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- PDB-3fyp: Crystal structure of the quadruple mutant (N23C/C246S/D247E/P249A... -

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Basic information

Entry
Database: PDB / ID: 3fyp
TitleCrystal structure of the quadruple mutant (N23C/C246S/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 / Resolution: 1.85 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 31, 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 / struct_conn ...database_2 / 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 / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr1_label_alt_id / _struct_conn.ptnr1_auth_asym_id / _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_asym_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_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.end_auth_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)122,44512
Polymers121,9214
Non-polymers5248
Water10,629590
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12690 Å2
ΔGint-114 kcal/mol
Surface area35450 Å2
MethodPISA
Unit cell
Length a, b, c (Å)81.781, 85.679, 163.828
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

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDRefine codeAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ILEILEILEILE5AA3 - 503 - 50
21ILEILEILEILE5BB3 - 503 - 50
31ILEILEILEILE5CC3 - 503 - 50
41ILEILEILEILE5DD3 - 503 - 50
12ILEILETHRTHR5AA77 - 19877 - 198
22ILEILETHRTHR5BB77 - 19877 - 198
32ILEILETHRTHR5CC77 - 19877 - 198
42ILEILETHRTHR5DD77 - 19877 - 198
13ASPASPPHEPHE6AA220 - 234220 - 234
23ASPASPPHEPHE6BB220 - 234220 - 234
33ASPASPPHEPHE6CC220 - 234220 - 234
43ASPASPPHEPHE6DD220 - 234220 - 234
14GLUGLUGLNGLN5AA258 - 274258 - 274
24GLUGLUGLNGLN5BB258 - 274258 - 274
34GLUGLUGLNGLN5CC258 - 274258 - 274
44GLUGLUGLNGLN5DD258 - 274258 - 274

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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: 30480.291 Da / Num. of mol.: 4 / Mutation: N23C, C246S, 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 , 6 types, 598 molecules

#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-PEP / PHOSPHOENOLPYRUVATE


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


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


Mass: 18.015 Da / Num. of mol.: 590 / 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: 2.35 Å3/Da / Density % sol: 47.74 %
Crystal growTemperature: 297 K / Method: vapor diffusion, hanging drop / pH: 5.4
Details: 27.7 mg/mL protein mixed 1:1 with reservoir liquor containing 100 mM NaOAc (pH 5.0), 2 mM PEP, 850 microM MnSO4 and 2.0 M NaCl. Immediately prior to data collection, crystals were harvested ...Details: 27.7 mg/mL protein mixed 1:1 with reservoir liquor containing 100 mM NaOAc (pH 5.0), 2 mM PEP, 850 microM MnSO4 and 2.0 M NaCl. Immediately prior to data collection, crystals were harvested and soaked briefly in cryoprotectant solution, comprising 20% glycerol and the resevoir solution, vapor diffusion, hanging drop, temperature 297K, pH 5.4

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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: Jan 1, 2007 / Details: Osmic blue optic
RadiationMonochromator: Osmic blue optic / Protocol: SINGLE WAVELENGTH / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.85→37.96 Å / Num. all: 98477 / Num. obs: 98477 / % possible obs: 99.7 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 4.88 % / Biso Wilson estimate: 31.8 Å2 / Rmerge(I) obs: 0.046 / Χ2: 0.91 / Net I/σ(I): 14.1 / Scaling rejects: 17788
Reflection shellResolution: 1.85→1.92 Å / Redundancy: 4.7 % / Rmerge(I) obs: 0.343 / Mean I/σ(I) obs: 3.4 / Num. measured all: 47031 / Num. unique all: 9740 / Χ2: 1.24 / % possible all: 99.9

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Processing

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

2qkf


Resolution: 1.85→37.96 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.941 / WRfactor Rfree: 0.245 / WRfactor Rwork: 0.203 / Occupancy max: 1 / Occupancy min: 0.25 / FOM work R set: 0.839 / SU B: 6.71 / SU ML: 0.091 / SU R Cruickshank DPI: 0.147 / SU Rfree: 0.141 / TLS residual ADP flag: LIKELY RESIDUAL / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.147 / ESU R Free: 0.141 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES RESIDUAL ONLY. THESE CLOSE CONTACTS ARE A CONSEQUENCE OF UNRESOLVED DISORDER IN SIDE CHAINS AND/OR WATER MOLECULES. CLOSE WATER- ...Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES RESIDUAL ONLY. THESE 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
Rfree0.247 4964 5 %RANDOM
Rwork0.205 ---
all0.207 98469 --
obs0.207 98469 99.65 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso max: 82.82 Å2 / Biso mean: 25.785 Å2 / Biso min: 6.93 Å2
Baniso -1Baniso -2Baniso -3
1-0.47 Å20 Å20 Å2
2--0.3 Å20 Å2
3----0.77 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.141 Å0.147 Å
Refinement stepCycle: LAST / Resolution: 1.85→37.96 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7786 0 27 590 8403
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.0228154
X-RAY DIFFRACTIONr_bond_other_d0.0010.025468
X-RAY DIFFRACTIONr_angle_refined_deg1.2571.98111071
X-RAY DIFFRACTIONr_angle_other_deg1.628313517
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.84451072
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.81224.969326
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.417151455
X-RAY DIFFRACTIONr_dihedral_angle_4_deg21.7561531
X-RAY DIFFRACTIONr_chiral_restr0.0810.21316
X-RAY DIFFRACTIONr_gen_planes_refined0.0150.0219004
X-RAY DIFFRACTIONr_gen_planes_other0.0060.021533
X-RAY DIFFRACTIONr_mcbond_it2.87335174
X-RAY DIFFRACTIONr_mcbond_other1.31332112
X-RAY DIFFRACTIONr_mcangle_it3.96848381
X-RAY DIFFRACTIONr_scbond_it2.44322980
X-RAY DIFFRACTIONr_scangle_it3.66832663
Refine LS restraints NCS

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

Auth asym-IDNumberTypeRms dev position (Å)Weight position
A1048MEDIUM POSITIONAL0.170.5
B1048MEDIUM POSITIONAL0.330.5
C1048MEDIUM POSITIONAL0.20.5
D1048MEDIUM POSITIONAL0.30.5
A1309LOOSE POSITIONAL0.495
B1309LOOSE POSITIONAL0.685
C1309LOOSE POSITIONAL0.595
D1309LOOSE POSITIONAL0.775
A1048MEDIUM THERMAL3.573
B1048MEDIUM THERMAL2.973
C1048MEDIUM THERMAL3.63
D1048MEDIUM THERMAL2.813
A1309LOOSE THERMAL2.9810
B1309LOOSE THERMAL2.6310
C1309LOOSE THERMAL2.9910
D1309LOOSE THERMAL2.4710
LS refinement shellResolution: 1.85→1.898 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.471 387 -
Rwork0.435 6817 -
all-7204 -
obs-6817 99.93 %
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)
10.1836-0.17210.05140.23510.0530.4233-0.0199-0.02610.02930.00570.00540.01880.02-0.01990.01450.0465-0.01080.00910.07560.00280.032914.17614.88580.62
20.1457-0.1339-0.19940.23650.1270.30080.010.0295-0.012-0.0714-0.00590.015-0.0028-0.0869-0.00410.0679-0.0079-0.01110.09780.00030.020622.5478.38140.999
30.3686-0.6195-0.3211.60350.28290.5679-0.14850.0326-0.00540.34150.07950.05080.19790.06560.0690.16580.03530.00860.0135-0.00560.009737.511-11.33373.033
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1B1 - 279
2X-RAY DIFFRACTION2C1 - 277
3X-RAY DIFFRACTION3D1 - 277

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