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- PDB-4flr: Crystal structure of Amylosucrase double mutant A289P-F290L from ... -

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Basic information

Entry
Database: PDB / ID: 4flr
TitleCrystal structure of Amylosucrase double mutant A289P-F290L from Neisseria polysaccharea
ComponentsAmylosucrase
KeywordsTRANSFERASE / BETA/ALPHA-BARREL / GLYCOSIDE HYDROLASE / AMYLOSE SYNTHESIS / SUCROSE ISOMERIZATION / GLUCOSYLTRANSFERASE / CARBOHYDRATE
Function / homology
Function and homology information


amylosucrase / amylosucrase activity / carbohydrate metabolic process / extracellular region
Similarity search - Function
: / Amylosucrase, C-terminal domain / Amylosucrase, catalytic domain / RNA polymerase sigma factor, region 2, helix turn helix motif / Oligo-1,6-glucosidase; domain 2 / Oligo-1,6-glucosidase; Domain 2 / Rna Polymerase Sigma Factor; Chain: A / Oligo-1,6-glucosidase, domain 2 / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain ...: / Amylosucrase, C-terminal domain / Amylosucrase, catalytic domain / RNA polymerase sigma factor, region 2, helix turn helix motif / Oligo-1,6-glucosidase; domain 2 / Oligo-1,6-glucosidase; Domain 2 / Rna Polymerase Sigma Factor; Chain: A / Oligo-1,6-glucosidase, domain 2 / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Golgi alpha-mannosidase II / Glycosyl hydrolase, all-beta / Glycosidases / Glycoside hydrolase superfamily / TIM Barrel / Alpha-Beta Barrel / Alpha-Beta Complex / Immunoglobulin-like / Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Biological speciesNeisseria polysaccharea (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å
AuthorsGuerin, F. / Champion, E. / Moulis, C. / Barbe, S. / Tran, T.H. / Morel, S. / Descroix, K. / Monsan, P. / Mulard, L.A. / Remaud-Simeon, M. ...Guerin, F. / Champion, E. / Moulis, C. / Barbe, S. / Tran, T.H. / Morel, S. / Descroix, K. / Monsan, P. / Mulard, L.A. / Remaud-Simeon, M. / Andre, I. / Mourey, L. / Tranier, S.
CitationJournal: J.Am.Chem.Soc. / Year: 2012
Title: Applying pairwise combinations of amino Acid mutations for sorting out highly efficient glucosylation tools for chemo-enzymatic synthesis of bacterial oligosaccharides.
Authors: Champion, E. / Guerin, F. / Moulis, C. / Barbe, S. / Tran, T.H. / Morel, S. / Descroix, K. / Monsan, P. / Mourey, L. / Mulard, L.A. / Tranier, S. / Remaud-Simeon, M. / Andre, I.
History
DepositionJun 15, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 31, 2012Provider: repository / Type: Initial release
Revision 1.1Jan 2, 2013Group: Database references
Revision 1.2Sep 13, 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_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _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: Amylosucrase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,57421
Polymers71,5561
Non-polymers2,01820
Water4,648258
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)96.020, 116.350, 60.170
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212
Components on special symmetry positions
IDModelComponents
11A-999-

HOH

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Components

#1: Protein Amylosucrase


Mass: 71556.094 Da / Num. of mol.: 1 / Fragment: Amylosucrase (unp residues 13-636) / Mutation: A289P, F290L,
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria polysaccharea (bacteria) / Gene: ams / Plasmid: pGEX6p3 / Production host: Escherichia coli (E. coli) / Strain (production host): JM109 / References: UniProt: Q9ZEU2, amylosucrase
#2: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400


Mass: 238.278 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H22O6 / Comment: precipitant*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 258 / Source method: isolated from a natural source / Formula: H2O

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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: 2.35 Å3/Da / Density % sol: 47.63 %
Crystal growTemperature: 285 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 30% PEG 6000, 0.1 M HEPES, pH 7.0, VAPOR DIFFUSION, HANGING DROP, temperature 285.0K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-2 / Wavelength: 0.933 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Jul 31, 2009
RadiationMonochromator: Si 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.933 Å / Relative weight: 1
ReflectionResolution: 2.4→38.78 Å / Num. all: 27091 / Num. obs: 26360 / % possible obs: 97.3 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3 % / Rmerge(I) obs: 0.098 / Net I/σ(I): 6.8
Reflection shellResolution: 2.4→2.54 Å / Redundancy: 2.9 % / Rmerge(I) obs: 0.337 / Mean I/σ(I) obs: 2 / Num. unique all: 3664 / % possible all: 94.8

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Processing

Software
NameVersionClassification
DNAdata collection
PHASERphasing
REFMAC5.6.0117refinement
MOSFLMdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1G5A

1g5a


Resolution: 2.4→11.99 Å / Cor.coef. Fo:Fc: 0.939 / Cor.coef. Fo:Fc free: 0.9 / SU B: 8.211 / SU ML: 0.189 / Cross valid method: THROUGHOUT / ESU R: 0.803 / ESU R Free: 0.285 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.23554 2624 10.1 %RANDOM
Rwork0.18234 ---
obs0.18774 26103 96.42 %-
all-27072 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 23.012 Å2
Baniso -1Baniso -2Baniso -3
1--0.22 Å20 Å20 Å2
2---0.15 Å20 Å2
3---0.37 Å2
Refinement stepCycle: LAST / Resolution: 2.4→11.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5008 0 132 258 5398
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.025289
X-RAY DIFFRACTIONr_angle_refined_deg0.9991.9497170
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.0275635
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.77924270
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.25915799
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.3281535
X-RAY DIFFRACTIONr_chiral_restr0.0710.2764
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.0214114
LS refinement shellResolution: 2.4→2.46 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.317 166 -
Rwork0.233 1492 -
obs-1492 93.67 %

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