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- PDB-4bwl: Structure of the Y137A mutant of E. coli N-acetylneuraminic acid ... -

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Basic information

Entry
Database: PDB / ID: 4bwl
TitleStructure of the Y137A mutant of E. coli N-acetylneuraminic acid lyase in complex with pyruvate, N-acetyl-D-mannosamine and N- acetylneuraminic acid
Components(N-ACETYLNEURAMINATE ...) x 2
KeywordsLYASE / DIRECTED EVOLUTION / SUBSTRATE SPECIFICITY / PROTEIN ENGINEERING
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
2-(ACETYLAMINO)-2-DEOXY-D-MANNOSE / Chem-SI3 / N-acetylneuraminate lyase
Similarity search - Component
Biological speciesESCHERICHIA COLI (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsCampeotto, I. / Phillips, S.E.V. / Pearson, A.R. / Nelson, A. / Berry, A.
CitationJournal: Acs Chem.Biol. / Year: 2014
Title: The Reaction Mechanism of N-Acetylneuraminic Acid Lyase Revealed by a Combination of Crystallography, Qm/Mm Simulation and Mutagenesis.
Authors: Daniels, A.D. / Campeotto, I. / Van Der Kamp, M.W. / Bolt, A.H. / Trinh, C.H. / Phillips, S.E.V. / Pearson, A.R. / Nelson, A. / Mulholland, A.J. / Berry, A.
History
DepositionJul 3, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 19, 2014Provider: repository / Type: Initial release
Revision 1.1Feb 26, 2014Group: Database references
Revision 1.2Apr 30, 2014Group: Database references
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_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "BA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "CA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "DA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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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)135,66410
Polymers134,0384
Non-polymers1,6266
Water1,838102
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11270 Å2
ΔGint-63.1 kcal/mol
Surface area40390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)55.970, 143.220, 83.410
Angle α, β, γ (deg.)90.00, 109.51, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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N-ACETYLNEURAMINATE ... , 2 types, 4 molecules ABDC

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


Mass: 33492.273 Da / Num. of mol.: 3 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: SCHIFF BASE BETWEEN LYS165 AND N-ACETYLNEURAMINIC ACID
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: Protein N-ACETYLNEURAMINATE LYASE / N-ACETYLNEURAMINATE PYRUVATE-LYASE / N-ACETYLNEURAMINIC ACID ALDOLASE / NALASE / SIALATE LYASE / ...N-ACETYLNEURAMINATE PYRUVATE-LYASE / N-ACETYLNEURAMINIC ACID ALDOLASE / NALASE / SIALATE LYASE / SIALIC ACID ALDOLASE / SIALIC ACID LYASE / N-ACETYLNEURAMINIC ACID LYASE


Mass: 33561.312 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Details: SCHIFF BASE BETWEEN LYS165 AND PYRUVATE / 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

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Sugars , 1 types, 3 molecules

#3: Sugar ChemComp-SI3 / 5-(acetylamino)-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid / N-acetylneuraminic acid, ketone form


Type: D-saccharide / Mass: 309.270 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C11H19NO9

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Non-polymers , 3 types, 105 molecules

#4: Chemical ChemComp-1PE / PENTAETHYLENE GLYCOL / PEG400


Mass: 238.278 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H22O6 / Comment: precipitant*YM
#5: Chemical ChemComp-MN9 / 2-(ACETYLAMINO)-2-DEOXY-D-MANNOSE


Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 102 / 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: 2.42 Å3/Da / Density % sol: 49.29 % / Description: NONE
Crystal growpH: 8.2 / Details: 100MM TRIS-HCL PH 8.2, 200MM NACL, 18% PEG3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I02 / Wavelength: 0.97
DetectorType: ADSC CCD / Detector: CCD / Date: Jul 8, 2009
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97 Å / Relative weight: 1
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H, K, L10.512
11-H, -K, H+L20.488
ReflectionResolution: 2→49.5 Å / Num. obs: 82202 / % possible obs: 98.7 % / Observed criterion σ(I): 2 / Redundancy: 3.6 % / Biso Wilson estimate: 21.4 Å2 / Rmerge(I) obs: 0.09 / Net I/σ(I): 9.6
Reflection shellResolution: 2→2.11 Å / Redundancy: 3.5 % / Rmerge(I) obs: 0.41 / Mean I/σ(I) obs: 2.8 / % possible all: 95.7

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Processing

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

2wnn


Resolution: 2→49.46 Å / Cor.coef. Fo:Fc: 0.952 / Cor.coef. Fo:Fc free: 0.92 / SU B: 4.557 / SU ML: 0.117 / Cross valid method: THROUGHOUT / ESU R: 0.037 / ESU R Free: 0.035 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2349 4084 5 %RANDOM
Rwork0.17964 ---
obs0.18247 78080 98.07 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 27.7 Å2
Baniso -1Baniso -2Baniso -3
1-50.39 Å20 Å2-7.26 Å2
2---24.13 Å20 Å2
3----26.26 Å2
Refinement stepCycle: LAST / Resolution: 2→49.46 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9166 0 92 102 9360
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0229419
X-RAY DIFFRACTIONr_bond_other_d0.0010.026299
X-RAY DIFFRACTIONr_angle_refined_deg1.3421.9812745
X-RAY DIFFRACTIONr_angle_other_deg0.9013.00115368
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.43951184
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.23824.793411
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.342151621
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.9031547
X-RAY DIFFRACTIONr_chiral_restr0.0750.21458
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.02110459
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021800
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_it0.5061.55880
X-RAY DIFFRACTIONr_mcbond_other0.1171.52436
X-RAY DIFFRACTIONr_mcangle_it0.86329430
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it1.38333539
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it2.0594.53315
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: 1.996→2.048 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.379 255 -
Rwork0.306 5127 -
obs--88.37 %

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