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- PDB-4img: Crystal Structure of Pasteurella multocida N-Acetyl-D-Neuraminic ... -

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Basic information

Entry
Database: PDB / ID: 4img
TitleCrystal Structure of Pasteurella multocida N-Acetyl-D-Neuraminic acid lyase K164 mutant complexed with N-Glycolylneuraminic acid
ComponentsN-acetylneuraminate lyase
KeywordsLYASE / Tim barrel / Schiff base
Function / homology
Function and homology information


N-acetylneuraminate lyase / N-acetylneuraminate lyase activity / N-acetylneuraminate catabolic process / carbohydrate metabolic process / 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
1-ETHOXY-2-(2-METHOXYETHOXY)ETHANE / Chem-NGF / N-acetylneuraminate lyase
Similarity search - Component
Biological speciesPasteurella multocida subsp. gallicida (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.85 Å
AuthorsFisher, A.J. / Huynh, N.
CitationJournal: Biochemistry / Year: 2013
Title: Structural Basis for Substrate Specificity and Mechanism of N-Acetyl-d-neuraminic Acid Lyase from Pasteurella multocida.
Authors: Huynh, N. / Aye, A. / Li, Y. / Yu, H. / Cao, H. / Tiwari, V.K. / Shin, D.W. / Chen, X. / Fisher, A.J.
History
DepositionJan 2, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 6, 2013Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2013Group: Database references
Revision 1.2Feb 28, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,98712
Polymers65,1512
Non-polymers1,83610
Water11,620645
1
A: N-acetylneuraminate lyase
B: N-acetylneuraminate lyase
hetero molecules

A: N-acetylneuraminate lyase
B: N-acetylneuraminate lyase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,97424
Polymers130,3024
Non-polymers3,67220
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_655-x+1,y,-z+1/21
Buried area15000 Å2
ΔGint28 kcal/mol
Surface area39630 Å2
MethodPISA
Unit cell
Length a, b, c (Å)82.202, 149.997, 112.456
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

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Components

#1: Protein N-acetylneuraminate lyase / N-acetylneuraminate pyruvate-lyase / N-acetylneuraminic acid aldolase / Sialate lyase / Sialic acid ...N-acetylneuraminate pyruvate-lyase / N-acetylneuraminic acid aldolase / Sialate lyase / Sialic acid aldolase / Sialic acid lyase


Mass: 32575.414 Da / Num. of mol.: 2 / Mutation: K164A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pasteurella multocida subsp. gallicida (bacteria)
Strain: P1059 / Gene: nanA, PM1715 / Plasmid: pET22b(+) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: Q9CKB0, N-acetylneuraminate lyase
#2: Sugar ChemComp-NGF / 3,5-dideoxy-5-[(hydroxyacetyl)amino]-D-glycero-D-galacto-non-2-ulosonic acid / N-glycolylneuraminic acid, ketone form


Type: D-saccharide / Mass: 325.269 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C11H19NO10
#3: Chemical
ChemComp-ME2 / 1-ETHOXY-2-(2-METHOXYETHOXY)ETHANE


Mass: 148.200 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C7H16O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 645 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsUNIPROT SEQUENCE REFERENCE Q9CKB0 IS FOR A DIFFERENT STRAIN (PM70).

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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: 2.66 Å3/Da / Density % sol: 53.77 %
Crystal growTemperature: 298 K / Method: vapor diffusion / pH: 6.5
Details: 38% PEG300, 0.01 M calcium chloride, 0.1 M sodium cacodylate, pH 6.5, 5 mM Neu5Gc, VAPOR DIFFUSION, temperature 298K

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Data collection

DiffractionMean temperature: 90 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RUH3R / Wavelength: 1.54 Å
DetectorType: BRUKER SMART 6000 / Detector: CCD / Date: Sep 20, 2012 / Details: Mirrors
RadiationMonochromator: mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.85→100 Å / Num. all: 59552 / Num. obs: 59552 / % possible obs: 100 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Redundancy: 9.6 % / Biso Wilson estimate: 18.6 Å2 / Rmerge(I) obs: 0.074 / Net I/σ(I): 18
Reflection shellResolution: 1.85→1.94 Å / Redundancy: 5 % / Rmerge(I) obs: 0.428 / Mean I/σ(I) obs: 2.48 / Num. unique all: 7821 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHASERphasing
REFMACrefinement
PDB_EXTRACT3.11data extraction
PROTEUM PLUSPLUSdata collection
SAINTdata reduction
PROTEUM PLUSPLUSdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.85→75 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.939 / WRfactor Rfree: 0.2026 / WRfactor Rwork: 0.1683 / Occupancy max: 1 / Occupancy min: 0.5 / FOM work R set: 0.8578 / SU B: 5.618 / SU ML: 0.086 / SU R Cruickshank DPI: 0.1321 / SU Rfree: 0.1259 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.132 / ESU R Free: 0.126 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.2163 2997 5 %RANDOM
Rwork0.1785 ---
all0.1804 59424 --
obs0.1804 59424 99.8 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 60.03 Å2 / Biso mean: 21.259 Å2 / Biso min: 5.28 Å2
Baniso -1Baniso -2Baniso -3
1--1.72 Å20 Å20 Å2
2--2.15 Å20 Å2
3----0.43 Å2
Refinement stepCycle: LAST / Resolution: 1.85→75 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4590 0 124 645 5359
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.0194822
X-RAY DIFFRACTIONr_angle_refined_deg1.85326474
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.255594
X-RAY DIFFRACTIONr_dihedral_angle_2_deg40.32225.266207
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.83715861
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.0271514
X-RAY DIFFRACTIONr_chiral_restr0.1310.2719
X-RAY DIFFRACTIONr_gen_planes_refined0.010.023528
Refine LS restraints NCS

Dom-ID: 1 / Auth asym-ID: A / Ens-ID: 1 / Refine-ID: X-RAY DIFFRACTION

NumberTypeRms dev position (Å)Weight position
1121MEDIUM POSITIONAL0.250.5
1167TIGHT THERMAL0.830.5
1121MEDIUM THERMAL1.372
LS refinement shellResolution: 1.85→1.898 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.286 199 -
Rwork0.233 4162 -
all-4361 -
obs--100 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.6083-0.1421-0.04160.57260.11670.50740.01640.0002-0.07010.0176-0.01330.03180.028-0.0779-0.0030.1069-0.0251-0.01980.01620.00230.089815.72323.82217.871
20.41790.00670.07950.28950.14540.5382-0.0043-0.054-0.03940.02250.01790.00140.0273-0.0696-0.01360.089-0.0169-0.00150.02530.0230.078617.89730.32632.72
30.4627-0.1280.2040.08780.03060.58340.03930.0088-0.10070.00430.02060.01770.04510.018-0.05990.1117-0.0102-0.03290.01420.00010.104230.39620.7820.022
40.9597-1.7826-0.08985.1726-1.21012.38430.09340.1645-0.0175-0.0839-0.0829-0.07940.03050.1612-0.01040.10760.014-0.0330.1558-0.03150.066631.11922.6851.516
50.3993-0.08880.15980.5174-0.08890.45270.0057-0.03890.0721-0.0563-0.01730.0479-0.0253-0.06910.01160.09930.02380.01130.0165-0.00390.113814.03460.65631.422
60.4158-0.0663-0.04850.32420.07270.5018-0.03110.05040.0505-0.03410.02390.0105-0.0122-0.04680.00730.09970.0034-0.00320.01530.01110.087619.89654.17517.595
70.57350.1087-0.11820.07970.06320.27920.014-0.01860.0936-0.0118-0.0024-0.0016-0.039-0.0239-0.01160.11430.00890.02110.0074-0.00580.121828.74763.67833.123
81.63143.0774-1.08289.0668-1.77010.76120.196-0.279-0.01510.2221-0.2409-0.0587-0.15450.18010.04490.1387-0.00270.01520.1235-0.03590.086224.6861.66251.17
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 57
2X-RAY DIFFRACTION2A58 - 167
3X-RAY DIFFRACTION3A168 - 277
4X-RAY DIFFRACTION4A278 - 293
5X-RAY DIFFRACTION5B1 - 57
6X-RAY DIFFRACTION6B58 - 167
7X-RAY DIFFRACTION7B168 - 277
8X-RAY DIFFRACTION8B278 - 293

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