+Open data
-Basic information
Entry | Database: PDB / ID: 8adi | ||||||
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Title | Cryo-EM structure of Darobactin 9 bound BAM complex | ||||||
Components |
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Keywords | STRUCTURAL PROTEIN / BAM Complex / Darobactin 9 | ||||||
Function / homology | Function and homology information Bam protein complex / Gram-negative-bacterium-type cell outer membrane assembly / protein insertion into membrane / cell outer membrane / protein-macromolecule adaptor activity / cell adhesion / response to antibiotic / cell surface / identical protein binding / membrane Similarity search - Function | ||||||
Biological species | Escherichia coli (E. coli) Escherichia coli K-12 (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||
Authors | Yuan, B. / Marlovits, T.C. | ||||||
Funding support | Germany, 1items
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Citation | Journal: Angew Chem Int Ed Engl / Year: 2023 Title: Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering. Authors: Carsten E Seyfert / Christoph Porten / Biao Yuan / Selina Deckarm / Fabian Panter / Chantal D Bader / Janetta Coetzee / Felix Deschner / Kamaleddin H M E Tehrani / Paul G Higgins / Harald ...Authors: Carsten E Seyfert / Christoph Porten / Biao Yuan / Selina Deckarm / Fabian Panter / Chantal D Bader / Janetta Coetzee / Felix Deschner / Kamaleddin H M E Tehrani / Paul G Higgins / Harald Seifert / Thomas C Marlovits / Jennifer Herrmann / Rolf Müller / Abstract: Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic ...Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8adi.cif.gz | 489.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8adi.ent.gz | 403.7 KB | Display | PDB format |
PDBx/mmJSON format | 8adi.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8adi_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8adi_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 8adi_validation.xml.gz | 56.2 KB | Display | |
Data in CIF | 8adi_validation.cif.gz | 83.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ad/8adi ftp://data.pdbj.org/pub/pdb/validation_reports/ad/8adi | HTTPS FTP |
-Related structure data
Related structure data | 15363MC 8adgC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Outer membrane protein assembly factor ... , 5 types, 5 molecules ABCDE
#1: Protein | Mass: 90918.711 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: bamA, yaeT, yzzN, yzzY, b0177, JW0172 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A940 |
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#2: Protein | Mass: 43478.750 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: bamB, yfgL, b2512, JW2496 / Production host: Escherichia coli (E. coli) / References: UniProt: P77774 |
#3: Protein | Mass: 37150.602 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: bamC, dapX, nlpB, b2477, JW2462 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A903 |
#4: Protein | Mass: 28133.678 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: bamD, yfiO, b2595, JW2577 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AC02 |
#5: Protein | Mass: 13657.521 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: bamE, smpA, b2617, JW2598 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A937 |
-Protein/peptide , 1 types, 1 molecules F
#6: Protein/peptide | Type: Peptide-like / Class: Antibiotic / Mass: 1010.083 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) / References: BIRD: PRD_002430 |
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-Details
Compound details | The native darobactin A producer Photorhabdus khanii HGB1456 shows very limited production under ...The native darobactin A producer Photorhabdus khanii HGB1456 shows very limited production under laboratory cultivation conditions. Hence scientists designed heterologous expression of the darobactin biosynthetic gene cluster (BGC) in Escherichia coli. Rational design of darA variant resulted in the production of new 'non-natural' darobactin derivatives. One of the non-natural compounds, darobactin 9, was more potent than darobactin A. |
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Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: E.coli BAM complex / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Escherichia coli (E. coli) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid type: Quantifoil R2/1 |
Vitrification | Cryogen name: ETHANE-PROPANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Image recording | Average exposure time: 3 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 |
EM imaging optics | Energyfilter slit width: 20 eV |
-Processing
Software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 406142 / Symmetry type: POINT |