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- PDB-3wfz: Crystal structure of Galacto-N-Biose/Lacto-N-Biose I Phosphorylas... -

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Basic information

Entry
Database: PDB / ID: 3wfz
TitleCrystal structure of Galacto-N-Biose/Lacto-N-Biose I Phosphorylase C236Y Mutant
ComponentsLacto-N-biose phosphorylase
KeywordsTRANSFERASE / beta-alpha-barrel / TIM barrel / phosphorylase
Function / homology
Function and homology information


1,3-beta-galactosyl-N-acetylhexosamine phosphorylase / 1,3-beta-galactosyl-N-acetylhexosamine phosphorylase activity / 1,4-alpha-oligoglucan phosphorylase activity / carbohydrate metabolic process
Similarity search - Function
Lacto-N-biose phosphorylase/D-galactosyl-beta-1->4-L-rhamnose phosphorylase / Lacto-N-biose phosphorylase-like, N-terminal TIM barrel domain / Lacto-N-biose phosphorylase, C-terminal domain / Lacto-N-biose phosphorylase, central domain / Lacto-N-biose phosphorylase N-terminal TIM barrel domain / Lacto-N-biose phosphorylase central domain / Lacto-N-biose phosphorylase C-terminal domain / Class I glutamine amidotransferase (GATase) domain / Class I glutamine amidotransferase-like / Golgi alpha-mannosidase II ...Lacto-N-biose phosphorylase/D-galactosyl-beta-1->4-L-rhamnose phosphorylase / Lacto-N-biose phosphorylase-like, N-terminal TIM barrel domain / Lacto-N-biose phosphorylase, C-terminal domain / Lacto-N-biose phosphorylase, central domain / Lacto-N-biose phosphorylase N-terminal TIM barrel domain / Lacto-N-biose phosphorylase central domain / Lacto-N-biose phosphorylase C-terminal domain / Class I glutamine amidotransferase (GATase) domain / Class I glutamine amidotransferase-like / Golgi alpha-mannosidase II / Glycosyl hydrolase, all-beta / Glycosidases / Immunoglobulins / TIM Barrel / Alpha-Beta Barrel / Immunoglobulin-like fold / Immunoglobulin-like / Sandwich / Rossmann fold / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
2-acetamido-2-deoxy-alpha-D-glucopyranose / 1,3-beta-galactosyl-N-acetylhexosamine phosphorylase
Similarity search - Component
Biological speciesBifidobacterium longum (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsKoyama, Y. / Hidaka, M. / Kawakami, M. / Nishimoto, M. / Kitaoka, M.
CitationJournal: Protein Eng.Des.Sel. / Year: 2013
Title: Directed evolution to enhance thermostability of galacto-N-biose/lacto-N-biose I phosphorylase.
Authors: Koyama, Y. / Hidaka, M. / Nishimoto, M. / Kitaoka, M.
History
DepositionJul 25, 2013Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 9, 2013Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_ref_seq_dif / struct_site / struct_site_gen
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Aug 24, 2022Group: Database references / Structure summary / Category: chem_comp / citation / database_2
Item: _chem_comp.pdbx_synonyms / _citation.journal_volume ..._chem_comp.pdbx_synonyms / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 2.2Nov 8, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lacto-N-biose phosphorylase
B: Lacto-N-biose phosphorylase
C: Lacto-N-biose phosphorylase
D: Lacto-N-biose phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)343,0618
Polymers342,1764
Non-polymers8854
Water16,214900
1
A: Lacto-N-biose phosphorylase
B: Lacto-N-biose phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,5314
Polymers171,0882
Non-polymers4422
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7540 Å2
ΔGint-6 kcal/mol
Surface area51710 Å2
MethodPISA
2
C: Lacto-N-biose phosphorylase
D: Lacto-N-biose phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,5314
Polymers171,0882
Non-polymers4422
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7530 Å2
ΔGint-5 kcal/mol
Surface area51880 Å2
MethodPISA
Unit cell
Length a, b, c (Å)68.617, 112.996, 118.934
Angle α, β, γ (deg.)105.48, 90.88, 106.73
Int Tables number1
Space group name H-MP1

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Components

#1: Protein
Lacto-N-biose phosphorylase / Galacto-N-biose/lacto-N-biose I phosphorylase


Mass: 85544.094 Da / Num. of mol.: 4 / Mutation: C236Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bifidobacterium longum (bacteria) / Strain: JCM1217 / Gene: BLLJ_1623, lnpA / Plasmid: pET28 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: E8MF13, 1,3-beta-galactosyl-N-acetylhexosamine phosphorylase
#2: Sugar
ChemComp-NDG / 2-acetamido-2-deoxy-alpha-D-glucopyranose / N-acetyl-alpha-D-glucosamine / 2-acetamido-2-deoxy-alpha-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / 2-(ACETYLAMINO)-2-DEOXY-A-D-GLUCOPYRANOSE


Type: D-saccharide, alpha linking / Mass: 221.208 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-a-D-glucopyranosamineCOMMON NAMEGMML 1.0
a-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 900 / Source method: isolated from a natural source / Formula: H2O

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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: 2.47 Å3/Da / Density % sol: 50.3 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Na cacodyrate, Mg(NO3)2, PEG 4000, GlcNAc, pH 6.5, VAPOR DIFFUSION, SITTING DROP, temperature 277K

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Data collection

DiffractionMean temperature: 95 K
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: AR-NW12A / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Jun 10, 2013
RadiationMonochromator: Si 111 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.6→50 Å / Num. all: 100835 / Num. obs: 97558 / % possible obs: 96.8 % / Redundancy: 2 % / Biso Wilson estimate: 28.4 Å2 / Rmerge(I) obs: 0.083 / Net I/σ(I): 10.9
Reflection shellResolution: 2.6→2.64 Å / Redundancy: 2 % / Rmerge(I) obs: 0.213 / Mean I/σ(I) obs: 2.5 / Num. unique all: 4960 / % possible all: 97.4

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Processing

Software
NameVersionClassification
HKL-2000data collection
MOLREPphasing
REFMAC5.7.0032refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2zuu

2zuu


Resolution: 2.6→36.09 Å / Cor.coef. Fo:Fc: 0.943 / Cor.coef. Fo:Fc free: 0.887 / SU B: 10.927 / SU ML: 0.232 / Cross valid method: THROUGHOUT / ESU R Free: 0.342 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2486 4869 5 %RANDOM
Rwork0.17931 ---
obs0.18279 92682 96.58 %-
all-100835 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 30.014 Å2
Baniso -1Baniso -2Baniso -3
1-2.38 Å20.11 Å2-2.15 Å2
2---0.39 Å2-1.83 Å2
3----0.91 Å2
Refinement stepCycle: LAST / Resolution: 2.6→36.09 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms23590 0 60 900 24550
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.01924324
X-RAY DIFFRACTIONr_bond_other_d0.0010.0221989
X-RAY DIFFRACTIONr_angle_refined_deg1.4681.93633133
X-RAY DIFFRACTIONr_angle_other_deg0.824350543
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.27552953
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.48823.7581240
X-RAY DIFFRACTIONr_dihedral_angle_3_deg14.989153676
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.8615164
X-RAY DIFFRACTIONr_chiral_restr0.0820.23494
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.02127995
X-RAY DIFFRACTIONr_gen_planes_other0.0010.025953
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_it1.7732.96511836
X-RAY DIFFRACTIONr_mcbond_other1.7712.96511835
X-RAY DIFFRACTIONr_mcangle_it2.8374.4414781
X-RAY DIFFRACTIONr_mcangle_other2.8374.4414782
X-RAY DIFFRACTIONr_scbond_it1.6783.09612488
X-RAY DIFFRACTIONr_scbond_other1.6783.09612488
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other2.7464.58618353
X-RAY DIFFRACTIONr_long_range_B_refined4.43623.74829202
X-RAY DIFFRACTIONr_long_range_B_other4.40923.77229005
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.6→2.668 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.349 350 -
Rwork0.255 6824 -
obs--96.46 %

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