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- PDB-2e9b: Crystal structure of pullulanase type I from Bacillus subtilis st... -

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Basic information

Entry
Database: PDB / ID: 2e9b
TitleCrystal structure of pullulanase type I from Bacillus subtilis str. 168 complexed with maltose
ComponentsAmyX protein
KeywordsHYDROLASE / smultiple domain / beta-alpha-barrel / alpha-amylase-family maltose
Function / homology
Function and homology information


pullulanase / pullulanase activity / carbohydrate metabolic process
Similarity search - Function
Immunoglobulin-like - #2320 / Pullulanase, N1 domain / Pullulanase N1-terminal domain / : / Pullulanase, all-beta domain / Pullulanase, type I / Glycoside hydrolase, family 13, N-terminal / Carbohydrate-binding module 48 (Isoamylase N-terminal domain) / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain ...Immunoglobulin-like - #2320 / Pullulanase, N1 domain / Pullulanase N1-terminal domain / : / Pullulanase, all-beta domain / Pullulanase, type I / Glycoside hydrolase, family 13, N-terminal / Carbohydrate-binding module 48 (Isoamylase N-terminal domain) / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Golgi alpha-mannosidase II / Glycosyl hydrolase, all-beta / Glycosidases / Immunoglobulin E-set / Glycoside hydrolase superfamily / Immunoglobulins / TIM Barrel / Alpha-Beta Barrel / Immunoglobulin-like fold / Immunoglobulin-like / Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
alpha-maltose / alpha-maltotriose / ACETATE ION / Pullulanase / Pullulanase
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 Å
AuthorsMikami, B. / Malle, D. / Utsumi, S. / Iwamoto, H. / Katsuya, Y.
Citation
Journal: To be Published
Title: Crystal structure of pullulanase type I from Bacillus subtilis str. 168 in complex with maltose and alpha-cyclodextrin
Authors: Malle, D. / Iwamoto, H. / Katsuya, Y. / Utsumi, S. / Mikami, B.
#1: Journal: ACTA CRYSTALLOGR.,SECT.F / Year: 2006
Title: Overexpression, purification and preliminary X-ray analysis of pullulanase from Bacillus subtilis strain 168
Authors: Malle, D. / Itoh, T. / Hashimoto, W. / Murata, K. / Utsumi, S. / Mikami, B.
History
DepositionJan 24, 2007Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 19, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Non-polymer description / Version format compliance
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 / 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_struct_assembly_gen / pdbx_struct_conn_angle / struct_asym / struct_conn / struct_conn_type / 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 / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _struct_conn.conn_type_id / _struct_conn.id / _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_type.id / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Oct 25, 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

Downloads & links

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Assembly

Deposited unit
A: AmyX protein
B: AmyX protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,20612
Polymers162,4132
Non-polymers1,79310
Water8,935496
1
A: AmyX protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)82,3047
Polymers81,2071
Non-polymers1,0976
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: AmyX protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)81,9025
Polymers81,2071
Non-polymers6964
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)71.031, 129.178, 192.060
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein AmyX protein / Pullulanase


Mass: 81206.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Strain: strain 168 / Gene: amyX / Plasmid: pET21d / Production host: Escherichia coli (E. coli) / Strain (production host): HMS 174
References: UniProt: O34587, UniProt: C0SPA0*PLUS, pullulanase

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Sugars , 3 types, 3 molecules

#2: 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
#3: Polysaccharide alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose / alpha-maltose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1a_1-5]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}LINUCSPDB-CARE
#4: Polysaccharide beta-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose


Type: oligosaccharide / Mass: 504.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpb1-4DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1a_1-5][a2122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][b-D-Glcp]{}}}LINUCSPDB-CARE

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Non-polymers , 4 types, 503 molecules

#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#6: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H3O2
#7: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 496 / 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: 2.71 Å3/Da / Density % sol: 54.64 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.2
Details: 10% PEG 4000, 0.1M sodium acetate buffer, 0.2M magnesium acetate, 0.2M maltose, pH 5.2, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 173 K
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL44XU / Wavelength: 0.915 Å
DetectorType: Bruker DIP-6040 / Detector: CCD / Date: Dec 12, 2005 / Details: mirrors
RadiationMonochromator: Si 111 CHANNEL / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.915 Å / Relative weight: 1
ReflectionResolution: 2.2→40 Å / Num. all: 90084 / Num. obs: 81525 / % possible obs: 90.5 % / Observed criterion σ(I): 1 / Redundancy: 4.4 % / Biso Wilson estimate: 19.3 Å2 / Rmerge(I) obs: 0.082 / Net I/σ(I): 14.3
Reflection shellResolution: 2.2→2.28 Å / Redundancy: 4.3 % / Rmerge(I) obs: 0.485 / Num. unique all: 8890 / % possible all: 83.3

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Processing

Software
NameVersionClassification
CNS1.1refinement
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2FGZ

2fgz


Resolution: 2.3→14.97 Å / Rfactor Rfree error: 0.003 / Data cutoff high absF: 207608.34 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.242 6750 10 %RANDOM
Rwork0.194 ---
obs0.194 67209 85.1 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 43.0189 Å2 / ksol: 0.344012 e/Å3
Displacement parametersBiso mean: 36.5 Å2
Baniso -1Baniso -2Baniso -3
1-14.66 Å20 Å20 Å2
2---1.96 Å20 Å2
3----12.71 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.34 Å0.26 Å
Luzzati d res low-5 Å
Luzzati sigma a0.34 Å0.26 Å
Refinement stepCycle: LAST / Resolution: 2.3→14.97 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11348 0 117 496 11961
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.3
X-RAY DIFFRACTIONc_dihedral_angle_d24.2
X-RAY DIFFRACTIONc_improper_angle_d0.79
X-RAY DIFFRACTIONc_mcbond_it1.291.5
X-RAY DIFFRACTIONc_mcangle_it2.092
X-RAY DIFFRACTIONc_scbond_it1.982
X-RAY DIFFRACTIONc_scangle_it2.892.5
LS refinement shellResolution: 2.3→2.44 Å / Rfactor Rfree error: 0.01 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.309 976 10.3 %
Rwork0.25 8463 -
obs--72.6 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramprotein.top
X-RAY DIFFRACTION2water_rep.paramwater.top
X-RAY DIFFRACTION3ion.paramion.top
X-RAY DIFFRACTION4carbohydrate.paramcarbohydrate.top
X-RAY DIFFRACTION5act.paramact.top

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