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- EMDB-32215: Cryo-EM structure of Depo32, a Klebsiella phage depolymerase targ... -

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Basic information

Entry
Database: EMDB / ID: EMD-32215
TitleCryo-EM structure of Depo32, a Klebsiella phage depolymerase targets the K2 serotype K. pneumoniae
Map data
Sample
  • Complex: Tail spike protein Depo32
    • Protein or peptide: Depolymerase
KeywordsDepolymerase / Klebsiella pneumoniae / K2 capsular type / Capsular polysaccharides / LYASE
Function / homologybiological process involved in interaction with host / Pectin lyase fold/virulence factor / viral life cycle / virion component / Depolymerase
Function and homology information
Biological speciesKlebsiella phage 020009 (virus) / Klebsiella phage GH-K3 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.32 Å
AuthorsCai R / Ren Z
Funding support China, 3 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32072824 China
National Natural Science Foundation of China (NSFC)31872505 China
National Natural Science Foundation of China (NSFC)U19A2038 China
CitationJournal: Microbiol Spectr / Year: 2023
Title: Structural biology and functional features of phage-derived depolymerase Depo32 on with K2 serotype capsular polysaccharides.
Authors: Ruopeng Cai / Zhuolu Ren / Rihong Zhao / Yan Lu / Xinwu Wang / Zhimin Guo / Jinming Song / Wentao Xiang / Rui Du / Xiaokang Zhang / Wenyu Han / Heng Ru / Jingmin Gu /
Abstract: Hypervirulent with capsular polysaccharides (CPSs) causes severe nosocomial- and community-acquired infections. Phage-derived depolymerases can degrade CPSs from to attenuate bacterial virulence, ...Hypervirulent with capsular polysaccharides (CPSs) causes severe nosocomial- and community-acquired infections. Phage-derived depolymerases can degrade CPSs from to attenuate bacterial virulence, but their antimicrobial mechanisms and clinical potential are not well understood. In the present study, phage GH-K3-derived depolymerase Depo32 (encoded by gene ) was identified to exhibit high efficiency in specifically degrading the CPSs of K2 serotype . The cryo-electron microscopy structure of trimeric Depo32 at a resolution up to 2.32 Å revealed potential catalytic centers in the cleft of each of the two adjacent subunits. subjected to Depo32 became more sensitive to phagocytosis by RAW264.7 cells and activated the cells by the mitogen-activated protein kinase signaling pathway. In addition, intranasal inoculation with Depo32 (a single dose of 200 µg, 20 µg daily for 3 days, or in combination with gentamicin) rescued all C57BL/6J mice infected with a lethal dose of K7 without interference from its neutralizing antibody. In summary, this work elaborates on the mechanism by which Depo32 targets the degradation of K2 serotype CPSs and its potential as an antivirulence agent. IMPORTANCE Depolymerases specific to more than 20 serotypes of spp. have been identified, but most studies only evaluated the single-dose treatment of depolymerases with relatively simple clinical evaluation indices and did not reveal the anti-infection mechanism of these depolymerases in depth. On the basis of determining the biological characteristics, the structure of Depo32 was analyzed by cryo-electron microscopy, and the potential active center was further identified. In addition, the effects of Depo32 on macrophage phagocytosis, signaling pathway activation, and serum killing were revealed, and the efficacy of the depolymerase (single treatment, multiple treatments, or in combination with gentamicin) against acute pneumonia caused by was evaluated. Moreover, the roles of the active sites of Depo32 were also elucidated in the and studies. Therefore, through structural biology, cell biology, and experiments, this study demonstrated the mechanism by which Depo32 targets K2 serotype . infection.
History
DepositionNov 15, 2021-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32215.png

Downloads & links

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Map

FileDownload / File: emd_32215.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 256 pix.
= 278.272 Å		1.09 Å/pix.
x 256 pix.
= 278.272 Å		1.09 Å/pix.
x 256 pix.
= 278.272 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.087 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0136
Minimum - Maximum-0.10526239 - 0.2379665
Average (Standard dev.)-0.000010317065 (±0.006657631)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 278.272 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharpened by B-factor -50

Fileemd_32215_additional_1.map
Annotationsharpened by B-factor -50
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Half map: #2

Fileemd_32215_half_map_1.map
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Half map: #1

Fileemd_32215_half_map_2.map
Projections & Slices
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Sample components

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Entire : Tail spike protein Depo32

EntireName: Tail spike protein Depo32
Components
  • Complex: Tail spike protein Depo32
    • Protein or peptide: Depolymerase

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Supramolecule #1: Tail spike protein Depo32

SupramoleculeName: Tail spike protein Depo32 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Klebsiella phage 020009 (virus) / Strain: vB_KpnS_GH-K3
Molecular weightTheoretical: 300 KDa

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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: 98.645289 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MALYREGKAA MAADGTVTGT GTKWQSSLSL IRPGATIMFL SSPIQMAVVN KVVSDTEIKA ITTNGAVVAS TDYAILLSDS LTVDGLAQD VAETLRYYQS QETVIADAVE FFKEFDFESL QNLANQIKAD SEASESSAAA AAASESKAKT SEDNAKSSEN A AKNSEVAA ...String:
MALYREGKAA MAADGTVTGT GTKWQSSLSL IRPGATIMFL SSPIQMAVVN KVVSDTEIKA ITTNGAVVAS TDYAILLSDS LTVDGLAQD VAETLRYYQS QETVIADAVE FFKEFDFESL QNLANQIKAD SEASESSAAA AAASESKAKT SEDNAKSSEN A AKNSEVAA ETTRDQIQQI IDNAGDQSTL VVLAQPDGFD SIGRVSSFAA LRNLKPKKSG QHVLLTSYYD GWAAENKMPT GG GEFISSI GTATDDGGYI AAGPGYYWTR VVNNNSFTAE DFGCKTTATP PPNFNVLPAE LFDNTARMQA AFNLAISKSF KLN LSAGTY YFESSDTLRI TGPIHIEGRP GTVFYHNPSN KANPKTDAFM NISGCSMGRI SSINCFSNSY LGKGINFDRS VGDN RKLVL EHVYVDTFRW GFYVGEPECI NQIEFHSCRA QSNYFQGIFI ESFKEGQEYG HSAPVHFFNT ICNGNGPTSF ALGAT YKTT KNEYIKVMDS VNDVGCQAYF QGLSNVQYIG GQLSGHGSPR NTSLATITQC NSFIIYGTDL EDINGFTTDG TAITAD NID TIESNYLKDI SGAAIVVSSC LGFKIDSPHI FKIKTLSTIK LMNNTYNYEI GGFTPDEALK YNVWDANGLA TNRISGV IH PRLVNSRLGI NSVAFDNMSN KLDVSSLIHN ETSQIIGLTP STGSNVPHTR IMWSNGAMYS 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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMNH2C(CH2OH)3tris(hydroxymethyl)aminomethane
150.0 mMNaClsodium chloride
0.5 mMC9H15O6Ptris(2-carboxyethyl)phosphine
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 285.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot 3.5 to 4 seconds before plunging.
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 2.56 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 462388
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-7vyv:
Cryo-EM structure of Depo32, a Klebsiella phage depolymerase targets the K2 serotype K. pneumoniae

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