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- PDB-7t3h: MicroED structure of Dynobactin -

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Basic information

Entry
Database: PDB / ID: 7t3h
TitleMicroED structure of Dynobactin
ComponentsTRP-ASN-SER-ASN-VAL-HIS-SER-TYR-ARG-PHE
KeywordsANTIBIOTIC / antibiotics / drug development / natural product / Bam A / MicroED
Biological speciesPhotorhabdus australis (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / Resolution: 1.05 Å
AuthorsYoo, B.-K. / Kaiser, J.T. / Rees, D.C. / Miller, R.D. / Iinishi, A. / Lewis, K. / Bowman, S.
Funding support2items
OrganizationGrant numberCountry
Swiss National Science Foundation177084
Swiss National Science Foundation187170
CitationJournal: Nat Microbiol / Year: 2022
Title: Computational identification of a systemic antibiotic for gram-negative bacteria.
Authors: Ryan D Miller / Akira Iinishi / Seyed Majed Modaresi / Byung-Kuk Yoo / Thomas D Curtis / Patrick J Lariviere / Libang Liang / Sangkeun Son / Samantha Nicolau / Rachel Bargabos / Madeleine ...Authors: Ryan D Miller / Akira Iinishi / Seyed Majed Modaresi / Byung-Kuk Yoo / Thomas D Curtis / Patrick J Lariviere / Libang Liang / Sangkeun Son / Samantha Nicolau / Rachel Bargabos / Madeleine Morrissette / Michael F Gates / Norman Pitt / Roman P Jakob / Parthasarathi Rath / Timm Maier / Andrey G Malyutin / Jens T Kaiser / Samantha Niles / Blake Karavas / Meghan Ghiglieri / Sarah E J Bowman / Douglas C Rees / Sebastian Hiller / Kim Lewis /
Abstract: Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an ...Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its β-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.
History
DepositionDec 7, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 19, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TRP-ASN-SER-ASN-VAL-HIS-SER-TYR-ARG-PHE


Theoretical massNumber of molelcules
Total (without water)1,3111
Polymers1,3111
Non-polymers00
Water1448
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)42.230, 9.730, 19.070
Angle α, β, γ (deg.)90.000, 112.000, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Protein/peptide TRP-ASN-SER-ASN-VAL-HIS-SER-TYR-ARG-PHE


Mass: 1311.405 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Photorhabdus australis (bacteria)
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Microcrystals / Type: CELL / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Photorhabdus australis (bacteria)
Buffer solutionpH: 8.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm
Image recordingElectron dose: 0.0165 e/Å2 / Film or detector model: FEI CETA (4k x 4k)
EM diffractionCamera length: 641 mm
EM diffraction shellResolution: 1.05→9.5 Å / Fourier space coverage: 98.6 % / Multiplicity: 6.96 / Num. of structure factors: 5002 / Phase residual: 15 °
EM diffraction statsFourier space coverage: 98 % / High resolution: 1.05 Å / Num. of intensities measured: 59418 / Num. of structure factors: 6485 / Phase error rejection criteria: NULL / Rmerge: 0.205
ReflectionHighest resolution: 0.9 Å / Num. obs: 3490 / % possible obs: 97.8 % / Redundancy: 17.162 % / Biso Wilson estimate: 11.208 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.205 / Rrim(I) all: 0.211 / Χ2: 0.849 / Net I/σ(I): 8.2 / Num. measured all: 59896 / Scaling rejects: 65
Reflection shell

Diffraction-ID: 1

Resolution (Å)Highest resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
1.05-1.0818.7190.6723.1648672652600.920.68998.1
1.08-1.1118.6930.4784.0445612462440.970.49199.2
1.11-1.1417.7080.4394.7243032462430.9760.45198.8
1.14-1.1718.8120.4145.2143082322290.9820.42498.7
1.17-1.2118.1210.415.5344942482480.9740.421100
1.21-1.2618.2190.3986.1738262132100.9770.40898.6
1.26-1.317.4830.3596.1335142042010.9840.36998.5
1.3-1.3618.1350.3766.6240262292220.9830.38696.9
1.36-1.4218.1620.3557.8734691941910.970.36598.5
1.42-1.4916.9380.3068.1930151821780.9810.31597.8
1.49-1.5716.7310.2859.0631121861860.9850.294100
1.57-1.6616.9840.26810.6331421881850.9760.27698.4
1.66-1.7715.7550.22811.2824421581550.9940.23598.1
1.77-1.9216.3120.19913.3725611611570.9870.20597.5
1.92-2.115.2390.17713.9920421351340.9790.18399.3
2.1-2.3515.0160.18114.2218771321250.9760.18794.7
2.35-2.7114.2520.15815.1916391181150.9910.16497.5
2.71-3.3214.670.14216.2814671051000.9930.14695.2
3.32-4.713.1250.13816.4194577720.9860.14393.5
4.78.1710.16112.7428650350.9770.1770

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
XSCALEdata scaling
SHELXrefinement
PDB_EXTRACT3.27data extraction
EM 3D crystal entity∠α: 90 ° / ∠β: 112 ° / ∠γ: 90 ° / A: 42.23 Å / B: 9.73 Å / C: 19.07 Å / Space group name: C2 / Space group num: 5
CTF correctionType: NONE
3D reconstructionResolution: 1.05 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 1.05→9.5 Å / Cross valid method: NONE
Details: Data was merged from 19 crystals of varying resolution limits up to 0.9A. Overall resolution limit was determined to be 1.05A and applied in the refinement program.
RfactorNum. reflection% reflection
all0.1545 72645 -
obs0.1294 4731 89.6 %
Displacement parametersBiso max: 64.25 Å2 / Biso mean: 13.9631 Å2 / Biso min: 5.55 Å2

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