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

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Basic information

Entry
Database: PDB / ID: 4imf
TitleCrystal Structure of Pasteurella multocida N-Acetyl-D-Neuraminic acid lyase K164 mutant complexed with N-Acetylneuraminic 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
ISOPROPYL ALCOHOL / 1-ETHOXY-2-(2-METHOXYETHOXY)ETHANE / Chem-SI3 / N-acetylneuraminate lyase
Similarity search - Component
Biological speciesPasteurella multocida subsp. gallicida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.9 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)66,44112
Polymers65,1512
Non-polymers1,29010
Water4,558253
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)132,88124
Polymers130,3024
Non-polymers2,57920
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_655-x+1,y,-z+1/21
Buried area13730 Å2
ΔGint-62 kcal/mol
Surface area38670 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.710, 150.230, 113.090
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

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Protein / Sugars , 2 types, 4 molecules AB

#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-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.: 2
Source method: isolated from a genetically manipulated source
Formula: C11H19NO9

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Non-polymers , 4 types, 261 molecules

#3: Chemical ChemComp-ME2 / 1-ETHOXY-2-(2-METHOXYETHOXY)ETHANE


Mass: 148.200 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C7H16O3
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#5: Chemical ChemComp-IPA / ISOPROPYL ALCOHOL / 2-PROPANOL


Mass: 60.095 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O / Comment: alkaloid*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 253 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 53.82 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 38% PEG300, 0.01 M calcium chloride, 0.1 M sodium cacodylate, pH 6.5, 5 mM Neu5Ac, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL7-1 / Wavelength: 0.97946 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Dec 15, 2011
RadiationMonochromator: Side scattering I-beam bent single crystal, asymmetric cut 4.9650 degrees
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 1.9→50 Å / Num. all: 54641 / Num. obs: 54641 / % possible obs: 99.2 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Biso Wilson estimate: 38.323 Å2 / Rmerge(I) obs: 0.031 / Net I/σ(I): 25.73
Reflection shell

Diffraction-ID: 1

Resolution (Å)Highest resolution (Å)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. unique obs% possible all
1.9-1.950.5062.9414798402799.8
1.95-20.3773.8514335388599.8
2-2.060.2884.9314142383299.6
2.06-2.120.2166.5413587367799.8
2.12-2.190.1558.9813351361899.8
2.19-2.270.11611.5412795348399.8
2.27-2.360.09413.7712460337599.9
2.36-2.450.07417.2311962323799.8
2.45-2.560.05820.9511470310799.8
2.56-2.690.04725.5411007299699.8
2.69-2.830.03929.9110405283199.8
2.83-30.0336.099854268199.8
3-3.210.02444.189210252899.6
3.21-3.470.0251.638573236499.6
3.47-3.80.01860.287791217699.4
3.8-4.250.01767.296951197599.1
4.25-4.910.01670.365797172497.9
4.91-6.010.01471.155359150799.5
6.01-8.50.01370.063896114996.4
8.50.01266.63132546966.7

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Processing

Software
NameVersionClassificationNB
XSCALEdata scaling
REFMACrefinement
PDB_EXTRACT3.11data extraction
Blu-Icedata collection
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.9→35.92 Å / Cor.coef. Fo:Fc: 0.967 / Cor.coef. Fo:Fc free: 0.957 / WRfactor Rfree: 0.1928 / WRfactor Rwork: 0.1618 / Occupancy max: 1 / Occupancy min: 0.5 / FOM work R set: 0.8773 / SU B: 5.828 / SU ML: 0.087 / SU R Cruickshank DPI: 0.1269 / SU Rfree: 0.12 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.127 / ESU R Free: 0.12 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.2005 2728 5 %RANDOM
Rwork0.1667 ---
obs0.1684 54613 99.18 %-
all-54613 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 87.3 Å2 / Biso mean: 41.031 Å2 / Biso min: 5.94 Å2
Baniso -1Baniso -2Baniso -3
1--0.28 Å20 Å20 Å2
2--0.38 Å20 Å2
3----0.1 Å2
Refinement stepCycle: LAST / Resolution: 1.9→35.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4574 0 83 253 4910
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0170.0194775
X-RAY DIFFRACTIONr_angle_refined_deg1.7611.9936430
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.4265592
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.43625.22205
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.615857
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.2751514
X-RAY DIFFRACTIONr_chiral_restr0.1410.2719
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.023528
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
1151MEDIUM POSITIONAL0.070.5
1118LOOSE POSITIONAL0.385
1151MEDIUM THERMAL12
1118LOOSE THERMAL1.7210
LS refinement shellResolution: 1.9→1.949 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.295 210 -
Rwork0.218 3805 -
all-4015 -
obs--99.83 %
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)
11.01780.3571-0.21931.47620.27221.67190.24850.0921-0.1372-0.015-0.02380.27820.4017-0.4149-0.22470.3101-0.173-0.17220.34120.07010.172615.26923.10318.416
21.0892-0.02460.12611.29030.05921.59060.2247-0.1526-0.11090.2002-0.03280.17150.3068-0.3445-0.19190.1993-0.1618-0.04390.27390.09030.064717.68730.08533.173
31.2224-0.26940.64890.20710.21732.05410.30920.1169-0.3030.01360.04030.04450.6106-0.0749-0.34950.408-0.089-0.21620.20920.02880.137930.11320.50520.282
42.6339-3.02510.9659.0558-1.10078.9360.34730.5547-0.0057-0.2999-0.1062-0.05410.36420.2821-0.24110.3855-0.0172-0.19750.4714-0.03690.142130.23122.9881.824
50.96310.3810.09112.07820.21652.09340.1686-0.01620.32440.08110.00880.4977-0.2529-0.7357-0.17740.1810.12480.16570.41740.10970.256513.81161.11332.171
61.16950.4374-0.01311.5081-0.09011.64030.10130.15460.1954-0.14340.0910.2524-0.115-0.4475-0.19230.13120.04630.02840.26770.11090.089319.43654.16318.275
70.89430.3760.05560.88970.15741.63550.1408-0.05610.25680.07660.02760.1565-0.3098-0.2916-0.16840.2420.0660.17030.15990.02930.152528.69463.61933.623
82.51034.9137-2.878112.5818-3.45365.37390.3881-0.20640.15810.5864-0.3110.2619-0.4541-0.1601-0.07710.3922-0.03360.24430.514-0.07710.171524.64860.99451.651
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 57
2X-RAY DIFFRACTION2A58 - 167
3X-RAY DIFFRACTION3A168 - 277
4X-RAY DIFFRACTION4A278 - 293
5X-RAY DIFFRACTION5B2 - 57
6X-RAY DIFFRACTION6B58 - 167
7X-RAY DIFFRACTION7B168 - 277
8X-RAY DIFFRACTION8B278 - 293

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