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- PDB-6m4m: X-ray crystal structure of the E249Q mutan of alpha-amylase I and... -

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Basic information

Entry
Database: PDB / ID: 6m4m
TitleX-ray crystal structure of the E249Q mutan of alpha-amylase I and maltohexaose complex 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 / Glycoside hydrolase superfamily
Similarity search - Domain/homology
alpha-maltotetraose / alpha-maltohexaose / TRIETHYLENE GLYCOL / alpha-amylase
Similarity search - Component
Biological speciesEisenia fetida (common brandling worm)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 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 23, 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)61,00313
Polymers58,3491
Non-polymers2,65412
Water9,206511
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 area17390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)96.809, 96.809, 121.233
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-1140-

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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Sugars , 2 types, 2 molecules

#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 / alpha-maltohexaose


Type: oligosaccharide, Oligosaccharide / Class: Substrate analog / Mass: 990.860 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltohexaose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,6,5/[a2122h-1a_1-5]/1-1-1-1-1-1/a4-b1_b4-c1_c4-d1_d4-e1_e4-f1WURCSPDB2Glycan 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]{}}}}}}LINUCSPDB-CARE
#3: Polysaccharide alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose / alpha-maltotetraose


Type: oligosaccharide, Oligosaccharide / Class: Substrate analog / Mass: 666.578 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: alpha-maltotetraose
DescriptorTypeProgram
DGlcpa1-4DGlcpa1-4DGlcpa1-4DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,4,3/[a2122h-1a_1-5]/1-1-1-1/a4-b1_b4-c1_c4-d1WURCSPDB2Glycan 1.1.0
[][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{[(4+1)][a-D-Glcp]{}}}}LINUCSPDB-CARE

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Non-polymers , 6 types, 521 molecules

#4: Chemical ChemComp-CA / CALCIUM ION


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


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


Mass: 96.063 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: SO4
#7: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#8: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


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


Mass: 18.015 Da / Num. of mol.: 511 / 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: 2.8 Å3/Da / Density % sol: 56.13 %
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 acetat, 10 mM maltohexaose

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: AR-NE3A / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Dec 16, 2018
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.7→48.4 Å / Num. obs: 72722 / % possible obs: 100 % / Redundancy: 10 % / Biso Wilson estimate: 18.9779772494 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.081 / Rrim(I) all: 0.09 / Net I/σ(I): 18.2
Reflection shellResolution: 1.7→1.73 Å / Redundancy: 9.9 % / Rmerge(I) obs: 0.952 / Mean I/σ(I) obs: 2.3 / Num. unique obs: 3840 / CC1/2: 0.822 / Rrim(I) all: 1.064 / % possible all: 100

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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: 1.7→41.92 Å / SU ML: 0.183574193813 / Cross valid method: FREE R-VALUE / σ(F): 1.33660349133 / Phase error: 17.4980157405
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.180042596777 3586 4.93524724405 %
Rwork0.157639917028 69075 -
obs0.158744131505 72661 99.9766091527 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 20.7025488004 Å2
Refinement stepCycle: LAST / Resolution: 1.7→41.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3906 0 167 511 4584
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.006134424172944628
X-RAY DIFFRACTIONf_angle_d0.8970914612756364
X-RAY DIFFRACTIONf_chiral_restr0.0556372218182643
X-RAY DIFFRACTIONf_plane_restr0.00570268566783856
X-RAY DIFFRACTIONf_dihedral_angle_d10.87443011682595
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.7-1.72240.2972076052281330.2714208643782646X-RAY DIFFRACTION99.964028777
1.7224-1.7460.2719788447231330.2532700329032625X-RAY DIFFRACTION99.9637549837
1.746-1.7710.2742020836631300.2384005807542637X-RAY DIFFRACTION99.9638728324
1.771-1.79740.2666459395321490.2238380765532621X-RAY DIFFRACTION99.9639119451
1.7974-1.82550.2197296796321630.2151375008142625X-RAY DIFFRACTION100
1.8255-1.85540.2337014167461520.1960252977022580X-RAY DIFFRACTION100
1.8554-1.88740.238383138071250.1807622381972638X-RAY DIFFRACTION100
1.8874-1.92170.20864980011460.1737055373392602X-RAY DIFFRACTION100
1.9217-1.95870.2170438854921410.1705126476142642X-RAY DIFFRACTION100
1.9587-1.99870.2075576478061350.1679586552872636X-RAY DIFFRACTION100
1.9987-2.04210.20891510411490.1739798381992640X-RAY DIFFRACTION100
2.0421-2.08960.2129600842561270.164989805552648X-RAY DIFFRACTION100
2.0896-2.14190.1930301889391180.1633710487212636X-RAY DIFFRACTION100
2.1419-2.19980.1746469443711280.1572096585532684X-RAY DIFFRACTION100
2.1998-2.26450.1780288861241430.1488215658622630X-RAY DIFFRACTION100
2.2645-2.33760.1545887855271340.1531927334732627X-RAY DIFFRACTION100
2.3376-2.42120.187256243881040.1525640226552706X-RAY DIFFRACTION100
2.4212-2.51810.1847965860991300.155257590192641X-RAY DIFFRACTION100
2.5181-2.63270.195273937441380.1584017532122684X-RAY DIFFRACTION100
2.6327-2.77140.2000984832381460.159084212462646X-RAY DIFFRACTION100
2.7714-2.9450.1763447786831440.1618621822662666X-RAY DIFFRACTION100
2.945-3.17240.1817763827971490.1587010417012657X-RAY DIFFRACTION100
3.1724-3.49150.1562242645211590.1509442732552666X-RAY DIFFRACTION100
3.4915-3.99640.1250819722781320.1224847872722714X-RAY DIFFRACTION100
3.9964-5.03360.1304244640111410.1148782948732724X-RAY DIFFRACTION99.9651081647
5.0336-41.920.1813734515831370.1661566249782854X-RAY DIFFRACTION99.6335776149

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