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- PDB-6m4l: X-ray crystal structure of the E249Q mutant of alpha-amylase I fr... -

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Basic information

Entry
Database: PDB / ID: 6m4l
TitleX-ray crystal structure of the E249Q mutant of alpha-amylase I from Eisenia fetida
ComponentsAlpha-amylase
KeywordsHYDROLASE / TIM barrel / GH family 13 / carbohydrase / cold-active
Function / homology
Function and homology information


alpha-amylase / alpha-amylase activity / carbohydrate metabolic process / metal ion binding
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 / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycosyl hydrolase, all-beta / Glycosidases / Glycoside hydrolase superfamily ...Alpha-amylase, C-terminal domain / Aamy_C / Alpha-amylase/branching enzyme, C-terminal all beta / Alpha amylase, C-terminal all-beta domain / Alpha amylase / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycosyl hydrolase, all-beta / Glycosidases / Glycoside hydrolase superfamily / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
ACETATE ION / TRIETHYLENE GLYCOL / alpha-amylase
Similarity search - Component
Biological speciesEisenia fetida (common brandling worm)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsHirano, Y. / Tsukamoto, K. / Ariki, S. / Naka, Y. / Ueda, M. / Tamada, T.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2020
Title: X-ray crystallographic structural studies of alpha-amylase I from Eisenia fetida.
Authors: Hirano, Y. / Tsukamoto, K. / Ariki, S. / Naka, Y. / Ueda, M. / Tamada, T.
History
DepositionMar 7, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 16, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha-amylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,67815
Polymers58,3491
Non-polymers1,32914
Water9,278515
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area170 Å2
ΔGint-13 kcal/mol
Surface area17570 Å2
MethodPISA
Unit cell
Length a, b, c (Å)96.897, 96.897, 121.957
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z
Components on special symmetry positions
IDModelComponents
11A-833-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Alpha-amylase


Mass: 58348.934 Da / Num. of mol.: 1 / Mutation: E249Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Eisenia fetida (common brandling worm) / Gene: alpha-amylase / Production host: Komagataella pastoris (fungus) / References: UniProt: A0A173N065, alpha-amylase

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Non-polymers , 9 types, 529 molecules

#2: Chemical ChemComp-CA / CALCIUM ION


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


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#5: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#6: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#7: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#8: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#9: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 515 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
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: 2.83 Å3/Da / Density % sol: 56.47 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 1.3 M lithium sulfate, 3% (v/v) PEG 400, 0.1 M magnesium sulfate, 0.1 M sodium acetate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-17A / Wavelength: 0.98 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 23, 2018
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 1.6→49.33 Å / Num. obs: 87577 / % possible obs: 100 % / Redundancy: 7.9 % / Biso Wilson estimate: 19.8550860307 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.05 / Rrim(I) all: 0.058 / Net I/σ(I): 20.1
Reflection shellResolution: 1.6→1.63 Å / Redundancy: 7.8 % / Rmerge(I) obs: 0.528 / Mean I/σ(I) obs: 3.5 / Num. unique obs: 4250 / CC1/2: 0.897 / Rrim(I) all: 0.609 / % possible all: 99.6

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Processing

Software
NameVersionClassification
PHENIX1.10.1_2155refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6M4K

6m4k


Resolution: 1.6→41.958 Å / SU ML: 0.135450576335 / Cross valid method: FREE R-VALUE / σ(F): 1.33473009263 / Phase error: 19.1629862456
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.190331477853 4353 4.97354980976 %
Rwork0.167694673064 83170 -
obs0.168807930266 87523 99.9474699951 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 23.2213397278 Å2
Refinement stepCycle: LAST / Resolution: 1.6→41.958 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3906 0 81 515 4502
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.006036301035674617
X-RAY DIFFRACTIONf_angle_d0.8370402915686321
X-RAY DIFFRACTIONf_chiral_restr0.053588863238611
X-RAY DIFFRACTIONf_plane_restr0.0061971743403869
X-RAY DIFFRACTIONf_dihedral_angle_d11.07175797772645
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.61860.2435255303371250.2348781325332737X-RAY DIFFRACTION99.3405067685
1.6186-1.63770.2707607900281580.2205253888132737X-RAY DIFFRACTION100
1.6377-1.65760.2543231694821650.2286196592382718X-RAY DIFFRACTION100
1.6576-1.67860.2343830035811500.2114111996522715X-RAY DIFFRACTION100
1.6786-1.70070.2191673119531450.2049562681242795X-RAY DIFFRACTION99.9659979599
1.7007-1.7240.2212357942731500.2147616611672704X-RAY DIFFRACTION100
1.724-1.74860.2147012037221330.2151866750042770X-RAY DIFFRACTION100
1.7486-1.77470.2605703143711310.2129208294862771X-RAY DIFFRACTION100
1.7747-1.80250.254635632321600.2088718781352704X-RAY DIFFRACTION100
1.8025-1.8320.2406238546851670.2050453839662787X-RAY DIFFRACTION100
1.832-1.86360.2350756130641590.2052885919352716X-RAY DIFFRACTION99.9652294854
1.8636-1.89750.247874520351240.1928134947422760X-RAY DIFFRACTION100
1.8975-1.9340.2167464220541560.1894790366492734X-RAY DIFFRACTION100
1.934-1.97350.2085005547351650.185989708382762X-RAY DIFFRACTION99.9658469945
1.9735-2.01640.2017991390741290.1850900775912736X-RAY DIFFRACTION100
2.0164-2.06330.2126206545761460.1801806845342754X-RAY DIFFRACTION100
2.0633-2.11490.1986515663121390.1799332421272779X-RAY DIFFRACTION99.9657416924
2.1149-2.17210.2027419597931130.1748251088852764X-RAY DIFFRACTION100
2.1721-2.2360.1920068316021520.1723087844752767X-RAY DIFFRACTION100
2.236-2.30820.2062623481451400.1643071119282784X-RAY DIFFRACTION100
2.3082-2.39060.2058951902331310.1742447111482794X-RAY DIFFRACTION100
2.3906-2.48630.1605340557251260.1642757544132800X-RAY DIFFRACTION100
2.4863-2.59950.2048781934161280.1692096089982793X-RAY DIFFRACTION100
2.5995-2.73650.2028154300081590.1720795837572753X-RAY DIFFRACTION100
2.7365-2.90790.2021336112091490.1766179674282783X-RAY DIFFRACTION99.9659052165
2.9079-3.13240.203246176671610.1685267609232803X-RAY DIFFRACTION99.9662731872
3.1324-3.44750.1779049728121670.1646710748222776X-RAY DIFFRACTION99.8642687479
3.4475-3.9460.1358607483361360.1296546300612833X-RAY DIFFRACTION99.7983193277
3.946-4.97030.13279824781430.1223596677162861X-RAY DIFFRACTION100
4.9703-41.950.1849132836511460.1631454625482980X-RAY DIFFRACTION99.6493465094

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