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- PDB-5w8s: Lipid A Disaccharide Synthase (LpxB)-7 solubilizing mutations -

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Basic information

Entry
Database: PDB / ID: 5w8s
TitleLipid A Disaccharide Synthase (LpxB)-7 solubilizing mutations
ComponentsLipid-A-disaccharide synthase
KeywordsTRANSFERASE / glycosyltransferase B / Rossmann-like / C-terminal swap / dimer / lipid A disaccharide synthase / Raetz pathway / lipid A synthesis pathway / lipiopolysaccharide synthesis
Function / homology
Function and homology information


lipid-A-disaccharide synthase / lipid-A-disaccharide synthase activity / lipid A biosynthetic process / extrinsic component of plasma membrane / extrinsic component of cytoplasmic side of plasma membrane / phospholipid binding / identical protein binding / cytoplasm
Similarity search - Function
Glycosyl transferase, family 19 / Lipid-A-disaccharide synthetase
Similarity search - Domain/homology
: / Lipid-A-disaccharide synthase / Lipid-A-disaccharide synthase
Similarity search - Component
Biological speciesEscherichia coli BL21 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.1 Å
AuthorsBohl, T.E. / Aihara, H. / Shi, K. / Lee, J.K.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118047 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41-GM103403 United States
Department of Energy (DOE, United States)DE-AC02-06CH11357 United States
CitationJournal: Nat Commun / Year: 2018
Title: Crystal structure of lipid A disaccharide synthase LpxB from Escherichia coli.
Authors: Bohl, T.E. / Shi, K. / Lee, J.K. / Aihara, H.
History
DepositionJun 22, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lipid-A-disaccharide synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,3354
Polymers42,2821
Non-polymers533
Water2,702150
1
A: Lipid-A-disaccharide synthase
hetero molecules

A: Lipid-A-disaccharide synthase
hetero molecules


  • defined by author&software
  • Evidence: equilibrium centrifugation, assay for oligomerization, Enzymatic activity assay showed that LpxB with mutations in the swapped portion of the dimer can be complemented by LpxB with mutation ...Evidence: equilibrium centrifugation, assay for oligomerization, Enzymatic activity assay showed that LpxB with mutations in the swapped portion of the dimer can be complemented by LpxB with mutation in the unswapped portion by forming an LpxB mutant heterodimer with more activity than individual LpxB mutants. Thus, C-terminal swapped dimerization occurs and creates one intact active site in each LpxB mutant heterodimer.
  • 84.7 kDa, 2 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)84,6708
Polymers84,5642
Non-polymers1066
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_465y-1,x+1,-z1
Buried area12980 Å2
ΔGint-177 kcal/mol
Surface area30010 Å2
MethodPISA
Unit cell
Length a, b, c (Å)68.016, 68.016, 155.139
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z
DetailsEnzymatic activity assay showed that LpxB with mutations in the swapped portion of the dimer can be complemented by LpxB with mutation in the unswapped portion by forming an LpxB mutant heterodimer with more activity than individual LpxB mutants. Thus, C-terminal swapped dimerization occurs and creates one intact active site in each LpxB mutant heterodimer.

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Components

#1: Protein Lipid-A-disaccharide synthase


Mass: 42281.875 Da / Num. of mol.: 1 / Mutation: V66S, V68S, L69S, L72S, L75S, L76S, M207S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli BL21(DE3) (bacteria) / Gene: lpxB, ECBD_3437 / Plasmid: pE-SUMO
Details (production host): cleavable His-tagged N-terminal SUMO
Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A140NAT1, UniProt: P10441*PLUS, lipid-A-disaccharide synthase
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#3: Chemical ChemComp-LI / LITHIUM ION


Mass: 6.941 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Li
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 150 / 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: 2.45 Å3/Da / Density % sol: 49.86 % / Description: bipyramidal
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 8.6
Details: 39% PEG 4000, 0.1 M Tris-HCl pH 8.6, 0.7 M LiCl mixed 1:1 with 8 g/L protein in 0.3 M NaCl, 5% glycerol, 20 mM Tris-HCl pH 7.4, 5 mM DTT with 100 mM trimethylammonium chloride additive used ...Details: 39% PEG 4000, 0.1 M Tris-HCl pH 8.6, 0.7 M LiCl mixed 1:1 with 8 g/L protein in 0.3 M NaCl, 5% glycerol, 20 mM Tris-HCl pH 7.4, 5 mM DTT with 100 mM trimethylammonium chloride additive used at 10 mM or 10% of drop volume

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97918 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jul 20, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.1→155.14 Å / Num. obs: 24865 / % possible obs: 99.4 % / Redundancy: 3.5 % / Biso Wilson estimate: 40.03 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.066 / Rpim(I) all: 0.041 / Net I/σ(I): 13.5
Reflection shellResolution: 2.1→2.16 Å / Redundancy: 3.6 % / Rmerge(I) obs: 0.997 / Mean I/σ(I) obs: 1.6 / Num. unique obs: 2012 / CC1/2: 0.55 / Rpim(I) all: 0.615 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIXv1.10_2155refinement
XDSJune 17, 2015data reduction
Cootv0.8.2model building
PHENIXv1.10_2155phasing
Aimless0.1.27data scaling
RefinementMethod to determine structure: SAD / Resolution: 2.1→55.07 Å / SU ML: 0.3 / Cross valid method: FREE R-VALUE / Phase error: 23.86
RfactorNum. reflection% reflection
Rfree0.2226 2007 8 %
Rwork0.1951 --
obs0.1973 24788 98.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 45.6 Å2
Refinement stepCycle: LAST / Resolution: 2.1→55.07 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2800 0 3 150 2953
Refine LS restraintsType: GEOSTD + MON.LIB. + CDL v1.2
LS refinement shellResolution: 2.1→2.15 Å
RfactorNum. reflection% reflection
Rfree0.3789 141 -
Rwork0.3344 1737 -
obs--99 %

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