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- PDB-5n6v: Crystal structure of Neisseria polysaccharea amylosucrase mutant ... -

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Basic information

Entry
Database: PDB / ID: 5n6v
TitleCrystal structure of Neisseria polysaccharea amylosucrase mutant derived from Neutral genetic Drift-based engineering
ComponentsAmylosucrase
KeywordsHYDROLASE / Glycoside hydrolase Glucosyl transferase variant neutral drift
Function / homology
Function and homology information


amylosucrase / amylosucrase activity / carbohydrate metabolic process / extracellular region
Similarity search - Function
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 ...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 / beta-D-fructofuranose / alpha-D-glucopyranose / TRIETHYLENE GLYCOL / Amylosucrase
Similarity search - Component
Biological speciesNeisseria polysaccharea (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsDaude, D. / Verges, A. / Tranier, S.
CitationJournal: Acs Catalysis / Year: 2019
Title: Neutral Genetic Drift-Based Engineering of a Sucrose-Utilizing Enzyme toward Glycodiversification.
Authors: Daude, D. / Verges, A. / Cambon, E. / Emond, S. / Tranier, S. / Andre, I. / Remaud-Simeon, M.
History
DepositionFeb 16, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 7, 2018Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2019Group: Data collection / Database references / Category: citation / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...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 / 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.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 / _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)73,59010
Polymers71,6161
Non-polymers1,9749
Water21,0601169
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3030 Å2
ΔGint21 kcal/mol
Surface area23910 Å2
MethodPISA
Unit cell
Length a, b, c (Å)60.100, 96.150, 116.920
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-1409-

HOH

21A-1728-

HOH

31A-1812-

HOH

41A-1933-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Amylosucrase /


Mass: 71616.133 Da / Num. of mol.: 1 / Mutation: D37H, V331D, D526N, V609I
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) / Variant (production host): TOP10 / References: UniProt: Q9ZEU2, amylosucrase

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Sugars , 3 types, 5 molecules

#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
#4: Sugar ChemComp-GLC / alpha-D-glucopyranose / alpha-D-glucose / D-glucose / glucose / Glucose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
IdentifierTypeProgram
DGlcpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-glucopyranoseCOMMON NAMEGMML 1.0
a-D-GlcpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#7: Sugar ChemComp-FRU / beta-D-fructofuranose / beta-D-fructose / D-fructose / fructose / Fructose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
IdentifierTypeProgram
DFrufbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-fructofuranoseCOMMON NAMEGMML 1.0
b-D-FrufIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
FruSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 5 types, 1173 molecules

#3: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER / Tris


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#5: Chemical ChemComp-EPE / 4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID / HEPES / HEPES


Mass: 238.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18N2O4S / Comment: pH buffer*YM
#6: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL / Polyethylene glycol


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#8: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400 / Polyethylene glycol


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


Mass: 18.015 Da / Num. of mol.: 1169 / 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: 2.39 Å3/Da / Density % sol: 49 %
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: ID29 / Wavelength: 0.97625 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Apr 24, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97625 Å / Relative weight: 1
ReflectionResolution: 1.6→48.075 Å / Num. obs: 89712 / % possible obs: 99.6 % / Observed criterion σ(I): -3 / Redundancy: 7.374 % / Biso Wilson estimate: 13.05 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.101 / Rrim(I) all: 0.108 / Χ2: 0.916 / Net I/σ(I): 14.89
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.6-1.646.9290.5613.1865180.8960.60798.9
1.64-1.697.3870.4893.8863660.9310.52699.2
1.69-1.747.620.4334.5861770.9490.46599.2
1.74-1.797.5580.3735.3360350.9560.40199.4
1.79-1.857.4420.3056.4658520.9680.32899.5
1.85-1.917.2780.2637.5256630.9760.28399.6
1.91-1.986.8080.2188.7155010.9790.23699.5
1.98-2.077.640.18510.8553040.9880.19899.8
2.07-2.167.6520.16812.7850700.990.1899.7
2.16-2.267.6360.14814.948850.9920.15999.8
2.26-2.397.4830.1316.7646270.9940.13999.9
2.39-2.536.8960.10718.544210.9930.11799.9
2.53-2.77.7470.09621.8541400.9960.10399.9
2.7-2.927.7410.08124.8338710.9970.08799.9
2.92-3.27.5890.06828.9535800.9980.07399.9
3.2-3.586.9780.05533.7232550.9980.059100
3.58-4.137.3080.04539.6628810.9980.04899.8
4.13-5.067.5110.04242.6824880.9980.04599.9
5.06-7.166.7860.04537.8419290.9980.04999.5
7.16-48.0756.9030.03847.0911490.9990.04198.6

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Processing

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

1g5a


Resolution: 1.6→48.075 Å / SU ML: 0.15 / Cross valid method: FREE R-VALUE / σ(F): 1.99 / Phase error: 17.33
RfactorNum. reflection% reflectionSelection details
Rfree0.18 4500 5.02 %random selection
Rwork0.1484 ---
obs0.15 89654 99.58 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 67.62 Å2 / Biso mean: 17.5553 Å2 / Biso min: 6.51 Å2
Refinement stepCycle: final / Resolution: 1.6→48.075 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5056 0 131 1169 6356
Biso mean--30.56 28.63 -
Num. residues----628
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0065407
X-RAY DIFFRACTIONf_angle_d1.2627358
X-RAY DIFFRACTIONf_chiral_restr0.141778
X-RAY DIFFRACTIONf_plane_restr0.005954
X-RAY DIFFRACTIONf_dihedral_angle_d13.6371956
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.6-1.61820.2381610.20992750291199
1.6182-1.63720.2551400.20862801294199
1.6372-1.65720.24881400.20172797293799
1.6572-1.67820.23841450.19512816296199
1.6782-1.70030.23561640.18952760292499
1.7003-1.72360.20891480.18412799294799
1.7236-1.74820.20461500.18052814296499
1.7482-1.77430.24231390.17472802294199
1.7743-1.8020.21481370.17022802293999
1.802-1.83150.2161560.16662811296799
1.8315-1.86310.19891430.158727892932100
1.8631-1.8970.17021680.15628102978100
1.897-1.93350.20081460.154828392985100
1.9335-1.9730.18011450.155727952940100
1.973-2.01590.21251480.156328352983100
2.0159-2.06280.20071400.15228372977100
2.0628-2.11430.14651560.146627992955100
2.1143-2.17150.16771300.140228743004100
2.1715-2.23540.18181360.136528693005100
2.2354-2.30760.1551610.138828172978100
2.3076-2.390.15641480.141428272975100
2.39-2.48570.1761390.141828713010100
2.4857-2.59880.19711820.144928142996100
2.5988-2.73580.18541450.144928733018100
2.7358-2.90720.17461630.149428673030100
2.9072-3.13170.15931560.140828673023100
3.1317-3.44670.15331630.138328763039100
3.4467-3.94530.14461380.124929203058100
3.9453-4.96980.14981470.121229603107100
4.9698-48.09670.20581660.15713063322999

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