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- PDB-1q4n: Structural studies of Phe256Trp of human salivary alpha-amylase: ... -

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Basic information

Entry
Database: PDB / ID: 1q4n
TitleStructural studies of Phe256Trp of human salivary alpha-amylase: implications for the role of a conserved water molecule and its associated chain in enzyme activity
ComponentsAlpha-amylase, salivary
KeywordsHYDROLASE / AMYLASE / MUTAGENESIS / TRIS / INHIBITOR
Function / homology
Function and homology information


Digestion of dietary carbohydrate / oligosaccharide metabolic process / alpha-amylase / alpha-amylase activity / chloride ion binding / carbohydrate metabolic process / calcium ion binding / extracellular space / extracellular exosome
Similarity search - Function
Alpha-amylase, C-terminal domain / Aamy_C / Alpha-amylase/branching enzyme, C-terminal all beta / Alpha amylase, C-terminal all-beta domain / Alpha amylase / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Golgi alpha-mannosidase II / Glycosyl hydrolase, all-beta ...Alpha-amylase, C-terminal domain / Aamy_C / Alpha-amylase/branching enzyme, C-terminal all beta / Alpha amylase, C-terminal all-beta domain / Alpha amylase / 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 / Immunoglobulin-like / Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Alpha-amylase 1B / Alpha-amylase 1C
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.07 Å
AuthorsRamasubbu, N.
CitationJournal: Arch.Biochem.Biophys. / Year: 2004
Title: Structural studies of a Phe256Trp mutant of human salivary alpha-amylase: implications for the role of a conserved water molecule in enzyme activity
Authors: Ramasubbu, N. / Sundar, K. / Ragunath, C. / Rafi, M.M.
History
DepositionAug 4, 2003Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 16, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Jan 31, 2018Group: Experimental preparation / Category: exptl_crystal_grow / Item: _exptl_crystal_grow.temp
Revision 2.0Dec 25, 2019Group: Database references / Derived calculations / Polymer sequence
Category: entity_poly / pdbx_struct_mod_residue ...entity_poly / pdbx_struct_mod_residue / struct_conn / struct_ref_seq_dif
Item: _entity_poly.pdbx_seq_one_letter_code_can / _pdbx_struct_mod_residue.parent_comp_id / _struct_conn.pdbx_leaving_atom_flag
Revision 2.1Oct 27, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / 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.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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 2.2Aug 16, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 2.3Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
X: Alpha-amylase, salivary
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,2334
Polymers55,9941
Non-polymers2393
Water5,837324
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)51.90, 74.20, 134.82
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Alpha-amylase, salivary / 1 / 4-alpha-D-glucan glucanohydrolase


Mass: 55994.297 Da / Num. of mol.: 1 / Mutation: F256W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AMY1A OR AMY1 / Plasmid: pVL1392 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P04745, UniProt: P0DTE8*PLUS, alpha-amylase
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-TAM / TRIS(HYDROXYETHYL)AMINOMETHANE


Mass: 163.215 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H17NO3 / Comment: pH buffer*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 324 / 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: 2.32 Å3/Da / Density % sol: 46.94 %
Crystal growMethod: vapor diffusion, hanging drop / pH: 9
Details: MPD, Calcium Chloride, Tris, pH 9.00, VAPOR DIFFUSION, HANGING DROP, temperature 100K
Crystal grow
*PLUS
Method: unknown / Details: Ramasubbu, N., (1991) Proteins, 11, 230.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU300 / Wavelength: 1.5418 / Wavelength: 1.5418 Å
DetectorType: MAR scanner 345 mm plate / Detector: IMAGE PLATE / Date: Dec 1, 2001 / Details: MIRRORS
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.07→41.1 Å / Num. all: 89732 / Num. obs: 30610 / % possible obs: 94.5 % / Observed criterion σ(F): 1 / Observed criterion σ(I): 1 / Redundancy: 3 % / Biso Wilson estimate: 28 Å2 / Rmerge(I) obs: 0.06 / Net I/σ(I): 13
Reflection shellResolution: 2.07→2.14 Å / % possible all: 60
Reflection
*PLUS
Highest resolution: 2.1 Å / Lowest resolution: 20 Å / Redundancy: 3 % / Num. measured all: 89732 / Rmerge(I) obs: 0.06
Reflection shell
*PLUS
% possible obs: 60 %

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Processing

Software
NameVersionClassification
DENZOdata reduction
SCALEPACKdata scaling
REFMAC5.1.24refinement
AMoREphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1MFV

1mfv


Resolution: 2.07→41.17 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.946 / SU B: 4.831 / SU ML: 0.121 / Cross valid method: THROUGHOUT / ESU R: 0.202 / ESU R Free: 0.17 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.20585 1551 5.1 %RANDOM
Rwork0.15623 ---
obs0.15874 29010 94.04 %-
all-30610 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso mean: 24.753 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refine analyzeLuzzati coordinate error obs: 0.216 Å
Refinement stepCycle: LAST / Resolution: 2.07→41.17 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3949 0 13 324 4286
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0214080
X-RAY DIFFRACTIONr_bond_other_d0.0020.023457
X-RAY DIFFRACTIONr_angle_refined_deg1.5471.9135542
X-RAY DIFFRACTIONr_angle_other_deg0.9238038
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9495495
X-RAY DIFFRACTIONr_dihedral_angle_2_deg
X-RAY DIFFRACTIONr_chiral_restr0.0980.2554
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.024658
X-RAY DIFFRACTIONr_gen_planes_other0.0060.02908
X-RAY DIFFRACTIONr_nbd_refined0.1990.2861
X-RAY DIFFRACTIONr_nbd_other0.2460.24394
X-RAY DIFFRACTIONr_nbtor_other0.0870.22285
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1650.2266
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.0740.26
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2990.252
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1640.212
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_mcbond_it0.7911.52456
X-RAY DIFFRACTIONr_mcangle_it1.43223946
X-RAY DIFFRACTIONr_scbond_it2.22531624
X-RAY DIFFRACTIONr_scangle_it3.4774.51596
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.07→2.124 Å / Total num. of bins used: 20 /
RfactorNum. reflection
Rfree0.421 73
Rwork0.371 1221
Refinement
*PLUS
Highest resolution: 2.1 Å / Lowest resolution: 20 Å / Rfactor Rfree: 0.204 / Rfactor Rwork: 0.158
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONr_bond_d0.017
X-RAY DIFFRACTIONr_angle_d
X-RAY DIFFRACTIONr_angle_deg1.5

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