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- EMDB-15786: Cryo-EM structure of apolipoprotein N-acyltransferase Lnt from E.... -

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Basic information

Entry
Database: EMDB / ID: EMD-15786
TitleCryo-EM structure of apolipoprotein N-acyltransferase Lnt from E. coli (Apo form)
Map datasharpened map
Sample
  • Organelle or cellular component: Apolipoprotein N-acyltransferase
    • Protein or peptide: Apolipoprotein N-acyltransferase
KeywordsLnt / apolipoprotein N-acyltransferase / bacterial lipoprotein / transferase / 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 / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
Apolipoprotein N-acyltransferase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsDegtjarik O / Smithers L / Boland C / Caffrey M / Shalev Benami M
Funding support Ireland, 2 items
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, 2022-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15786.png

Downloads & links

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Map

FileDownload / File: emd_15786.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 240 pix.
= 198.24 Å		0.83 Å/pix.
x 240 pix.
= 198.24 Å		0.83 Å/pix.
x 240 pix.
= 198.24 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.26
Minimum - Maximum-1.1532431 - 2.185187
Average (Standard dev.)0.0002456825 (±0.051937383)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 198.23999 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened map

Fileemd_15786_additional_1.map
Annotationunsharpened map
Projections & Slices
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Half map: half map

Fileemd_15786_half_map_1.map
Annotationhalf map
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Histogram

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Half map: half map

Fileemd_15786_half_map_2.map
Annotationhalf map
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Sample components

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Entire : Apolipoprotein N-acyltransferase

EntireName: Apolipoprotein N-acyltransferase
Components
  • Organelle or cellular component: Apolipoprotein N-acyltransferase
    • Protein or peptide: Apolipoprotein N-acyltransferase

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Supramolecule #1: Apolipoprotein N-acyltransferase

SupramoleculeName: Apolipoprotein N-acyltransferase / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Macromolecule #1: Apolipoprotein N-acyltransferase

MacromoleculeName: Apolipoprotein N-acyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Acyltransferases; Transferring groups other than aminoacyl groups
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 59.280766 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MAFASLIERQ RIRLLLALLF GACGTLAFSP YDVWPAAIIS LMGLQALTFN RRPLQSAAIG FCWGFGLFG SGINWVYVSI ATFGGMPGPV NIFLVVLLAA YLSLYTGLFA GVLSRLWPKT TWLRVAIAAP ALWQVTEFLR G WVLTGFPW ...String:
MGSSHHHHHH SSGLVPRGSH MAFASLIERQ RIRLLLALLF GACGTLAFSP YDVWPAAIIS LMGLQALTFN RRPLQSAAIG FCWGFGLFG SGINWVYVSI ATFGGMPGPV NIFLVVLLAA YLSLYTGLFA GVLSRLWPKT TWLRVAIAAP ALWQVTEFLR G WVLTGFPW LQFGYSQIDG PLKGLAPIMG VEAINFLLMM VSGLLALALV KRNWRPLVVA VVLFALPFPL RYIQWFTPQP EK TIQVSMV QGDIPQSLKW DEGQLLNTLK IYYNATAPLM GKSSLIIWPE SAITDLEINQ QPFLKALDGE LRDKGSSLVT GIV DARLNK QNRYDTYNTI ITLGKGAPYS YESADRYNKN HLVPFGEFVP LESILRPLAP FFDLPMSSFS RGPYIQPPLS ANGI ELTAA ICYEIILGEQ VRDNFRPDTD YLLTISNDAW FGKSIGPWQH FQMARMRALE LARPLLRSTN NGITAVIGPQ GEIQA MIPQ FTREVLTTNV TPTTGLTPYA RTGNWPLWVL TALFGFAAVL MSLRQRRK

UniProtKB: Apolipoprotein N-acyltransferase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration14 mg/mL
BufferpH: 6
Component:
ConcentrationName
20.0 mMSodium citrate
250.0 mMSodium chloride
0.001 %Lauryl Maltose Neopentyl Glycol (LMNG)
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 33.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5n6l

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 111235
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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