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- EMDB-15362: Cryo-EM structure of Darobactin 22 bound BAM complex -

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Basic information

Entry
Database: EMDB / ID: EMD-15362
TitleCryo-EM structure of Darobactin 22 bound BAM complex
Map data
Sample
  • Complex: E.coli BAM complex
    • Protein or peptide: Outer membrane protein assembly factor BamA
    • Protein or peptide: Outer membrane protein assembly factor BamB
    • Protein or peptide: Outer membrane protein assembly factor BamC
    • Protein or peptide: Outer membrane protein assembly factor BamD
    • Protein or peptide: Outer membrane protein assembly factor BamE
  • Protein or peptide: Darobactin 22
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 / membrane / identical protein binding
Similarity search - Function
Outer membrane protein assembly factor BamB / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamC, C-terminal / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane lipoprotein BamD-like / SmpA / OmlA family ...Outer membrane protein assembly factor BamB / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamC, C-terminal / NlpB/DapX lipoprotein / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamE / Lipoprotein SmpA/OmlA / Outer membrane lipoprotein BamD-like / SmpA / OmlA family / Outer membrane lipoprotein / BamE-like / Outer membrane protein assembly factor BamA / Pyrrolo-quinoline quinone repeat / PQQ-like domain / POTRA domain, BamA/TamA-like / Surface antigen variable number repeat / POTRA domain / POTRA domain profile. / Pyrrolo-quinoline quinone beta-propeller repeat / beta-propeller repeat / Surface antigen D15-like / Bacterial surface antigen (D15) / Omp85 superfamily domain / Quinoprotein alcohol dehydrogenase-like superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile. / Tetratricopeptide-like helical domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
Outer membrane protein assembly factor BamC / Outer membrane protein assembly factor BamE / Outer membrane protein assembly factor BamA / Outer membrane protein assembly factor BamD / Outer membrane protein assembly factor BamB
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli K-12 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsYuan B / Marlovits TC
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)INST152/772-1 Germany
CitationJournal: 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
DepositionJul 8, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15362.png

Downloads & links

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Map

FileDownload / File: emd_15362.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.)
0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-2.381543 - 3.0405667
Average (Standard dev.)0.0012008437 (±0.050703783)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15362_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15362_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E.coli BAM complex

EntireName: E.coli BAM complex
Components
  • Complex: E.coli BAM complex
    • Protein or peptide: Outer membrane protein assembly factor BamA
    • Protein or peptide: Outer membrane protein assembly factor BamB
    • Protein or peptide: Outer membrane protein assembly factor BamC
    • Protein or peptide: Outer membrane protein assembly factor BamD
    • Protein or peptide: Outer membrane protein assembly factor BamE
  • Protein or peptide: Darobactin 22

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Supramolecule #1: E.coli BAM complex

SupramoleculeName: E.coli BAM complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Outer membrane protein assembly factor BamA

MacromoleculeName: Outer membrane protein assembly factor BamA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 90.918711 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSMAMKKLL IASLLFSSAT VYGAEGFVVK DIHFEGLQRV AVGAALLSMP VRTGDTVNDE DISNTIRALF ATGNFEDVRV LRDGDTLLV QVKERPTIAS ITFSGNKSVK DDMLKQNLEA SGVRVGESLD RTTIADIEKG LEDFYYSVGK YSASVKAVVT P LPRNRVDL ...String:
MGSMAMKKLL IASLLFSSAT VYGAEGFVVK DIHFEGLQRV AVGAALLSMP VRTGDTVNDE DISNTIRALF ATGNFEDVRV LRDGDTLLV QVKERPTIAS ITFSGNKSVK DDMLKQNLEA SGVRVGESLD RTTIADIEKG LEDFYYSVGK YSASVKAVVT P LPRNRVDL KLVFQEGVSA EIQQINIVGN HAFTTDELIS HFQLRDEVPW WNVVGDRKYQ KQKLAGDLET LRSYYLDRGY AR FNIDSTQ VSLTPDKKGI YVTVNITEGD QYKLSGVEVS GNLAGHSAEI EQLTKIEPGE LYNGTKVTKM EDDIKKLLGR YGY AYPRVQ SMPEINDADK TVKLRVNVDA GNRFYVRKIR FEGNDTSKDA VLRREMRQME GAWLGSDLVD QGKERLNRLG FFET VDTDT QRVPGSPDQV DVVYKVKERN TGSFNFGIGY GTESGVSFQA GVQQDNWLGT GYAVGINGTK NDYQTYAELS VTNPY FTVD GVSLGGRLFY NDFQADDADL SDYTNKSYGT DVTLGFPINE YNSLRAGLGY VHNSLSNMQP QVAMWRYLYS MGEHPS TSD QDNSFKTDDF TFNYGWTYNK LDRGYFPTDG SRVNLTGKVT IPGSDNEYYK VTLDTATYVP IDDDHKWVVL GRTRWGY GD GLGGKEMPFY ENFYAGGSST VRGFQSNTIG PKAVYFPHQA SNYDPDYDYE CATQDGAKDL CKSDDAVGGN AMAVASLE F ITPTPFISDK YANSVRTSFF WDMGTVWDTN WDSSQYSGYP DYSDPSNIRM SAGIALQWMS PLGPLVFSYA QPFKKYDGD KAEQFQFNIG KTW

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Macromolecule #2: Outer membrane protein assembly factor BamB

MacromoleculeName: Outer membrane protein assembly factor BamB / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 43.47875 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSMQLRKLL LPGLLSVTLL SGCSLFNSEE DVVKMSPLPT VENQFTPTTA WSTSVGSGIG NFYSNLHPAL ADNVVYAADR AGLVKALNA DDGKEIWSVS LAEKDGWFSK EPALLSGGVT VSGGHVYIGS EKAQVYALNT SDGTVAWQTK VAGEALSRPV V SDGLVLIH ...String:
MGSMQLRKLL LPGLLSVTLL SGCSLFNSEE DVVKMSPLPT VENQFTPTTA WSTSVGSGIG NFYSNLHPAL ADNVVYAADR AGLVKALNA DDGKEIWSVS LAEKDGWFSK EPALLSGGVT VSGGHVYIGS EKAQVYALNT SDGTVAWQTK VAGEALSRPV V SDGLVLIH TSNGQLQALN EADGAVKWTV NLDMPSLSLR GESAPTTAFG AAVVGGDNGR VSAVLMEQGQ MIWQQRISQA TG STEIDRL SDVDTTPVVV NGVVFALAYN GNLTALDLRS GQIMWKRELG SVNDFIVDGN RIYLVDQNDR VMALTIDGGV TLW TQSDLL HRLLTSPVLY NGNLVVGDSE GYLHWINVED GRFVAQQKVD SSGFQTEPVA ADGKLLIQAK DGTVYSITRK LWSH PQFEK

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Macromolecule #3: Outer membrane protein assembly factor BamC

MacromoleculeName: Outer membrane protein assembly factor BamC / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 37.150602 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSMAYSVQK SRLAKVAGVS LVLLLAACSS DSRYKRQVSG DEAYLEAAPL AELHAPAGMI LPVTSGDYAI PVTNGSGAVG KALDIRPPA QPLALVSGAR TQFTGDTASL LVENGRGNTL WPQVVSVLQA KNYTITQRDD AGQTLTTDWV QWNRLDEDEQ Y RGRYQISV ...String:
MGSMAYSVQK SRLAKVAGVS LVLLLAACSS DSRYKRQVSG DEAYLEAAPL AELHAPAGMI LPVTSGDYAI PVTNGSGAVG KALDIRPPA QPLALVSGAR TQFTGDTASL LVENGRGNTL WPQVVSVLQA KNYTITQRDD AGQTLTTDWV QWNRLDEDEQ Y RGRYQISV KPQGYQQAVT VKLLNLEQAG KPVADAASMQ RYSTEMMNVI SAGLDKSATD AANAAQNRAS TTMDVQSAAD DT GLPMLVV RGPFNVVWQR LPAALEKVGM KVTDSTRSQG NMAVTYKPLS DSDWQELGAS DPGLASGDYK LQVGDLDNRS SLQ FIDPKG HTLTQSQNDA LVAVFQAAFS K

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Macromolecule #4: Outer membrane protein assembly factor BamD

MacromoleculeName: Outer membrane protein assembly factor BamD / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightTheoretical: 28.133678 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSMTRMKYL VAAATLSLFL AGCSGSKEEV PDNPPNEIYA TAQQKLQDGN WRQAITQLEA LDNRYPFGPY SQQVQLDLIY AYYKNADLP LAQAAIDRFI RLNPTHPNID YVMYMRGLTN MALDDSALQG FFGVDRSDRD PQHARAAFSD FSKLVRGYPN S QYTTDATK ...String:
MGSMTRMKYL VAAATLSLFL AGCSGSKEEV PDNPPNEIYA TAQQKLQDGN WRQAITQLEA LDNRYPFGPY SQQVQLDLIY AYYKNADLP LAQAAIDRFI RLNPTHPNID YVMYMRGLTN MALDDSALQG FFGVDRSDRD PQHARAAFSD FSKLVRGYPN S QYTTDATK RLVFLKDRLA KYEYSVAEYY TERGAWVAVV NRVEGMLRDY PDTQATRDAL PLMENAYRQM QMNAQAEKVA KI IAANSSN T

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Macromolecule #5: Outer membrane protein assembly factor BamE

MacromoleculeName: Outer membrane protein assembly factor BamE / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Molecular weightTheoretical: 13.657521 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MGSMRCKTLT AAAAVLLMLT AGCSTLERVV YRPDINQGNY LTANDVSKIR VGMTQQQVAY ALGTPLMSDP FGTNTWFYVF RQQPGHEGV TQQTLTLTFN SSGVLTNIDN KPALSGNKLH HHHHH

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Macromolecule #6: Darobactin 22

MacromoleculeName: Darobactin 22 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.094225 KDa
SequenceString:
WN(UX8)TKRW

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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

Concentration5.0 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
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:

7nri

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 499389
FSC plot (resolution estimation)

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