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- PDB-1s46: Covalent intermediate of the E328Q amylosucrase mutant -

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Basic information

Entry
Database: PDB / ID: 1s46
TitleCovalent intermediate of the E328Q amylosucrase mutant
Componentsamylosucrase
KeywordsTRANSFERASE / protein-glucopyranosyl covalent intermediate / (beta/alpha)8-barrel
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
beta-D-glucopyranose / Amylosucrase
Similarity search - Component
Biological speciesNeisseria polysaccharea (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsJensen, M.H. / Mirza, O. / Albenne, C. / Remaud-Simeon, M. / Monsan, P. / Gajhede, M. / Skov, L.K.
Citation
Journal: Biochemistry / Year: 2004
Title: Crystal structure of the covalent intermediate of amylosucrase from Neisseria polysaccharea.
Authors: Jensen, M.H. / Mirza, O. / Albenne, C. / Remaud-Simeon, M. / Monsan, P. / Gajhede, M. / Skov, L.K.
#1: Journal: J.Biol.Chem. / Year: 2001
Title: Amylosucrase, a Glucan-Synthesizing Enzyme from the Alpha-Amylase Family
Authors: Skov, L.K. / Mirza, O. / Henriksen, A. / De Montalk, G.P. / Remaud-Simeon, M. / Sarcabal, P. / Willemot, R.M. / Monsan, P. / Gajhede, M.
#2: Journal: Biochemistry / Year: 2001
Title: Crystal Structure Of Amylosucrase From Neisseria Polysaccharea In Complex With D-Glucose
Authors: Mirza, O. / Skov, L.K. / Remaud-Simeon, M. / De Montalk, G.P. / Albenne, C. / Monsan, P. / Gajhede, M.
History
DepositionJan 15, 2004Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 23, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 11, 2017Group: Data collection / Refinement description / Category: diffrn_source / software
Item: _diffrn_source.pdbx_synchrotron_site / _software.classification / _software.name
Revision 1.4Jan 24, 2018Group: Database references / Category: citation_author / Item: _citation_author.name
Revision 1.5Jul 29, 2020Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_ref_seq_dif / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.6Oct 27, 2021Group: Database references / Structure summary / Category: chem_comp / database_2 / struct_ref_seq_dif
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Remark 999 SEQUENCE THE DISCREPANCY BETWEEN RESIDUE TYR 131 AND THE GENBANK SEQUENCE DATABASE REFERENCE, ... SEQUENCE THE DISCREPANCY BETWEEN RESIDUE TYR 131 AND THE GENBANK SEQUENCE DATABASE REFERENCE, ACCESSION 4107260, RESIDUE HIS 139 IS DUE TO A PCR ERROR.

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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)71,7102
Polymers71,5301
Non-polymers1801
Water9,008500
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)96.253, 116.630, 60.539
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-1288-

HOH

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Components

#1: Protein amylosucrase /


Mass: 71530.086 Da / Num. of mol.: 1 / Mutation: E328Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria polysaccharea (bacteria) / Plasmid: pGEX-6-P-3 / Production host: Escherichia coli (E. coli) / Strain (production host): JM109 / References: UniProt: Q9ZEU2, amylosucrase
#2: Sugar ChemComp-BGC / beta-D-glucopyranose / beta-D-glucose / D-glucose / glucose / Glucose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
IdentifierTypeProgram
DGlcpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-glucopyranoseCOMMON NAMEGMML 1.0
b-D-GlcpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 500 / 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: 48.2 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7
Details: Sucrose, PEG 6000, SODIUM CHLORIDE, TRIS-HCL, EDTA, HEPES, DTT, Alpha-D-glucopyranosyl fluoride , pH 7.0, VAPOR DIFFUSION, HANGING DROP, temperature 277K
Crystal grow
*PLUS
Method: vapor diffusion, hanging drop
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
14 mg/mlprotein1drop
2150 mM1dropNaCl
350 mMTris-HCl1droppH7.0
41 mMEDTA1drop
51 mMalpha-dithiothreitol1drop
614 mMsucrose1reservoir
730 %PEG60001reservoir
80.1 MHEPES1reservoirpH7.0

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: SYNCHROTRON / Site: MAX II / Beamline: I711 / Wavelength: 1.011 Å
DetectorType: MARRESEARCH / Detector: CCD / Date: Nov 24, 2000
RadiationMonochromator: SI 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.011 Å / Relative weight: 1
ReflectionResolution: 2.2→20 Å / Num. all: 32544 / Num. obs: 32544 / % possible obs: 92 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.9 % / Biso Wilson estimate: 19.4 Å2 / Rsym value: 0.081 / Net I/σ(I): 18
Reflection shellResolution: 2.2→2.3 Å / Redundancy: 4 % / Mean I/σ(I) obs: 6 / Num. unique all: 3287 / Rsym value: 0.328 / % possible all: 94.7
Reflection
*PLUS
Num. measured all: 128590 / Rmerge(I) obs: 0.081
Reflection shell
*PLUS
Highest resolution: 2.2 Å / Lowest resolution: 2.28 Å / % possible obs: 94.7 % / Rmerge(I) obs: 0.328

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Processing

Software
NameVersionClassification
CNS1.1refinement
MAR345data collection
SCALEPACKdata scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.2→19.89 Å / Rfactor Rfree error: 0.006 / Data cutoff high absF: 844885.82 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.221 1539 4.9 %RANDOM
Rwork0.184 ---
all0.188 31129 --
obs0.184 31129 88.2 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 10 Å2 / ksol: 0.318055 e/Å3
Displacement parametersBiso mean: 15.6 Å2
Baniso -1Baniso -2Baniso -3
1--0.82 Å20 Å20 Å2
2---2.09 Å20 Å2
3---2.91 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.28 Å0.22 Å
Luzzati d res low-5 Å
Luzzati sigma a0.29 Å0.19 Å
Refinement stepCycle: LAST / Resolution: 2.2→19.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5060 0 11 500 5571
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.012
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d23.6
X-RAY DIFFRACTIONc_improper_angle_d1.43
LS refinement shellResolution: 2.2→2.34 Å / Rfactor Rfree error: 0.018 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.286 264 5.2 %
Rwork0.223 4810 -
obs--87.3 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1NYPROTEIN.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAM
X-RAY DIFFRACTION3NYG1I.PAR
Refinement
*PLUS
Highest resolution: 2.2 Å / Lowest resolution: 20 Å / % reflection Rfree: 5 %
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg23.6
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg1.43

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