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- PDB-1ob0: Kinetic stabilization of Bacillus licheniformis alpha-amylase thr... -

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Basic information

Entry
Database: PDB / ID: 1ob0
TitleKinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface
ComponentsALPHA-AMYLASE
KeywordsHYDROLASE / GLYCOSYLTRANSFERASE / STARCH DEGRADATION / THERMOSTABILITY / CALCIUM / SODIUM
Function / homology
Function and homology information


alpha-amylase / alpha-amylase activity / carbohydrate metabolic process / calcium ion binding / extracellular space
Similarity search - Function
Elongation Factor Tu (Ef-tu); domain 3 - #140 / Alpha-amylase, thermostable / Alpha-amylase C-terminal, prokaryotic / Alpha-amylase C-terminal / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Golgi alpha-mannosidase II / Elongation Factor Tu (Ef-tu); domain 3 / Glycosyl hydrolase, all-beta ...Elongation Factor Tu (Ef-tu); domain 3 - #140 / Alpha-amylase, thermostable / Alpha-amylase C-terminal, prokaryotic / Alpha-amylase C-terminal / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Golgi alpha-mannosidase II / Elongation Factor Tu (Ef-tu); domain 3 / Glycosyl hydrolase, all-beta / Glycosidases / Glycoside hydrolase superfamily / TIM Barrel / Alpha-Beta Barrel / Immunoglobulin-like / Beta Barrel / Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Biological speciesBACILLUS LICHENIFORMIS (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.83 Å
AuthorsMachius, M. / Declerck, N. / Huber, R. / Wiegand, G.
Citation
Journal: J.Biol.Chem. / Year: 2003
Title: Kinetic Stabilization of Bacillus Licheniformis Alpha-Amylase Through Introduction of Hydrophobic Residues at the Surface
Authors: Machius, M. / Declerck, N. / Huber, R. / Wiegand, G.
#1: Journal: Mol.Cells / Year: 1997
Title: Crystal Structure of Thermostable Alpha-Amylase from Bacillus Licheniformis Refined at 1.7 A Resolution
Authors: Hwang, K.Y. / Song, H.K. / Chang, C. / Lee, J. / Lee, S.Y. / Kim, K.K. / Choe, S. / Sweet, R.M. / Suh, S.W.
#2: Journal: Structure / Year: 1998
Title: Activation of Bacillus Licheniformis Alpha-Amylase Through a Disorder-->Order Transition of the Substrate-Binding Site Mediated by a Calcium-Sodium-Calcium Metal Triad
Authors: Machius, M. / Declerck, N. / Huber, R. / Wiegand, G.
#3: Journal: J.Mol.Biol. / Year: 1995
Title: Crystal Structure of Calcium-Depleted Bacillus Licheniformis Alpha-Amylase at 2.2 A Resolution
Authors: Machius, M. / Wiegand, G. / Huber, R.
History
DepositionJan 21, 2003Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 30, 2003Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2014Group: Atomic model / Derived calculations ...Atomic model / Derived calculations / Non-polymer description / Other / Refinement description / Structure summary / Version format compliance
Revision 1.2Sep 25, 2019Group: Data collection / Experimental preparation / Other / Category: exptl_crystal_grow / pdbx_database_status
Item: _exptl_crystal_grow.method / _pdbx_database_status.status_code_sf
Revision 1.3Dec 13, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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_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_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 9-STRANDED BARREL THIS IS REPRESENTED BY A 10-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. SHEET THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN THE SHEET RECORDS BELOW, TWO SHEETS ARE DEFINED.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ALPHA-AMYLASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)55,4575
Polymers55,3141
Non-polymers1434
Water5,837324
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)91.292, 91.292, 137.466
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number169
Space group name H-MP61

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Components

#1: Protein ALPHA-AMYLASE / / 1 / 4-ALPHA-D-GLUCAN-4-GLUCANOHYDROLASE


Mass: 55314.027 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) BACILLUS LICHENIFORMIS (bacteria) / Production host: BACILLUS SUBTILIS (bacteria) / References: UniProt: P06278, alpha-amylase
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 324 / Source method: isolated from a natural source / Formula: H2O
Compound detailsCATALYSES ENDOHYDROLYSIS OF 1,4-ALPHA-GLUCOSIDIC LINKAGES IN OLIGOSACCHARIDES AND POLYSACCHARIDES. ...CATALYSES ENDOHYDROLYSIS OF 1,4-ALPHA-GLUCOSIDIC LINKAGES IN OLIGOSACCHARIDES AND POLYSACCHARIDES. PRIMARILY USED IN THE FOOD INDUSTRY FOR HIGH TEMPERATURE LIQUEFACTION OF STARCH-CONTAINING MASHES AND IN THE DETERGENT INDUSTRY TO REMOVE STARCH. ACTIVE AT RELATIVELY HIGH PH VALUES (UP TO PH 11) AND AT HIGH TEMPERATURES (UP TO 100 DEGREE CELSIUS). ENGINEERED RESIDUES HIS (133) VAL ASN (190) PHE ALA (209) VAL GLN (264) SER ASN (265) TYR
Sequence detailsDATABASE ENTRY SWS P06278 CONTAINS A 29 RESIDUE PRECURSOR. NUMBERING IN THE PDB ENTRY STARTS WITH 1 ...DATABASE ENTRY SWS P06278 CONTAINS A 29 RESIDUE PRECURSOR. NUMBERING IN THE PDB ENTRY STARTS WITH 1 WHICH CORRESPONDS TO RESIDUE 30 IN THE DATABASE ENTRY.

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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: 3 Å3/Da / Density % sol: 59 %
Crystal growMethod: vapor diffusion / pH: 7
Details: PROTEIN WAS CRYSTALLIZED BY VAPOR DIFFUSION FROM DROPS CONTAINING 4 UL OF PROTEIN SOLUTION (10 MG/ML IN 50 MM TRIS/HCL, PH 8.0) PLUS 4 UL OF RESERVOIR SOLUTION (50 MM HEPES, 1 M AMMONIUM ...Details: PROTEIN WAS CRYSTALLIZED BY VAPOR DIFFUSION FROM DROPS CONTAINING 4 UL OF PROTEIN SOLUTION (10 MG/ML IN 50 MM TRIS/HCL, PH 8.0) PLUS 4 UL OF RESERVOIR SOLUTION (50 MM HEPES, 1 M AMMONIUM SULFATE, 1% (V/V) PEG 500, PH 7.0) EQUILIBRATED AGAINST 1 ML OF RESERVOIR SOLUTION.
Crystal grow
*PLUS
Temperature: 20 ℃ / pH: 8 / Method: vapor diffusion
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
110 mg/mlprotein1drop
250 mMTris-HCl1droppH8.0
350 mMHEPES1reservoir
41 Mammonium sulfate1reservoir
51 %(v/v)PEG5001reservoirpH7.0

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Data collection

DiffractionMean temperature: 278 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: May 15, 1997 / Details: BEAM FOCUSSING MIRROR SYSTEM (MSC, USA)
RadiationMonochromator: NI FILTER / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.83→25.9 Å / Num. obs: 55286 / % possible obs: 96.9 % / Observed criterion σ(I): 0 / Redundancy: 4 % / Biso Wilson estimate: 19.7 Å2 / Rmerge(I) obs: 0.067 / Net I/σ(I): 19
Reflection shellResolution: 1.83→1.85 Å / Redundancy: 2.6 % / Rmerge(I) obs: 0.444 / Mean I/σ(I) obs: 2.1 / % possible all: 96.1
Reflection
*PLUS
Highest resolution: 1.83 Å / Num. obs: 61355 / % possible obs: 83.8 % / Redundancy: 3.8 % / Num. measured all: 231592 / Rmerge(I) obs: 0.073
Reflection shell
*PLUS
Highest resolution: 1.7 Å / Lowest resolution: 1.79 Å / % possible obs: 25 % / Rmerge(I) obs: 0.31

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Processing

Software
NameVersionClassification
CNS1.1refinement
DENZOdata reduction
SCALEPACKdata scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1BPL

1bpl


Resolution: 1.83→25.89 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 987507.06 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.154 1448 2.7 %RANDOM
Rwork0.147 ---
obs0.147 53205 93 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 48.3911 Å2 / ksol: 0.357256 e/Å3
Displacement parametersBiso mean: 23.7 Å2
Baniso -1Baniso -2Baniso -3
1--0.34 Å20.23 Å20 Å2
2---0.34 Å20 Å2
3---0.69 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.16 Å0.16 Å
Luzzati d res low-5 Å
Luzzati sigma a0.15 Å0.14 Å
Refinement stepCycle: LAST / Resolution: 1.83→25.89 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3909 0 4 324 4237
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.01
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d24
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.84
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.151.5
X-RAY DIFFRACTIONc_mcangle_it1.662
X-RAY DIFFRACTIONc_scbond_it2.042
X-RAY DIFFRACTIONc_scangle_it3.142.5
LS refinement shellResolution: 1.83→1.94 Å / Rfactor Rfree error: 0.015 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.223 215 2.6 %
Rwork0.228 8072 -
obs--87.1 %
Xplor file
Refine-IDSerial noParam file
X-RAY DIFFRACTION1PROTEIN_REP.PARAM
X-RAY DIFFRACTION2WATER_REP.PARAM
X-RAY DIFFRACTION3ION.PARAM
Refinement
*PLUS
Highest resolution: 1.83 Å / Lowest resolution: 25.89 Å / Num. reflection obs: 58628 / Num. reflection Rfree: 2345 / % reflection Rfree: 5 % / Rfactor Rfree: 0.174 / Rfactor Rwork: 0.156
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.009
X-RAY DIFFRACTIONc_angle_deg1.359
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.82
LS refinement shell
*PLUS
Highest resolution: 1.7 Å / Lowest resolution: 1.73 Å / Rfactor Rfree: 0.33 / Rfactor Rwork: 0.319

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