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2May 8, 2019Group: Data collection / Database references / Category: pdbx_database_related
Revision 1.3May 1, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
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: Phosphoenolpyruvate-protein phosphotransferase
B: Phosphoenolpyruvate-protein phosphotransferase


Theoretical massNumber of molelcules
Total (without water)126,8392
Polymers126,8392
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)2 / 120target function
RepresentativeModel #1closest to the average

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Components

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


Mass: 63419.344 Da / Num. of mol.: 2 / Fragment: UNP residues 1-573
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / 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, 100 mM sodium chloride, 10 mM DTT, 4 mM MgCl2, 1 mM EDTA, 10 % D2O, 1 tablet protease inhibitor, 0.15 mM EI dimer, 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
1 %protease inhibitor-71
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 scatter

Data analysis software list: GNOM / Protein length: 150 / Sample pH: 7.4 / Temperature: 298 K

TypeIDConc. range (mg/ml)Detector typeMean guiner radius (nm)Num. of time framesSource beamlineSource classSource type
x-ray12.5-5Gold CCD41.9202.12-IDCYALS
neutron2544.230M NG3NNIST NCNR

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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
Details: STRUCTURE STATISTICS: MODEL 1: SAXS CHI2 Q->0.44: 0.43 SAXS CHI2 FULL RANGE: 2.26 SANS CHI2: 0.39 RDC R-FACTOR: 18.03 % RDC DA: 14.5 HZ RDC RH: 0.49 MODEL 2: SAXS CHI2 Q->0.44: 0.40 SAXS ...Details: STRUCTURE STATISTICS: MODEL 1: SAXS CHI2 Q->0.44: 0.43 SAXS CHI2 FULL RANGE: 2.26 SANS CHI2: 0.39 RDC R-FACTOR: 18.03 % RDC DA: 14.5 HZ RDC RH: 0.49 MODEL 2: SAXS CHI2 Q->0.44: 0.40 SAXS CHI2 FULL RANGE: 1.99 SANS CHI2: 0.58 RDC R-FACTOR: 18.07 RDC DA: 14.5 HZ RDC RH: 0.48 AVERAGE OVER THE FULL 99-MEMBER ENSEMBLE: SAXS CHI2 Q->0.44: 0.29 +/- 0.05 SAXS CHI2 FULL RANGE: 1.34 +/- 0.27 SANS CHI2: 0.41 +/- 0.08 RDC R-FACTOR: 18.07 +/- 0.02 % RDC DA: 14.5 +/- 0.1 HZ RDC RH: 0.49 +/- 0.00
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: target function / Conformers calculated total number: 120 / Conformers submitted total number: 2
Soln scatter modelConformer selection criteria: REGULARIZED MEAN OF 99 MODELS
Details: The initial structure of the EI dimer was constructed as a hybrid of the crystal structure of phosphorylated EI intermediate captured by the inhibitor oxalate (PDB code 2HWG) and the NMR ...Details: The initial structure of the EI dimer was constructed as a hybrid of the crystal structure of phosphorylated EI intermediate captured by the inhibitor oxalate (PDB code 2HWG) and the NMR structure of the EIN-HPr complex (PDB code 3EZA). Throughout the structure determination, the backbone atomic coordinates of each EIN domain (residues 1-254) were treated as rigid bodies, with the two symmetry related EIC domains (residues 262- 573) held fixed in space. Coordinates in the linker region (residues 255-261) were allowed varying degrees of freedom during the calculation through the use of the internal variable module (IVM) of Xplor-NIH. This entry corresponds to the regularized mean of the 99 structures for which data was reported in the primary publication, with the B-factor column representing the per-atom spread (in B-factor units). The calculated structural statistics for the original 99 structures and for the regularized mean are shown below.
Entry fitting list: PDB ENTRIES 2HWG AND 3EZA / Num. of conformers calculated: 99 / Num. of conformers submitted: 1 / Representative conformer: 1 / Software list: GNOM,XPLOR-NIH

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