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- PDB-9fz2: Crystal structure of Amylase 5 (Amy5) from Ruminococcus bromii co... -

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Basic information

Entry
Database: PDB / ID: 9fz2
TitleCrystal structure of Amylase 5 (Amy5) from Ruminococcus bromii covalently bound to alpha-1,6 branched pseudo-trisaccharide activity-based probe
ComponentsAlpha-amylase
KeywordsHYDROLASE / GLYCOSIDE HYDROLASE / AMYLASE
Function / homology
Function and homology information


alpha-amylase / alpha-amylase activity / carbohydrate metabolic process / metal ion binding
Similarity search - Function
Oligo-1,6-glucosidase, domain 2 / Alpha amylase / Alpha amylase, catalytic domain / Glycosyl hydrolase, family 13, catalytic domain / Alpha-amylase domain / Glycosyl hydrolase, all-beta / Prokaryotic membrane lipoprotein lipid attachment site profile. / Glycoside hydrolase superfamily
Similarity search - Domain/homology
: / Chem-PBW / Alpha-amylase
Similarity search - Component
Biological speciesRuminococcus bromii (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.4 Å
AuthorsPickles, I.B. / Moroz, O. / Davies, G.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)951231European Union
CitationJournal: Angew.Chem.Int.Ed.Engl. / Year: 2025
Title: Precision Activity-Based alpha-Amylase Probes for Dissection and Annotation of Linear and Branched-Chain Starch-Degrading Enzymes.
Authors: Pickles, I.B. / Chen, Y. / Moroz, O. / Brown, H.A. / de Boer, C. / Armstrong, Z. / McGregor, N.G.S. / Artola, M. / Codee, J.D.C. / Koropatkin, N.M. / Overkleeft, H.S. / Davies, G.J.
History
DepositionJul 4, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.year / _citation_author.identifier_ORCID

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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)61,4337
Polymers60,6311
Non-polymers8026
Water10,647591
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1090 Å2
ΔGint-26 kcal/mol
Surface area19460 Å2
Unit cell
Length a, b, c (Å)49.869, 94.873, 49.904
Angle α, β, γ (deg.)90.00, 94.64, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Alpha-amylase


Mass: 60630.668 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ruminococcus bromii (bacteria) / Gene: RBL236_00453 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2N0UXJ4, alpha-amylase
#2: Polysaccharide alpha-D-glucopyranose-(1-6)-alpha-D-glucopyranose


Type: oligosaccharide / Mass: 342.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpa1-6DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1a_1-5]/1-1/a6-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-Glcp]{[(6+1)][a-D-Glcp]{}}LINUCSPDB-CARE

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Non-polymers , 4 types, 596 molecules

#3: Chemical ChemComp-PBW / (1~{S},4~{S},5~{R})-6-(hydroxymethyl)cyclohexane-1,2,3,4,5-pentol


Mass: 194.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H14O6 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-A1IHI / 8-azanyloctan-1-ol


Mass: 145.243 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H19NO / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 591 / 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: 2.05 Å3/Da / Density % sol: 40.1 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 9
Details: 0.1 M SPG pH 9.0, 25% w/v PEG 1500, 1 mM 4'-octylamine-a-D-maltotriose epicyclophellitol (compound 2a)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.7446 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Jul 1, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.7446 Å / Relative weight: 1
ReflectionResolution: 1.4→44.05 Å / Num. obs: 90248 / % possible obs: 99.4 % / Redundancy: 7 % / CC1/2: 1 / Rmerge(I) obs: 0.16 / Net I/σ(I): 6.9
Reflection shellResolution: 1.4→1.42 Å / Num. unique obs: 4525 / CC1/2: 0.41

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Processing

Software
NameVersionClassification
REFMAC5.8.0425refinement
DIALSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.4→44.05 Å / Cor.coef. Fo:Fc: 0.954 / Cor.coef. Fo:Fc free: 0.937 / SU B: 1.631 / SU ML: 0.062 / Cross valid method: THROUGHOUT / ESU R: 0.071 / ESU R Free: 0.074 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.22234 4612 5.1 %RANDOM
Rwork0.18739 ---
obs0.18915 85535 99.25 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 14.298 Å2
Baniso -1Baniso -2Baniso -3
1--0.22 Å20 Å2-0.46 Å2
2--0.39 Å2-0 Å2
3----0.09 Å2
Refinement stepCycle: 1 / Resolution: 1.4→44.05 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4027 0 46 591 4664
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0124211
X-RAY DIFFRACTIONr_bond_other_d0.0010.0163830
X-RAY DIFFRACTIONr_angle_refined_deg1.691.8145688
X-RAY DIFFRACTIONr_angle_other_deg0.6321.7798887
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.6615523
X-RAY DIFFRACTIONr_dihedral_angle_2_deg7.437512
X-RAY DIFFRACTIONr_dihedral_angle_3_deg12.25510715
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.2690.2625
X-RAY DIFFRACTIONr_gen_planes_refined0.010.024907
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02923
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.0741.2762069
X-RAY DIFFRACTIONr_mcbond_other1.0651.2752063
X-RAY DIFFRACTIONr_mcangle_it1.5332.2932575
X-RAY DIFFRACTIONr_mcangle_other1.5342.2942576
X-RAY DIFFRACTIONr_scbond_it1.7471.4162142
X-RAY DIFFRACTIONr_scbond_other1.7471.4172143
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.562.5273108
X-RAY DIFFRACTIONr_long_range_B_refined4.00317.295054
X-RAY DIFFRACTIONr_long_range_B_other3.81815.684883
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.4→1.436 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.301 316 -
Rwork0.295 6378 -
obs--99.41 %

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