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- PDB-1mw0: Amylosucrase mutant E328Q co-crystallized with maltoheptaose then... -

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Basic information

Entry
Database: PDB / ID: 1mw0
TitleAmylosucrase mutant E328Q co-crystallized with maltoheptaose then soaked with maltoheptaose.
Componentsamylosucrase
KeywordsTRANSFERASE / (beta-alpha)8 barrel / protein-sugar complex
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
2,3-DIHYDROXY-1,4-DITHIOBUTANE / : / Amylosucrase
Similarity search - Component
Biological speciesNeisseria polysaccharea (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.01 Å
AuthorsSkov, L.K. / Mirza, O. / Sprogoe, D. / Dar, I. / Remaud-Simeon, M. / Albenne, C. / Monsan, P. / Gajhede, M.
Citation
Journal: J.BIOL.CHEM. / Year: 2002
Title: Oligosaccharide and Sucrose Complexes of Amylosucrase. STRUCTURAL IMPLICATIONS FOR THE POLYMERASE ACTIVITY
Authors: Skov, L.K. / Mirza, O. / Sprogoe, D. / Dar, I. / Remaud-Simeon, M. / Albenne, C. / Monsan, P. / Gajhede, M.
#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 and the Active Site Mutant Glu328Gln in Complex with the Natural Substrate Sucrose
Authors: Mirza, O. / Skov, L.K. / Remaud-Simeon, M. / De Montalk, G.P. / Albenne, C. / Monsan, P. / Gajhede, M.
History
DepositionSep 27, 2002Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Dec 18, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 11, 2017Group: Refinement description / Category: software
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / 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_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / 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_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Nov 10, 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
Revision 2.2Nov 6, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_entry_details / pdbx_modification_feature

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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)74,6575
Polymers71,5301
Non-polymers3,1274
Water7,404411
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)96.067, 116.097, 60.745
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: 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-6P-3 / Production host: Escherichia coli (E. coli)
References: GenBank: 4107260, UniProt: Q9ZEU2*PLUS, amylosucrase
#2: Polysaccharide alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D- ...alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose


Type: oligosaccharide / Mass: 1153.001 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,7,6/[a2122h-1a_1-5]/1-1-1-1-1-1-1/a4-b1_b4-c1_c4-d1_d4-e1_e4-f1_f4-g1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}}}}}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose


Type: oligosaccharide / Mass: 666.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpb1-4DGlcpa1-4DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,4,3/[a2122h-1a_1-5][a2122h-1b_1-5]/1-1-1-2/a4-b1_b4-c1_c4-d1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][b-D-Glcp]{}}}}LINUCSPDB-CARE
#4: Chemical ChemComp-DTT / 2,3-DIHYDROXY-1,4-DITHIOBUTANE / 1,4-DITHIOTHREITOL


Mass: 154.251 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O2S2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 411 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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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: 2.37 Å3/Da / Density % sol: 48.03 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 7
Details: PEG6000, SODIUM CHLORIDE, TRIS-HCL, EDTA, HEPES, DTT, maltoheptaose, pH 7, VAPOR DIFFUSION, HANGING DROP, temperature 277K
Crystal grow
*PLUS
pH: 7 / Details: Skov, L.K., (2000) Acta Crystallogr., D56, 203.
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
14-6 mg/mlprotein1drop
2150 mM1dropNaCl
350 mMTris-HCl1droppH7.0
41 mMEDTA1drop
51 mMalpha-dithiothreitol1drop
630 %(w/v)PEG60001reservoir
7100 mMHEPES1reservoirpH7.0

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Data collection

DiffractionMean temperature: 120 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 1.008 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Feb 17, 2001
RadiationMonochromator: Si 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.008 Å / Relative weight: 1
ReflectionResolution: 2→20 Å / Num. all: 45913 / Num. obs: 45913 / % possible obs: 99.7 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Biso Wilson estimate: 10.8 Å2 / Rsym value: 0.071
Reflection shellResolution: 2→2.07 Å / Rsym value: 0.301 / % possible all: 99.6
Reflection
*PLUS
Lowest resolution: 20 Å / Num. measured all: 403150 / Rmerge(I) obs: 0.071
Reflection shell
*PLUS
% possible obs: 99.6 % / Rmerge(I) obs: 0.301

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Processing

Software
NameClassification
ProDCdata collection
SCALEPACKdata scaling
CNSrefinement
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.01→24.84 Å / Rfactor Rfree error: 0.005 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.223 2226 5 %RANDOM
Rwork0.185 ---
all-45913 --
obs-44662 96.5 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 41.0279 Å2 / ksol: 0.350515 e/Å3
Displacement parametersBiso mean: 19.8 Å2
Baniso -1Baniso -2Baniso -3
1--4.68 Å20 Å20 Å2
2---3 Å20 Å2
3---7.69 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.26 Å0.21 Å
Luzzati d res low-5 Å
Luzzati sigma a0.18 Å0.14 Å
Refinement stepCycle: LAST / Resolution: 2.01→24.84 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5050 0 209 411 5670
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_angle_deg1.2
X-RAY DIFFRACTIONc_dihedral_angle_d22.5
X-RAY DIFFRACTIONc_improper_angle_d0.75
X-RAY DIFFRACTIONc_mcbond_it0.651.5
X-RAY DIFFRACTIONc_mcangle_it1.042
X-RAY DIFFRACTIONc_scbond_it1.12
X-RAY DIFFRACTIONc_scangle_it1.72.5
LS refinement shellResolution: 2.01→2.13 Å / Rfactor Rfree error: 0.015 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.263 310 4.7 %
Rwork0.216 6220 -
obs--85.4 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION3CARBOHYDRATE.PARAMCARBOHYDRATE.TOP
X-RAY DIFFRACTION4ION.PARAM
X-RAY DIFFRACTION5DTTRED.PARAMDTTRED.TOP
Refinement
*PLUS
Highest resolution: 2 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_deg22.5
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.75

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