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- PDB-8b0m: Cryo-EM structure of apolipoprotein N-acyltransferase Lnt from E.... -

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Basic information

Entry
Database: PDB / ID: 8b0m
TitleCryo-EM structure of apolipoprotein N-acyltransferase Lnt from E. coli in complex with PE (C387S mutant)
ComponentsApolipoprotein N-acyltransferase
KeywordsTRANSFERASE / Lnt / apolipoprotein N-acyltransferase / bacterial lipoprotein / cryo-EM
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 / Apolipoprotein N-acyltransferase, N-terminal / Apolipoprotein N-acyltransferase N-terminal domain / Nitrilase/N-carbamoyl-D-aminoacid amidohydrolase / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase / 4-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHATIDYLETHANOLAMINE / Apolipoprotein N-acyltransferase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsDegtjarik, O. / Smithers, L. / Boland, C. / Caffrey, M. / Shalev Benami, 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
DepositionSep 7, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 12, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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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)59,9992
Polymers59,2651
Non-polymers7341
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 59264.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: lnt, cutE, b0657, JW0654 / Production host: Escherichia coli (E. coli)
References: UniProt: P23930, apolipoprotein N-acyltransferase
#2: Chemical ChemComp-PTY / PHOSPHATIDYLETHANOLAMINE


Mass: 734.039 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C40H80NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Apolipoprotein N-acyltransferase / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6
Buffer component
IDConc.NameBuffer-ID
120 mMSodium citrate1
2250 mMSodium chloride1
30.001 %Lauryl Maltose Neopentyl Glycol (LMNG)1
SpecimenConc.: 14 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 33 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123014 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 90.46 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00294128
ELECTRON MICROSCOPYf_angle_d0.48195632
ELECTRON MICROSCOPYf_chiral_restr0.0423634
ELECTRON MICROSCOPYf_plane_restr0.0048705
ELECTRON MICROSCOPYf_dihedral_angle_d10.44311484

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