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- PDB-5w8n: Lipid A Disaccharide Synthase (LpxB)-6 solubilizing mutations -

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Basic information

Entry
Database: PDB / ID: 5w8n
TitleLipid A Disaccharide Synthase (LpxB)-6 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 / extrinsic component of cytoplasmic side of plasma membrane / extrinsic component of plasma membrane / lipid A biosynthetic process / phospholipid binding / identical protein binding / membrane / 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: 2.02 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


Theoretical massNumber of molelcules
Total (without water)42,3261
Polymers42,3261
Non-polymers00
Water3,261181
1
A: Lipid-A-disaccharide synthase

A: Lipid-A-disaccharide synthase


  • 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)84,6522
Polymers84,6522
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation4_555y,x,-z1
Buried area12270 Å2
ΔGint-119 kcal/mol
Surface area30260 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.784, 67.784, 154.503
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
Components on special symmetry positions
IDModelComponents
11A-575-

HOH

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: 42325.988 Da / Num. of mol.: 1 / Mutation: V66S, V68S, L69S, L72S, L75S, L76S
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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 181 / 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: 2.42 Å3/Da / Density % sol: 49.26 % / Description: bipyramidal
Crystal growTemperature: 292 K / Method: vapor diffusion, hanging drop / pH: 8.6
Details: 40 % PEG 4000, 0.1 M Tris-HCl pH 8.6, 0.8 M LiCl combined 1:1 with 8 g/L protein in 0.3 M NaCl, 20 mM Tris-HCl pH 7.5, 5% glycerol, 5 mM DTT with 100 mM trimethylammonium chloride additive ...Details: 40 % PEG 4000, 0.1 M Tris-HCl pH 8.6, 0.8 M LiCl combined 1:1 with 8 g/L protein in 0.3 M NaCl, 20 mM Tris-HCl pH 7.5, 5% glycerol, 5 mM DTT with 100 mM trimethylammonium chloride additive used at 10% of drop volume ie. 10 mM final concentration

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97919 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jun 7, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97919 Å / Relative weight: 1
ReflectionResolution: 2.02→19.57 Å / Num. obs: 27746 / % possible obs: 99.6 % / Redundancy: 6.5 % / Biso Wilson estimate: 38.57 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.112 / Rpim(I) all: 0.047 / Net I/σ(I): 14.2
Reflection shellResolution: 2.02→2.07 Å / Redundancy: 6.1 % / Rmerge(I) obs: 1.686 / Num. unique obs: 1958 / CC1/2: 0.53 / Rpim(I) all: 0.717 / % possible all: 97

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Processing

Software
NameVersionClassification
PHENIXv1.11.1_2575refinement
XDSNovember 1, 2017data reduction
Cootv0.8.7-premodel building
PHENIXv1.11.1_2575phasing
Aimless0.5.23data scaling
RefinementMethod to determine structure: SAD / Resolution: 2.02→19.57 Å / SU ML: 0.23 / Cross valid method: FREE R-VALUE / Phase error: 23.8
RfactorNum. reflection% reflection
Rfree0.2129 1385 5 %
Rwork0.1872 --
obs0.1885 27680 99.62 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 46.06 Å2
Refinement stepCycle: LAST / Resolution: 2.02→19.57 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2841 0 0 181 3022
Refine LS restraintsType: GEOSTD + MON.LIB. + CDL v1.2
LS refinement shellResolution: 2.02→2.09 Å
RfactorNum. reflection% reflection
Rfree0.3439 131 -
Rwork0.3043 2519 -
obs-2650 98 %

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