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- PDB-1fxo: THE STRUCTURAL BASIS OF THE CATALYTIC MECHANISM AND REGULATION OF... -

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Basic information

Entry
Database: PDB / ID: 1fxo
TitleTHE STRUCTURAL BASIS OF THE CATALYTIC MECHANISM AND REGULATION OF GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE (RMLA). TMP COMPLEX.
ComponentsGLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
KeywordsTRANSFERASE / rhamnose / nucleotidyltransferase / pyrophosphorylase / thymidylyltransferase / allostery
Function / homology
Function and homology information


glucose-1-phosphate thymidylyltransferase / glucose-1-phosphate thymidylyltransferase activity / dTDP-rhamnose biosynthetic process / lipopolysaccharide core region biosynthetic process / nucleotide binding / metal ion binding
Similarity search - Function
Glucose-1-phosphate thymidylyltransferase, short form / Nucleotidyl transferase domain / Nucleotidyl transferase / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Nucleotide-diphospho-sugar transferases / Alpha-Beta Complex / Alpha Beta
Similarity search - Domain/homology
THYMIDINE-5'-PHOSPHATE / Glucose-1-phosphate thymidylyltransferase
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Phase extension in direct method / Resolution: 1.66 Å
AuthorsBlankenfeldt, W. / Lam, J.S. / Naismith, J.H.
Citation
Journal: EMBO J. / Year: 2000
Title: The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA).
Authors: Blankenfeldt, W. / Asuncion, M. / Lam, J.S. / Naismith, J.H.
#1: Journal: To be Published
Title: The Purification, Crystallisation and Preliminary Structural Characterisation of Glucose-1-phosphate Thymidylyltransferase (RmlA), the First Enzyme of the dTDP-L-rhamnose Synthesis Pathway ...Title: The Purification, Crystallisation and Preliminary Structural Characterisation of Glucose-1-phosphate Thymidylyltransferase (RmlA), the First Enzyme of the dTDP-L-rhamnose Synthesis Pathway from Pseudomonas aeruginosa
Authors: Blankenfeldt, W. / Giraud, M.F. / Leonard, G. / Rahim, R. / Lam, J.S. / Naismith, J.H.
#2: Journal: J.Biol.Chem. / Year: 1965
Title: The Nucleotide Specificity and Feedback Control of Thymidine Diphosphate D-glucose Pyrophosphorylase
Authors: Melo, A. / Glaser, L.
History
DepositionSep 26, 2000Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 27, 2000Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Aug 9, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
B: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
C: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
D: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
E: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
F: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
G: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
H: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)266,31537
Polymers259,9118
Non-polymers6,40429
Water60,6203365
1
A: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
B: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
C: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
D: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,30220
Polymers129,9554
Non-polymers3,34616
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19240 Å2
ΔGint-159 kcal/mol
Surface area42310 Å2
MethodPISA
2
E: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
F: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
G: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
H: GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,01317
Polymers129,9554
Non-polymers3,05813
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19020 Å2
ΔGint-144 kcal/mol
Surface area42360 Å2
MethodPISA
Unit cell
Length a, b, c (Å)71.494, 73.064, 134.738
Angle α, β, γ (deg.)89.93, 80.92, 81.11
Int Tables number1
Space group name H-MP1
DetailsThe biological assembly is a tetramer consisting of chains A,B,C and D or E,F,G and H, respectively.

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Components

#1: Protein
GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE / GLUCOSE-1-PHOSPHATE THYMIDYLYLTRANSFERASE (RMLA)


Mass: 32488.844 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Plasmid: PET23A(+) / Production host: Escherichia coli (E. coli)
References: UniProt: Q9HU22, glucose-1-phosphate thymidylyltransferase
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: SO4
#3: Chemical
ChemComp-TMP / THYMIDINE-5'-PHOSPHATE


Mass: 322.208 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C10H15N2O8P
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3365 / Source method: isolated from a natural source / Formula: H2O
Compound detailsRmlA in complex with thymidine-5'-phosphate (TMP). TMP binds the active centre in two alternative ...RmlA in complex with thymidine-5'-phosphate (TMP). TMP binds the active centre in two alternative conformations.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.54 Å3/Da / Density % sol: 54.9 %
Crystal growTemperature: 292 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 10 % PEG 6000, 0.1 M sodium citrate, 0.5 M lithium sulfate, 4 microlitre protein, 4 microlitre precipitant, 1 microlitre 50 mM TMP, pH 4.6, VAPOR DIFFUSION, SITTING DROP, temperature 292K
Crystal grow
*PLUS
Temperature: 293 K
Details: Blankenfeldt, W., (2000) Acta Crystallogr.,Sect.D, 56, 1501.
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
19-12 %(w/v)PEG60001reservoir
20.5 Mlithium sulfate1reservoir
30.1 Mcitrate/NaOH1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM14 / Wavelength: 0.978
DetectorType: MARRESEARCH / Detector: CCD / Date: Mar 3, 2000
RadiationMonochromator: SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978 Å / Relative weight: 1
ReflectionResolution: 1.66→32.12 Å / Num. all: 297160 / Num. obs: 1141819 / % possible obs: 94.2 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.8 % / Biso Wilson estimate: 19.4 Å2 / Rmerge(I) obs: 0.053 / Rsym value: 0.052 / Net I/σ(I): 10.9
Reflection shellResolution: 1.66→1.74 Å / Redundancy: 2 % / Rmerge(I) obs: 0.332 / Mean I/σ(I) obs: 2 / Num. unique all: 75372 / Rsym value: 0.332 / % possible all: 81.2
Reflection
*PLUS
Num. obs: 297160 / Num. measured all: 1141819
Reflection shell
*PLUS
% possible obs: 81.2 %

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Processing

Software
NameVersionClassification
SOLVEphasing
REFMACrefinement
MOSFLMdata reduction
CCP4(SCALA)data scaling
RefinementMethod to determine structure: Phase extension in direct method
Starting model: MAD structure (1G23)

1g23


Resolution: 1.66→73 Å / SU B: 5.39619 / SU ML: 0.09328 / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / ESU R: 0.68841 / ESU R Free: 0.12821 / Stereochemistry target values: Engh and Huber
Details: Used TLS-option in refmac5. Anisotropic B-factors were calculated but not refined (ncycle 0).
RfactorNum. reflection% reflectionSelection details
Rfree0.196 14902 5 %RANDOM
Rwork0.142 ---
obs0.145 281394 94.3 %-
Displacement parametersBiso mean: 14.14 Å2
Baniso -1Baniso -2Baniso -3
1-0.12 Å20.04 Å20.1 Å2
2--0.09 Å2-0.01 Å2
3----0.25 Å2
Refinement stepCycle: LAST / Resolution: 1.66→73 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms18858 0 548 3365 22771
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONp_bond_d0.0160.021
X-RAY DIFFRACTIONp_plane_restr0.0090.02
X-RAY DIFFRACTIONp_chiral_restr0.1290.2
X-RAY DIFFRACTIONp_mcbond_it2.0391.5
X-RAY DIFFRACTIONp_mcangle_it2.9462
X-RAY DIFFRACTIONp_scbond_it5.6943
X-RAY DIFFRACTIONp_scangle_it6.8744.5
Software
*PLUS
Name: REFMAC / Classification: refinement
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONp_angle_d
X-RAY DIFFRACTIONp_angle_deg2.32
LS refinement shell
*PLUS
Rfactor Rfree: 0.303 / Rfactor Rwork: 0.225

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