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- PDB-5n7j: Crystal structure of Neisseria polysaccharea amylosucrase mutant ... -

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Basic information

Entry
Database: PDB / ID: 5n7j
TitleCrystal structure of Neisseria polysaccharea amylosucrase mutant efficient for the synthesis of controlled size maltooligosaccharides
ComponentsAmylosucrase
KeywordsHYDROLASE / amylosucrase / Glycoside hydrolase / Glucosyl transferase / mutant / Neisseria polysaccharea / GH13
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 / alpha-maltotriose / Amylosucrase
Similarity search - Component
Biological speciesNeisseria polysaccharea (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.001 Å
AuthorsVerges, A. / Tranier, S.
CitationJournal: Carbohydr Polym / Year: 2017
Title: Engineering of anp efficient mutant of Neisseria polysaccharea amylosucrase for the synthesis of controlled size maltooligosaccharides.
Authors: Verges, A. / Barbe, S. / Cambon, E. / Moulis, C. / Tranier, S. / Remaud-Simeon, M. / Andre, I.
History
DepositionFeb 20, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Database references / Category: citation
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / 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 / pdbx_validate_close_contact / struct_asym / struct_conn / 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_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.occupancy / _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.pdbx_number_of_molecules / _entity.src_method / _entity.type / _pdbx_validate_close_contact.auth_asym_id_1 / _pdbx_validate_close_contact.auth_atom_id_1 / _pdbx_validate_close_contact.auth_comp_id_1 / _pdbx_validate_close_contact.auth_seq_id_1 / _struct_asym.entity_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Jan 17, 2024Group: 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

Downloads & links

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Assembly

Deposited unit
A: Amylosucrase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,8189
Polymers71,4941
Non-polymers2,3248
Water16,952941
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2970 Å2
ΔGint3 kcal/mol
Surface area24300 Å2
MethodPISA
Unit cell
Length a, b, c (Å)59.990, 97.570, 115.150
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP22121
Components on special symmetry positions
IDModelComponents
11A-1084-

HOH

21A-1698-

HOH

31A-1732-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Amylosucrase


Mass: 71494.000 Da / Num. of mol.: 1
Mutation: R226K, I228V, A289I, F290Y, E300I, V331T, Q437S, N439D, C445A, G537D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria polysaccharea (bacteria) / Gene: ams / Plasmid: pGEX-6P-3 / Production host: Escherichia coli (E. coli) / Strain (production host): TOP10 / References: UniProt: Q9ZEU2, amylosucrase

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Sugars , 2 types, 4 molecules

#2: Polysaccharide beta-D-fructofuranose-(2-1)-alpha-D-glucopyranose / sucrose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 3
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: Polysaccharide alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose / alpha-maltotriose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 504.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltotriose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,3,2/[a2122h-1a_1-5]/1-1-1/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}}LINUCSPDB-CARE

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Non-polymers , 4 types, 945 molecules

#4: 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
#5: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400


Mass: 238.278 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H22O6 / Comment: precipitant*YM
#6: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 941 / 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: 2.36 Å3/Da / Density % sol: 47.81 %
Crystal growTemperature: 285 K / Method: vapor diffusion, hanging drop / pH: 7.3 / Details: PEG 6000, HEPES

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID14-1 / Wavelength: 0.9334 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Oct 31, 2012
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9334 Å / Relative weight: 1
ReflectionResolution: 2→48.785 Å / Num. obs: 45528 / % possible obs: 98.2 % / Observed criterion σ(I): -3 / Redundancy: 4.028 % / Rmerge F obs: 0.152 / Rmerge(I) obs: 0.09 / Rrim(I) all: 0.103 / Χ2: 0.948 / Net I/σ(I): 15.51 / Num. measured all: 183402 / Scaling rejects: 16
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsRrim(I) all% possible all
2-2.053.4220.4512.8327800.53482.4
2.05-2.113.760.3793.6431090.44294.9
2.11-2.174.1310.3464.4732240.39899.8
2.17-2.244.1330.2985.1431020.34299.8
2.24-2.314.1250.254630260.29299.9
2.31-2.394.1440.2127.0729280.24399.9
2.39-2.484.1450.1878.1128080.21499.9
2.48-2.584.1310.169.3827340.184100
2.58-2.74.1250.13410.9226150.15499.9
2.7-2.834.120.111325070.12799.8
2.83-2.984.1060.09315.4123960.10799.9
2.98-3.164.1040.07219.1522790.082100
3.16-3.384.0960.05623.9321340.06499.8
3.38-3.654.090.04229.9219830.04899.8
3.65-44.050.03336.1318450.03899.7
4-4.474.0690.02840.2616740.03299.6
4.47-5.174.020.02841.4714930.03299.7
5.17-6.333.9630.03236.112780.03799.7
6.33-8.953.8450.02841.610190.03299.8
8.95-48.7853.5050.02155.375940.02497.9

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Processing

Software
NameVersionClassification
XSCALEdata scaling
PHENIXrefinement
PDB_EXTRACT3.22data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1G5A

1g5a


Resolution: 2.001→48.785 Å / SU ML: 0.19 / Cross valid method: FREE R-VALUE / σ(F): 1.99 / Phase error: 17.19
RfactorNum. reflection% reflectionSelection details
Rfree0.1896 2305 5.06 %Random selection
Rwork0.1421 ---
obs0.1444 45528 98.2 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 74.37 Å2 / Biso mean: 19.3667 Å2 / Biso min: 5.08 Å2
Refinement stepCycle: final / Resolution: 2.001→48.785 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5048 0 156 941 6145
Biso mean--41.94 28.23 -
Num. residues----628
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0075437
X-RAY DIFFRACTIONf_angle_d1.3167402
X-RAY DIFFRACTIONf_chiral_restr0.169794
X-RAY DIFFRACTIONf_plane_restr0.004954
X-RAY DIFFRACTIONf_dihedral_angle_d14.6421943
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 16

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.001-2.04450.28021230.19522183230682
2.0445-2.09210.26831320.18292495262792
2.0921-2.14440.23761590.171726912850100
2.1444-2.20240.22811440.161527082852100
2.2024-2.26720.24331520.161526982850100
2.2672-2.34040.19611370.152827252862100
2.3404-2.4240.20871440.148927242868100
2.424-2.52110.21841600.147827112871100
2.5211-2.63580.23221390.150527442883100
2.6358-2.77480.19061450.145327282873100
2.7748-2.94860.19481540.145627352889100
2.9486-3.17620.17121520.137327632915100
3.1762-3.49580.17571380.135427602898100
3.4958-4.00140.14631430.117827812924100
4.0014-5.04050.14621420.110528242966100
5.0405-48.79950.14781410.144329533094100

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