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- EMDB-14149: CryoEM structure of mammalian AAP in complex with Meropenem -

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Basic information

Entry
Database: EMDB / ID: EMD-14149
TitleCryoEM structure of mammalian AAP in complex with Meropenem
Map data
Sample
  • Complex: Homotetramer of aclyaminoacyl-peptidase
    • Protein or peptide: Acylamino-acid-releasing enzymeAcylaminoacyl-peptidase
  • Ligand: (2S,3R,4S)-4-{[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3-yl]sulfanyl}-2-[(2S,3R)-3-hydroxy-1-oxobutan-2-yl]-3-methyl-3,4-dihydro-2H-pyrrole-5-carboxylic acid
Function / homology
Function and homology information


acylaminoacyl-peptidase / serine-type endopeptidase activity / proteolysis / cytoplasm
Similarity search - Function
Acylamino-acid-releasing enzyme, N-terminal domain / Acylamino-acid-releasing enzyme, N-terminal domain / Prolyl endopeptidase family serine active site. / Peptidase S9, serine active site / Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / Six-bladed beta-propeller, TolB-like / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Acylamino-acid-releasing enzyme
Similarity search - Component
Biological speciesSus scrofa domesticus (domestic pig) / domestic pig (domestic pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsKiss-Szeman AJ / Harmat V / Straner P / Jakli I / Menyhard KD / Masiulis S / Perczel A
Funding support Hungary, 5 items
OrganizationGrant numberCountry
European Regional Development FundVEKOP-2.3.2-16-2017-00014 Hungary
European Regional Development FundVEKOP-2.3.3-15-2017-00018 Hungary
Ministry of Human Capacities2018-1.2.1-NKP-2018-00005 Hungary
National Research Development and Innovation Office (NKFIH)Thematic Excellence Program 2019, Synth+ Hungary
National Research Development and Innovation Office (NKFIH)K116305 Hungary
CitationJournal: Chem Sci / Year: 2022
Title: A carbapenem antibiotic inhibiting a mammalian serine protease: structure of the acylaminoacyl peptidase-meropenem complex.
Authors: Anna J Kiss-Szemán / Luca Takács / Zoltán Orgován / Pál Stráner / Imre Jákli / Gitta Schlosser / Simonas Masiulis / Veronika Harmat / Dóra K Menyhárd / András Perczel /
Abstract: The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem ...The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem antibiotic. AAP is a modulator of the ubiquitin-proteasome degradation system and the site of a drug-drug interaction between the widely used antipsychotic, valproate and carbapenems. The active form of pAAP - a toroidal tetramer - binds four meropenem molecules covalently linked to the catalytic Ser587 of the serine-protease triad, in an acyl-enzyme state. AAP is hindered from fully processing the antibiotic by the displacement and protonation of His707 of the catalytic triad. We show that AAP is made susceptible to the association by its unusually sheltered active pockets and flexible catalytic triads, while the carbapenems possess sufficiently small substituents on their β-lactam rings to fit into the shallow substrate-specificity pocket of the enzyme.
History
DepositionJan 18, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateFeb 1, 2023-
Current statusFeb 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14149.png

Downloads & links

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Map

FileDownload / File: emd_14149.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 360 pix.
= 345.6 Å		0.96 Å/pix.
x 360 pix.
= 345.6 Å		0.96 Å/pix.
x 360 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.6614008 - 3.218018
Average (Standard dev.)0.00039692695 (±0.09741245)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Homotetramer of aclyaminoacyl-peptidase

EntireName: Homotetramer of aclyaminoacyl-peptidase
Components
  • Complex: Homotetramer of aclyaminoacyl-peptidase
    • Protein or peptide: Acylamino-acid-releasing enzymeAcylaminoacyl-peptidase
  • Ligand: (2S,3R,4S)-4-{[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3-yl]sulfanyl}-2-[(2S,3R)-3-hydroxy-1-oxobutan-2-yl]-3-methyl-3,4-dihydro-2H-pyrrole-5-carboxylic acid

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Supramolecule #1: Homotetramer of aclyaminoacyl-peptidase

SupramoleculeName: Homotetramer of aclyaminoacyl-peptidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Sus scrofa domesticus (domestic pig)

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Macromolecule #1: Acylamino-acid-releasing enzyme

MacromoleculeName: Acylamino-acid-releasing enzyme / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: domestic pig (domestic pig)
Molecular weightTheoretical: 81.324391 KDa
SequenceString: MERQVLLSEP EEAAALYRGL SRQPALSAAC LGPEVTTQYG GRYRTVHTEW TQRDLERMEN IRFCRQYLVF HDGDSVVFAG PAGNSVETR GELLSRESPS GTMKAVLRKA GGTGTAEEKQ FLEVWEKNRK LKSFNLSALE KHGPVYEDDC FGCLSWSHSE T HLLYVADK ...String:
MERQVLLSEP EEAAALYRGL SRQPALSAAC LGPEVTTQYG GRYRTVHTEW TQRDLERMEN IRFCRQYLVF HDGDSVVFAG PAGNSVETR GELLSRESPS GTMKAVLRKA GGTGTAEEKQ FLEVWEKNRK LKSFNLSALE KHGPVYEDDC FGCLSWSHSE T HLLYVADK KRPKAESFFQ TKALDVTGSD DEMARTKKPD QAIKGDQFLF YEDWGENMVS KSTPVLCVLD IESGNISVLE GV PESVSPG QAFWAPGDTG VVFVGWWHEP FRLGIRFCTN RRSALYYVDL TGGKCELLSD ESVAVTSPRL SPDQCRIVYL RFP SLVPHQ QCGQLCLYDW YTRVTSVVVD IVPRQLGEDF SGIYCSLLPL GCWSADSQRV VFDSPQRSRQ DLFAVDTQMG SVTS LTAGG SGGSWKLLTI DRDLMVVQFS TPSVPPSLKV GFLPPAGKEQ AVSWVSLEEA EPFPDISWSI RVLQPPPQQE HVQYA GLDF EAILLQPSNS PEKTQVPMVV MPHGGPHSSF VTAWMLFPAM LCKMGFAVLL VNYRGSTGFG QDSILSLPGN VGHQDV KDV QFAVEQVLQE EHFDAGRVAL MGGSHGGFLS CHLIGQYPET YSACVVRNPV INIASMMGST DIPDWCMVEA GFSYSSD CL PDLSVWAAML DKSPIKYAPQ VKTPLLLMLG QEDRRVPFKQ GMEYYRVLKA RNVPVRLLLY PKSTHALSEV EVESDSFM N AVLWLCTHLG S

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Macromolecule #2: (2S,3R,4S)-4-{[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3-yl]sulfa...

MacromoleculeName: (2S,3R,4S)-4-{[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3-yl]sulfanyl}-2-[(2S,3R)-3-hydroxy-1-oxobutan-2-yl]-3-methyl-3,4-dihydro-2H-pyrrole-5-carboxylic acid
type: ligand / ID: 2 / Number of copies: 4 / Formula: DWZ
Molecular weightTheoretical: 385.478 Da
Chemical component information

ChemComp-DWZ:
(2S,3R,4S)-4-{[(3S,5S)-5-(dimethylcarbamoyl)pyrrolidin-3-yl]sulfanyl}-2-[(2S,3R)-3-hydroxy-1-oxobutan-2-yl]-3-methyl-3,4-dihydro-2H-pyrrole-5-carboxylic acid / antibiotic*YM / Meropenem

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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

Concentration1 mg/mL
BufferpH: 7.5 / Component - Concentration: 10.0 mM / Component - Formula: C4H11NO3 / Component - Name: TRIS
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsCovalent complex with carbapenem type antibiotic Meropenem

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 4 / Number real images: 11625 / Average exposure time: 5.93 sec. / Average electron dose: 41.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.2.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 654863

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