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- EMDB-23608: DpK2 bacteriophage tail spike depolymerase -

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Basic information

Entry
Database: EMDB / ID: EMD-23608
TitleDpK2 bacteriophage tail spike depolymerase
Map datapostprocessed map, C3 point group symmetry
Sample
  • Complex: DpK2 depolymerase tail spike complex
    • Protein or peptide: Depolymerase
Keywordsbateriophage tail spike / depolymerase / klebsiella targeting / SUGAR BINDING PROTEIN
Function / homologybiological process involved in interaction with host / Pectin lyase fold/virulence factor / viral life cycle / virion component / Depolymerase
Function and homology information
Biological speciesBacteriophage sp. (virus) / Klebsiella phage GH-K3 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBelousoff MJ / Bamert RS
Funding support Australia, 1 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1092262 Australia
CitationJournal: Microbiol Spectr / Year: 2021
Title: Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.
Authors: Rhys A Dunstan / Rebecca S Bamert / Matthew J Belousoff / Francesca L Short / Christopher K Barlow / Derek J Pickard / Jonathan J Wilksch / Ralf B Schittenhelm / Richard A Strugnell / Gordon ...Authors: Rhys A Dunstan / Rebecca S Bamert / Matthew J Belousoff / Francesca L Short / Christopher K Barlow / Derek J Pickard / Jonathan J Wilksch / Ralf B Schittenhelm / Richard A Strugnell / Gordon Dougan / Trevor Lithgow /
Abstract: The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages ...The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.
History
DepositionMar 10, 2021-
Header (metadata) releaseAug 25, 2021-
Map releaseAug 25, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lzj
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23608.png

Downloads & links

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Map

FileDownload / File: emd_23608.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocessed map, C3 point group symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 320 pix.
= 286.4 Å		0.9 Å/pix.
x 320 pix.
= 286.4 Å		0.9 Å/pix.
x 320 pix.
= 286.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.895 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.20291762 - 0.32955217
Average (Standard dev.)-0.000075933756 (±0.009701162)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 286.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8950.8950.895
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z286.400286.400286.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.2030.330-0.000

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Supplemental data

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Additional map: local resolution filtered map, C3 point group symmetry

Fileemd_23608_additional_1.map
Annotationlocal resolution filtered map, C3 point group symmetry
Projections & Slices
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Additional map: unprocessed map, C3 point group symmetry

Fileemd_23608_additional_2.map
Annotationunprocessed map, C3 point group symmetry
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Additional map: post-processed map, C1 point group symmetry

Fileemd_23608_additional_3.map
Annotationpost-processed map, C1 point group symmetry
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Additional map: unprocessed map, C1 point group symmetry

Fileemd_23608_additional_4.map
Annotationunprocessed map, C1 point group symmetry
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1, C3 point group symmetry

Fileemd_23608_half_map_1.map
Annotationhalf map 1, C3 point group symmetry
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2, C3 point group symmetry

Fileemd_23608_half_map_2.map
Annotationhalf map 2, C3 point group symmetry
Projections & Slices
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Sample components

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Entire : DpK2 depolymerase tail spike complex

EntireName: DpK2 depolymerase tail spike complex
Components
  • Complex: DpK2 depolymerase tail spike complex
    • Protein or peptide: Depolymerase

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Supramolecule #1: DpK2 depolymerase tail spike complex

SupramoleculeName: DpK2 depolymerase tail spike complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacteriophage sp. (virus)

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Macromolecule #1: Depolymerase

MacromoleculeName: Depolymerase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella phage GH-K3 (virus)
Molecular weightTheoretical: 98.437008 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MALYREGKAA MAADGTVTGT GTKWQSSLSL IRPGATIMFL SSPIQMAVVN KVVSDTEIKA ITTSGAVVAS TDYAILLSDS LTVDGLAQD VAETLRHYQS QETVIADAVE FFKSFDFDSL QNLANQIKAD SESAESSAAA AAASESKAKT SEDNAKSSEN A AKNSEVAA ...String:
MALYREGKAA MAADGTVTGT GTKWQSSLSL IRPGATIMFL SSPIQMAVVN KVVSDTEIKA ITTSGAVVAS TDYAILLSDS LTVDGLAQD VAETLRHYQS QETVIADAVE FFKSFDFDSL QNLANQIKAD SESAESSAAA AAASESKAKT SEDNAKSSEN A AKNSEVAA ETTRDQIQQI IDNAGDQSTL VVLAQPDGFD SIGRVSSFAA LRNLKPKKSG QHVLLTSYYD GWAAENKMPT GG GEFISSI GTATDDGGYI AAGPGYYWTR VVNNNSFTAE DFGCKTTATP PPNFNVLPAE LFDNTAMMQA AFNLAISKSF KLN LSTGTY YFESSDTLRI TGPIHIEGRP GTVFYHNPSN KANPKTDAFM NISGCSAGRI SSINCFSNSY LGKGINFDRS VGDN RKLVL EHVYVDTFRW GFYVGEPECI NQIEFHSCRA QSNYFQGIFI ESFKEGQEYG HSAPVHFFNT ICNGNGPTSF ALGAT YKTT KNEYIKVMDS VNDVGCQAYF QGLSNVQYIG GQLSGHGSPR NTSLATITQC NSFIIYGTDL EDINGFTTDG TAITAD NID AIESNYLKDI SGAAIVVSSC PGFKIDSPHI FKIKTLSTIK LMNNTYNYEI GGFTPDEALK YNVWDANGLA TNRISGV IH PRLVNSRLGI NSVAFDNMSN KLDVSSLIHN ETSQIVGLTP STGSNVPHTR KMWSNGAMYS STDLNNGFRL NYLSNHNE P LTPMHLYNEF SVSEFGGSVT ESNALDEIKY IFIQTTYANS GDGRFIIQAL DASGSVLSSN WYSPQSFNST FPISGFVRF DVPTGAKKIR YGFVNSANYT GSLRSHFMSG FAYNKRFFLK IYAVYNDLGR YGQFEPPYSV AIDRFRVGDN TTQMPSIPAS SATDVAGVN EVINSLLASL KANGFMSS

UniProtKB: Depolymerase

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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

Concentration0.4 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 331000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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