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Yorodumi- PDB-2xdf: Solution Structure of the Enzyme I Dimer Complexed with HPr Using... -
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-Basic information
Entry | Database: PDB / ID: 2xdf | ||||||
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Title | Solution Structure of the Enzyme I Dimer Complexed with HPr Using Residual Dipolar Couplings and Small Angle X-Ray Scattering | ||||||
Components |
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Keywords | TRANSFERASE / SUGAR TRANSPORT | ||||||
Function / homology | Function and homology information phosphotransferase activity, nitrogenous group as acceptor / phosphoenolpyruvate-protein phosphotransferase / phosphoenolpyruvate-protein phosphotransferase activity / regulation of carbon utilization / antisigma factor binding / positive regulation of glycogen catabolic process / phosphoenolpyruvate-dependent sugar phosphotransferase system / enzyme inhibitor activity / enzyme regulator activity / enzyme activator activity ...phosphotransferase activity, nitrogenous group as acceptor / phosphoenolpyruvate-protein phosphotransferase / phosphoenolpyruvate-protein phosphotransferase activity / regulation of carbon utilization / antisigma factor binding / positive regulation of glycogen catabolic process / phosphoenolpyruvate-dependent sugar phosphotransferase system / enzyme inhibitor activity / enzyme regulator activity / enzyme activator activity / kinase activity / identical protein binding / metal ion binding / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | ESCHERICHIA COLI (E. coli) | ||||||
Method | SOLUTION NMR / SOLUTION SCATTERING / simulated annealing | ||||||
Authors | Schwieters, C.D. / Suh, J.-Y. / Grishaev, A. / Guirlando, R. / Takayama, Y. / Clore, G.M. | ||||||
Citation | Journal: 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 |
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Remark 700 | SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AB" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AB" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 9-STRANDED BARREL THIS IS REPRESENTED BY A 10-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "BB" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 9-STRANDED BARREL THIS IS REPRESENTED BY A 10-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. |
-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 2xdf.cif.gz | 924.4 KB | Display | PDBx/mmCIF format |
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PDB format | pdb2xdf.ent.gz | 789.8 KB | Display | PDB format |
PDBx/mmJSON format | 2xdf.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 2xdf_validation.pdf.gz | 407.9 KB | Display | wwPDB validaton report |
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Full document | 2xdf_full_validation.pdf.gz | 617.3 KB | Display | |
Data in XML | 2xdf_validation.xml.gz | 74.4 KB | Display | |
Data in CIF | 2xdf_validation.cif.gz | 104.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/xd/2xdf ftp://data.pdbj.org/pub/pdb/validation_reports/xd/2xdf | HTTPS FTP |
-Related structure data
-Links
-Assembly
Deposited unit |
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1 |
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NMR ensembles |
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-Components
#1: Protein | Mass: 63419.344 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Strain: BL21 STAR (DE3) / Plasmid: PET11 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 STAR (DE3) References: UniProt: P08839, phosphoenolpyruvate-protein phosphotransferase #2: Protein | Mass: 9129.332 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Strain: BL21 STAR (DE3) / Plasmid: PET11 / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 STAR (DE3) References: UniProt: P0AA06, UniProt: P0AA04*PLUS, Transferases; Transferring phosphorus-containing groups; Protein-serine/threonine kinases |
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-Experimental details
-Experiment
Experiment |
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NMR experiment | Type: TROSY-BASED 1H-15N CORRELATION SPECTROSCOPY |
-Sample preparation
Details | Contents: 90% H2O/10% D2O |
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Sample conditions | pH: 7.4 / Pressure: 1.0 atm / Temperature: 310.0 K |
-Data collection
NMR spectrometer | Type: Bruker DRX / Manufacturer: Bruker / Model: DRX / Field strength: 800 MHz | |||||||||||||||||||||||||||
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Soln scatter | Data analysis software list: GNOM / Protein length: 16 / Temperature: 298 K
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-Processing
NMR software |
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Refinement | Method: simulated annealing / Software ordinal: 1 Details: REFINEMENT DETAILS CAN BE FOUND IN THE PRIMARY CITATION. THE INITIAL STRUCTURE OF THE EI DIMER COMPLEXED WITH HPR WAS CONSTRUCTED AS A HYBRID OF THE CRYSTAL STRUCTURE OF PHOSPHORYLATED EI ...Details: REFINEMENT DETAILS CAN BE FOUND IN THE PRIMARY CITATION. THE INITIAL STRUCTURE OF THE EI DIMER COMPLEXED WITH HPR 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-HPR COMPLEX (RESIDUES 1-254 AND 601-685) 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. THE ENSEMBLE OF CALCULATED STRUCTURES FELL IN TWO CLUSTERS, THE REGULARIZED REFINED MEAN OF EACH IS INCLUDED BELOW AS MODELS 1 AND 2, RESPECTIVELY. STRUCTURAL STATISTICS: CLUSTER 1: SAXS CHI2 Q->0.44 0.63+/-0.11 SAXS CHI2 FULL RANGE FIT 0.45+/-0.07 SANS CHI2 1.38+/-0.51 RDC R-FACTOR 16.30+/-0.03 % RDC DA 13.73+/-0.05 HZ RDC RHOMBICITY 0.63+/-0.00 MODEL 1: SAXS CHI2 Q->0.44 0.62 SAXS CHI2 FULL RANGE FIT 0.42 SANS CHI2 1.30 RDC R-FACTOR 16.27 % RDC DA 13.74 HZ RDC RHOMBICITY 0.63 STRUCTURAL STATISTICS: CLUSTER 2 SAXS CHI2 Q->0.44 0.76+/-0.07 SAXS CHI2 FULL RANGE FIT 0.48+/-0.06 SANS CHI2 2.97+/-0.62 RDC R-FACTOR 16.25+/-0.02 % RDC DA 13.83+/-0.08 HZ RDC RHOMBICITY 0.63+/-0.00 MODEL 2 SAXS CHI2 Q->0.44 0.78 SAXS CHI2 FULL RANGE FIT 0.49 SANS CHI2 2.82 RDC R-FACTOR 16.25 % RDC DA 13.85 HZ RDC RHOMBICITY 0.63 | |||||||||
NMR ensemble | Conformer selection criteria: BEST EXPERIMENT FIT, AND THEN LOWEST ENERGY Conformers calculated total number: 120 / Conformers submitted total number: 2 |