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- PDB-8aq4: In surfo structure of the membrane integral lipoprotein N-acyltra... -

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Basic information

Entry
Database: PDB / ID: 8aq4
TitleIn surfo structure of the membrane integral lipoprotein N-acyltransferase Lnt from E. coli in complex with TITC and lyso-PE
ComponentsApolipoprotein N-acyltransferase
KeywordsTRANSFERASE / Lnt / apolipoprotein N-acyltransferase / Bacterial lipoproteins.
Function / homology
Function and homology information


apolipoprotein N-acyltransferase / N-acyltransferase activity / lipoprotein biosynthetic process / outer membrane-bounded periplasmic space / plasma membrane
Similarity search - Function
Apolipoprotein N-acyltransferase, N-terminal / Apolipoprotein N-acyltransferase N-terminal domain / Apolipoprotein N-acyltransferase / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
Chem-6OU / ACETATE ION / 1-METHOXY-2-[2-(2-METHOXY-ETHOXY]-ETHANE / Chem-PG6 / ~{N}-tridecylmethanethioamide / Apolipoprotein N-acyltransferase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.62 Å
AuthorsHuang, C.-Y. / Weichert, D. / Boland, C. / Smithers, L. / Olieric, V. / Wang, M. / Caffrey, M.
Funding support Ireland, 2items
OrganizationGrant numberCountry
Science Foundation Ireland16/IA/4435 Ireland
Irish Research CouncilGOIPD/2021/40 Ireland
CitationJournal: Sci Adv / Year: 2023
Title: Structure snapshots reveal the mechanism of a bacterial membrane lipoprotein -acyltransferase.
Authors: Luke Smithers / Oksana Degtjarik / Dietmar Weichert / Chia-Ying Huang / Coilín Boland / Katherine Bowen / Abraham Oluwole / Corinne Lutomski / Carol V Robinson / Eoin M Scanlan / Meitian ...Authors: Luke Smithers / Oksana Degtjarik / Dietmar Weichert / Chia-Ying Huang / Coilín Boland / Katherine Bowen / Abraham Oluwole / Corinne Lutomski / Carol V Robinson / Eoin M Scanlan / Meitian Wang / Vincent Olieric / Moran Shalev-Benami / Martin Caffrey /
Abstract: Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP ...Bacterial lipoproteins (BLPs) decorate the surface of membranes in the cell envelope. They function in membrane assembly and stability, as enzymes, and in transport. The final enzyme in the BLP synthesis pathway is the apolipoprotein -acyltransferase, Lnt, which is proposed to act by a ping-pong mechanism. Here, we use x-ray crystallography and cryo-electron microscopy to chart the structural changes undergone during the progress of the enzyme through the reaction. We identify a single active site that has evolved to bind, individually and sequentially, substrates that satisfy structural and chemical criteria to position reactive parts next to the catalytic triad for reaction. This study validates the ping-pong mechanism, explains the molecular bases for Lnt's substrate promiscuity, and should facilitate the design of antibiotics with minimal off-target effects.
History
DepositionAug 11, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 12, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 7, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Apolipoprotein N-acyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,99423
Polymers59,2811
Non-polymers5,71322
Water39622
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4850 Å2
ΔGint6 kcal/mol
Surface area20790 Å2
MethodPISA
Unit cell
Length a, b, c (Å)160.111, 160.111, 90.934
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221

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Components

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Protein / Sugars , 2 types, 4 molecules A

#1: Protein Apolipoprotein N-acyltransferase / ALP N-acyltransferase / Copper homeostasis protein CutE


Mass: 59280.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: lnt, cutE, b0657, JW0654 / Production host: Escherichia coli (E. coli)
References: UniProt: P23930, Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
#9: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM

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Non-polymers , 9 types, 41 molecules

#2: Chemical
ChemComp-PG5 / 1-METHOXY-2-[2-(2-METHOXY-ETHOXY]-ETHANE / Triethylene glycol dimethyl ether


Mass: 178.226 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H18O4
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-6OU / [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate


Mass: 717.996 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C39H76NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#5: Chemical ChemComp-QGT / ~{N}-tridecylmethanethioamide / Tetradecyl-1-isothiocyanate (reacted form) / TITC (reacted form)


Mass: 243.452 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H29NS / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-PG6 / 1-(2-METHOXY-ETHOXY)-2-{2-[2-(2-METHOXY-ETHOXY]-ETHOXY}-ETHANE / Polyethylene glycol


Mass: 266.331 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H26O6
#7: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER / Tris


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#8: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#10: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#11: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 22 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 5.68 Å3/Da / Density % sol: 78.33 %
Crystal growTemperature: 291 K / Method: vapor diffusion
Details: 50 mM sodium acetate pH 5.0, 50 mM magnesium acetate and 28-36 %(v/v) PEG200

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.96862 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Sep 5, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.96862 Å / Relative weight: 1
ReflectionResolution: 2.62→55.13 Å / Num. obs: 24528 / % possible obs: 93.2 % / Redundancy: 9.8 % / CC1/2: 0.99 / Rrim(I) all: 0.26 / Net I/σ(I): 8
Reflection shellResolution: 2.66→3.01 Å / Redundancy: 9.2 % / Mean I/σ(I) obs: 1.7 / Num. unique obs: 1226 / CC1/2: 0.53 / Rrim(I) all: 1.55 / % possible all: 60.8

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3600refinement
XSCALEdata scaling
PDB_EXTRACT3.25data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5N6H

5n6h


Resolution: 2.62→55.13 Å / SU ML: 0.22 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 26.17
RfactorNum. reflection% reflection
Rfree0.229 1350 5.5 %
Rwork0.205 --
obs0.206 24528 60.1 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 159.79 Å2 / Biso mean: 56.3 Å2 / Biso min: 9.51 Å2
Refinement stepCycle: LAST / Resolution: 2.62→55.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4001 0 306 22 4329
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.62-2.710.350790.3648842
2.71-2.820.5357140.36042396
2.82-2.950.3488110.311951413
2.95-3.10.3223620.2984104227
3.1-3.30.2931440.2608209155
3.3-3.550.23942280.2408364795
3.55-3.910.25492430.20773823100
3.91-4.470.1972060.1813854100
4.47-5.630.21572250.17273883100
5.63-55.130.21612080.2096400199

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