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- PDB-4fls: Crystal structure of Amylosucrase inactive double mutant F290K-E3... -

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Basic information

Entry
Database: PDB / ID: 4fls
TitleCrystal structure of Amylosucrase inactive double mutant F290K-E328Q from Neisseria polysaccharea in complex with sucrose.
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
sucrose / Amylosucrase
Similarity search - Component
Biological speciesNeisseria polysaccharea (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 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 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / entity_name_com / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_molecule_features / pdbx_nonpoly_scheme / struct_conn / struct_ref_seq_dif / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.type / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Sep 13, 2023Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _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: Amylosucrase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,3575
Polymers71,5451
Non-polymers8124
Water5,044280
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)95.110, 114.700, 54.620
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein Amylosucrase


Mass: 71545.102 Da / Num. of mol.: 1 / Fragment: Amylosucrase (unp residues 13-636) / Mutation: F290K, E328Q
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: Polysaccharide beta-D-fructofuranose-(2-1)-alpha-D-glucopyranose / sucrose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: oligosaccharide with reducing-end-to-reducing-end glycosidic bond
References: sucrose
DescriptorTypeProgram
DFrufb2-1DGlcpaGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[ha122h-2b_2-5][a2122h-1a_1-5]/1-2/a2-b1WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(2+1)][a-D-Glcp]{}}LINUCSPDB-CARE
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 280 / 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: 2.08 Å3/Da / Density % sol: 40.92 %
Crystal growTemperature: 285 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 30% PEG 6000, 0.1 M HEPES, 0.020 M sucrose, 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: ID23-1 / Wavelength: 0.8 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Sep 12, 2010
RadiationMonochromator: Si 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.8 Å / Relative weight: 1
ReflectionResolution: 2.3→57.35 Å / Num. all: 27320 / Num. obs: 26993 / % possible obs: 98.8 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.3 % / Rmerge(I) obs: 0.142 / Net I/σ(I): 5.1
Reflection shellResolution: 2.3→2.42 Å / Redundancy: 3.2 % / Rmerge(I) obs: 0.418 / Mean I/σ(I) obs: 1.9 / Num. unique all: 3768 / % possible all: 96.7

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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: 2.3→11.98 Å / Cor.coef. Fo:Fc: 0.94 / Cor.coef. Fo:Fc free: 0.907 / SU B: 6.806 / SU ML: 0.164 / Cross valid method: THROUGHOUT / ESU R: 0.487 / ESU R Free: 0.239 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.22036 1340 5 %RANDOM
Rwork0.17604 ---
all0.17829 27284 --
obs0.17829 25382 97.94 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 17.361 Å2
Baniso -1Baniso -2Baniso -3
1--0.03 Å20 Å20 Å2
2---0.07 Å20 Å2
3---0.1 Å2
Refinement stepCycle: LAST / Resolution: 2.3→11.98 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5007 0 53 280 5340
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.025231
X-RAY DIFFRACTIONr_angle_refined_deg1.1251.9427132
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.2955635
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.19723.993268
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.69115799
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.2131535
X-RAY DIFFRACTIONr_chiral_restr0.080.2763
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.0214103
LS refinement shellResolution: 2.3→2.358 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.291 88 -
Rwork0.219 1606 -
obs-1606 95.81 %

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