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- PDB-2wpb: Crystal structure of the E192N mutant of E. Coli N-acetylneuramin... -

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Basic information

Entry
Database: PDB / ID: 2wpb
TitleCrystal structure of the E192N mutant of E. Coli N-acetylneuraminic acid lyase in complex with pyruvate and the inhibitor (2R,3R)-2,3,4- trihydroxy-N,N-dipropylbutanamide in space group P21 crystal form I
ComponentsN-ACETYLNEURAMINATE LYASE
KeywordsLYASE / SUBSTRATE SPECIFICITY / CARBOHYDRATE METABOLISM / PROTEIN ENGINEERING / ALDOLASE / SCHIFF BASE
Function / homology
Function and homology information


N-acetylneuraminate lyase / N-acetylneuraminate lyase activity / N-acetylneuraminate catabolic process / single-species biofilm formation / carbohydrate metabolic process / identical protein binding / cytosol
Similarity search - Function
N-acetylneuraminate lyase / Schiff base-forming aldolase, conserved site / Dihydrodipicolinate synthase signature 1. / Schiff base-forming aldolase, active site / Dihydrodipicolinate synthase signature 2. / DapA-like / Dihydrodipicolinate synthetase family / Dihydrodipicolinate synthetase family / Aldolase class I / Aldolase-type TIM barrel ...N-acetylneuraminate lyase / Schiff base-forming aldolase, conserved site / Dihydrodipicolinate synthase signature 1. / Schiff base-forming aldolase, active site / Dihydrodipicolinate synthase signature 2. / DapA-like / Dihydrodipicolinate synthetase family / Dihydrodipicolinate synthetase family / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
(2R,3R)-2,3,4-TRIHYDROXY-N,N-DIPROPYLBUTANAMIDE / N-acetylneuraminate lyase
Similarity search - Component
Biological speciesESCHERICHIA COLI (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.05 Å
AuthorsCampeotto, I. / Bolt, A.H. / Harman, T.A. / Trinh, C.H. / Dennis, C.A. / Phillips, S.E.V. / Pearson, A.R. / Nelson, A. / Berry, A.
CitationJournal: J.Mol.Biol. / Year: 2010
Title: Structural Insights Into Substrate Specificity in Variants of N-Acetylneuraminic Acid Lyase Produced by Directed Evolution.
Authors: Campeotto, I. / Bolt, A.H. / Harman, T.A. / Dennis, C.A. / Trinh, C.H. / Phillips, S.E.V. / Nelson, A. / Pearson, A.R. / Berry, A.
History
DepositionAug 3, 2009Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 25, 2010Provider: repository / Type: Initial release
Revision 1.1May 8, 2011Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Dec 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_sf / _struct_conn.pdbx_leaving_atom_flag / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: N-ACETYLNEURAMINATE LYASE
B: N-ACETYLNEURAMINATE LYASE
C: N-ACETYLNEURAMINATE LYASE
D: N-ACETYLNEURAMINATE LYASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,4318
Polymers134,5544
Non-polymers8774
Water9,728540
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8770 Å2
ΔGint-56.3 kcal/mol
Surface area40670 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.020, 143.741, 84.304
Angle α, β, γ (deg.)90.00, 109.90, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
N-ACETYLNEURAMINATE LYASE / N-ACETYL-NEURAMINIC ACID LYASE / N-ACETYLNEURAMINIC ACID ALDOLASE / N-ACETYLNEURAMINATE PYRUVATE- ...N-ACETYL-NEURAMINIC ACID LYASE / N-ACETYLNEURAMINIC ACID ALDOLASE / N-ACETYLNEURAMINATE PYRUVATE-LYASE / SIALIC ACID LYASE / SIALATE LYASE / SIALIC ACID ALDOLASE / NALASE


Mass: 33638.395 Da / Num. of mol.: 4 / Fragment: RESIDUES 2-297 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: SCHIFF BASE BETWEEN LYS165 AND PYRUVATE IN ALL CHAINS
Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Plasmid: PKNANA / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P0A6L4, N-acetylneuraminate lyase
#2: Chemical
ChemComp-ZZI / (2R,3R)-2,3,4-TRIHYDROXY-N,N-DIPROPYLBUTANAMIDE


Mass: 219.278 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H21NO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 540 / Source method: isolated from a natural source / Formula: H2O
Compound detailsENGINEERED RESIDUE IN CHAIN A, GLU 192 TO ASN ENGINEERED RESIDUE IN CHAIN B, GLU 192 TO ASN ...ENGINEERED RESIDUE IN CHAIN A, GLU 192 TO ASN ENGINEERED RESIDUE IN CHAIN B, GLU 192 TO ASN ENGINEERED RESIDUE IN CHAIN C, GLU 192 TO ASN ENGINEERED RESIDUE IN CHAIN D, GLU 192 TO ASN

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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: 2.5 Å3/Da / Density % sol: 50.81 % / Description: NONE
Crystal growpH: 8.2 / Details: 100MM TRIS-HCL PH 8.2, 200 MM NACL, 18% PEG 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.92
DetectorType: ADSC CCD / Detector: CCD / Date: May 13, 2008
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.92 Å / Relative weight: 1
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H,K,L10.938
11-H,-K,H+L20.062
ReflectionResolution: 2.05→79.31 Å / Num. obs: 78597 / % possible obs: 98.6 % / Observed criterion σ(I): 2 / Redundancy: 3.5 % / Biso Wilson estimate: 24.528 Å2 / Rmerge(I) obs: 0.09 / Net I/σ(I): 8.6
Reflection shellResolution: 2.05→2.16 Å / Redundancy: 3.4 % / Rmerge(I) obs: 0.44 / Mean I/σ(I) obs: 2.6 / % possible all: 96.3

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Processing

Software
NameVersionClassification
REFMAC5.5.0097refinement
MOSFLMdata reduction
SCALAdata scaling
REFMACphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2WNN

2wnn


Resolution: 2.05→71.88 Å / Cor.coef. Fo:Fc: 0.952 / Cor.coef. Fo:Fc free: 0.931 / SU B: 4.827 / SU ML: 0.122 / Cross valid method: THROUGHOUT / ESU R: 0.045 / ESU R Free: 0.038 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.23394 3966 5 %RANDOM
Rwork0.18951 ---
obs0.19176 74586 97.68 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 22.926 Å2
Baniso -1Baniso -2Baniso -3
1-36.75 Å20 Å25.88 Å2
2---8.33 Å20 Å2
3----28.42 Å2
Refinement stepCycle: LAST / Resolution: 2.05→71.88 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9192 0 60 540 9792
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0229495
X-RAY DIFFRACTIONr_bond_other_d0.0010.026364
X-RAY DIFFRACTIONr_angle_refined_deg1.2831.97712865
X-RAY DIFFRACTIONr_angle_other_deg0.9043.00115546
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.60951207
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.26124.81420
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.218151645
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.8271548
X-RAY DIFFRACTIONr_chiral_restr0.0730.21458
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.02110597
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021823
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_it0.5831.55910
X-RAY DIFFRACTIONr_mcbond_other0.1221.52444
X-RAY DIFFRACTIONr_mcangle_it1.04829497
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.60233585
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it2.5714.53357
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.045→2.098 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.337 271 -
Rwork0.281 4787 -
obs--85.34 %

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