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- PDB-3fyo: Crystal structure of the triple mutant (N23C/D247E/P249A) of 3-de... -

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Basic information

Entry
Database: PDB / ID: 3fyo
TitleCrystal structure of the triple mutant (N23C/D247E/P249A) of 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) from Neisseria meningitidis
Components3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase
KeywordsTRANSFERASE / MANNO-OCTULOSONATE / SYNTHASE / LIPOPOLYSACCHARIDE / KDOP / KDO8 KDOPS / KDO8PS / TIM BARREL / BIOSYNTHESIS / LYASE / Lipopolysaccharide biosynthesis
Function / homology
Function and homology information


3-deoxy-8-phosphooctulonate synthase / 3-deoxy-8-phosphooctulonate synthase activity / keto-3-deoxy-D-manno-octulosonic acid biosynthetic process / cytosol
Similarity search - Function
3-deoxy-8-phosphooctulonate synthase / DAHP synthetase I/KDSA / DAHP synthetase I family / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
: / PHOSPHOENOLPYRUVATE / 2-dehydro-3-deoxyphosphooctonate aldolase
Similarity search - Component
Biological speciesNeisseria meningitidis serogroup B (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.9 Å
AuthorsJameson, G.B. / Parker, E.J. / Cochrane, F.P. / Patchett, M.L.
CitationJournal: J.Mol.Biol. / Year: 2009
Title: Reversing evolution: re-establishing obligate metal ion dependence in a metal-independent KDO8P synthase
Authors: Cochrane, F.C. / Cookson, T.V. / Jameson, G.B. / Parker, E.J.
History
DepositionJan 22, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 24, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Refinement description / Version format compliance
Revision 1.2Oct 20, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_conn_angle ...database_2 / pdbx_struct_conn_angle / struct_conn / 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 / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Sep 6, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_comp_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_comp_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase
B: 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase
C: 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase
D: 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)122,3029
Polymers121,9854
Non-polymers3175
Water8,161453
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12360 Å2
ΔGint-105 kcal/mol
Surface area35520 Å2
MethodPISA
Unit cell
Length a, b, c (Å)82.740, 86.197, 163.891
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
31C
41D

NCS domain segments:

Ens-ID: 1 / Refine code: 5

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ASPASPILEILEAA2 - 502 - 50
21ASPASPILEILEBB2 - 502 - 50
31ASPASPILEILECC2 - 502 - 50
41ASPASPILEILEDD2 - 502 - 50
12GLYGLYHISHISAA70 - 19970 - 199
22GLYGLYHISHISBB70 - 19970 - 199
32GLYGLYHISHISCC70 - 19970 - 199
42GLYGLYHISHISDD70 - 19970 - 199
13ASPASPPHEPHEAA220 - 234220 - 234
23ASPASPPHEPHEBB220 - 234220 - 234
33ASPASPPHEPHECC220 - 234220 - 234
43ASPASPPHEPHEDD220 - 234220 - 234
14GLUGLUPROPROAA258 - 275258 - 275
24GLUGLUPROPROBB258 - 275258 - 275
34GLUGLUPROPROCC258 - 275258 - 275
44GLUGLUPROPRODD258 - 275258 - 275

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase / 2-dehydro-3-deoxyphosphooctonate aldolase / Phospho-2-dehydro-3-deoxyoctonate aldolase / KDO-8- ...2-dehydro-3-deoxyphosphooctonate aldolase / Phospho-2-dehydro-3-deoxyoctonate aldolase / KDO-8-phosphate synthetase / KDO 8-P synthase / KDOPS


Mass: 30496.354 Da / Num. of mol.: 4 / Mutation: N23C, D247E, P249A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis serogroup B (bacteria)
Gene: kdsA, NMB1283 / Plasmid: pT7-7 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
References: UniProt: Q9JZ55, 3-deoxy-8-phosphooctulonate synthase

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Non-polymers , 5 types, 458 molecules

#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#4: Chemical ChemComp-PEP / PHOSPHOENOLPYRUVATE / Phosphoenolpyruvic acid


Mass: 168.042 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H5O6P
#5: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 453 / 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: 2.4 Å3/Da / Density % sol: 48.65 %
Crystal growTemperature: 297 K / Method: vapor diffusion, hanging drop / pH: 5
Details: 21.0 mg/mL protein mixed 1:1 with reservoir liquor containing 100 mM NaOAc (pH 5.0), 2 mM PEP, 200 microM MnSO4 and 2.0 M NaCl. Immediately prior to data collection, crystal were harvested ...Details: 21.0 mg/mL protein mixed 1:1 with reservoir liquor containing 100 mM NaOAc (pH 5.0), 2 mM PEP, 200 microM MnSO4 and 2.0 M NaCl. Immediately prior to data collection, crystal were harvested and soaked briefly in cryoprotectant solution, comprising 20% glycerol and 100 mM NaOAc (pH 5.0) and 2.0 M NaCl, VAPOR DIFFUSION, HANGING DROP, temperature 297K

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Data collection

DiffractionMean temperature: 110 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Nov 15, 2006 / Details: Osmic blue optic
RadiationMonochromator: Osmic blue optic / Protocol: SINGLE WAVELENGTH / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.8→33.95 Å / Num. all: 108582 / Num. obs: 108582 / % possible obs: 99.5 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 4.15 % / Biso Wilson estimate: 32.4 Å2 / Rmerge(I) obs: 0.065 / Χ2: 0.94 / Net I/σ(I): 9 / Scaling rejects: 12568
Reflection shellResolution: 1.8→1.86 Å / Redundancy: 4.09 % / Rmerge(I) obs: 0.472 / Mean I/σ(I) obs: 2.3 / Num. measured all: 44603 / Num. unique all: 10737 / Χ2: 1.09 / % possible all: 100

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Phasing

PhasingMethod: molecular replacement
Phasing MRRfactor: 0.344 / Cor.coef. Fo:Fc: 0.75 / Cor.coef. Io to Ic: 0.748
Highest resolutionLowest resolution
Rotation3 Å15 Å
Translation3 Å15 Å

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Processing

Software
NameVersionClassificationNB
d*TREKdata scaling
AMoREphasing
REFMACrefinement
PDB_EXTRACT3.006data extraction
CrystalCleardata collection
d*TREKdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2QKF

2qkf


Resolution: 1.9→33.94 Å / Cor.coef. Fo:Fc: 0.953 / Cor.coef. Fo:Fc free: 0.94 / WRfactor Rfree: 0.264 / WRfactor Rwork: 0.225 / Occupancy max: 1 / Occupancy min: 0.25 / FOM work R set: 0.815 / SU B: 7.818 / SU ML: 0.103 / SU R Cruickshank DPI: 0.172 / SU Rfree: 0.157 / TLS residual ADP flag: LIKELY RESIDUAL / Isotropic thermal model: ISOTROPIC / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.168 / ESU R Free: 0.156 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES RESIDUAL ONLY. CLOSE CONTACTS ARE A CONSEQUENCE OF UNRESOLVED DISORDER IN SIDE CHAINS AND/OR WATER MOLECULES. CLOSE WATER-WATER ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES RESIDUAL ONLY. CLOSE CONTACTS ARE A CONSEQUENCE OF UNRESOLVED DISORDER IN SIDE CHAINS AND/OR WATER MOLECULES. CLOSE WATER-WATER CONTACTS MAY BE ASCRIBED TO POORLY DEFINED GLYCEROL (CRYOPROTECTANT) SPECIES. PHI/PSI VALUES FOR RESIDUES 6 AND 229 ARE HIGHLY CONSERVED IN KDO8PS AND ARE LOCATED IN WELL-DEFINED ELECTRON DENSITY. RESIDES 56 AND 172 (CHAINS B AND C) LIE IN WELL-DEFINED ELECTRON DENSITY. OTHER RESIDUES WITH POOR PHI/PSI VALUES LIE IN REGIONS OF POOR DENSITY IN ILL-DEFINED LOOPS OR AT FRAYED BEGINNINGS AND ENDS OF MISSING LOOPS.
RfactorNum. reflection% reflectionSelection details
Rfree0.257 4644 5 %RANDOM
Rwork0.216 ---
all0.218 92429 --
obs0.218 92429 99.42 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 67.84 Å2 / Biso mean: 27.845 Å2 / Biso min: 8.01 Å2
Baniso -1Baniso -2Baniso -3
1-0.6 Å20 Å20 Å2
2--0.25 Å20 Å2
3----0.85 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.156 Å0.168 Å
Refinement stepCycle: LAST / Resolution: 1.9→33.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7886 0 14 453 8353
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0228335
X-RAY DIFFRACTIONr_bond_other_d00.025586
X-RAY DIFFRACTIONr_angle_refined_deg1.0751.97711333
X-RAY DIFFRACTIONr_angle_other_deg0.946313794
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.74351096
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.12724.794340
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.356151476
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.3891534
X-RAY DIFFRACTIONr_chiral_restr0.1390.21334
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0219278
X-RAY DIFFRACTIONr_gen_planes_other0.0030.021584
X-RAY DIFFRACTIONr_mcbond_it1.3061.55288
X-RAY DIFFRACTIONr_mcbond_other0.5021.52147
X-RAY DIFFRACTIONr_mcangle_it2.16728573
X-RAY DIFFRACTIONr_scbond_it3.26533047
X-RAY DIFFRACTIONr_scangle_it5.1024.52737
Refine LS restraints NCS

Dom-ID: 1 / Ens-ID: 1 / Refine-ID: X-RAY DIFFRACTION

Auth asym-IDNumberTypeRms dev position (Å)Weight position
A1145MEDIUM POSITIONAL0.190.5
B1145MEDIUM POSITIONAL0.390.5
C1145MEDIUM POSITIONAL0.230.5
D1145MEDIUM POSITIONAL0.330.5
A1205LOOSE POSITIONAL0.585
B1205LOOSE POSITIONAL0.775
C1205LOOSE POSITIONAL0.575
D1205LOOSE POSITIONAL0.745
A1145MEDIUM THERMAL2.482
B1145MEDIUM THERMAL1.952
C1145MEDIUM THERMAL1.952
D1145MEDIUM THERMAL1.492
A1205LOOSE THERMAL2.110
B1205LOOSE THERMAL1.8710
C1205LOOSE THERMAL1.7810
D1205LOOSE THERMAL1.6410
LS refinement shellResolution: 1.9→1.949 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.409 355 -
Rwork0.353 6424 -
all-6779 -
obs-6424 99.97 %
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)
10.8407-0.0795-0.3280.2454-0.02410.5315-0.01460.2088-0.0109-0.0421-0.00020.0203-0.0095-0.14970.01480.04140.0146-0.00160.12360.00610.04148.51520.79848.654
20.2347-0.17420.0310.27090.02240.3641-0.0219-0.01430.0344-0.00310.02790.0040.026-0.0135-0.00610.0298-0.00480.01050.0629-0.00520.048916.26113.82684.142
30.0548-0.0739-0.00950.13560.01680.5126-0.0042-0.0471-0.0045-0.03330.0048-0.0126-0.0002-0.0408-0.00070.0586-0.0040.01570.0487-0.00960.043733.812-1.56938.195
40.175-0.2828-0.2010.99580.28730.4123-0.07980.0360.01020.20050.03460.02320.12820.04980.04530.11050.0390.00450.0155-0.00650.039537.91-11.58273.378
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 276
2X-RAY DIFFRACTION2B1 - 279
3X-RAY DIFFRACTION3C1 - 280
4X-RAY DIFFRACTION4D1 - 277

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