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- EMDB-42956: CryoEM Structure of Diffocin - precontracted - Baseplate - focuse... -

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Basic information

Entry
Database: EMDB / ID: EMD-42956
TitleCryoEM Structure of Diffocin - precontracted - Baseplate - focused refinement on triplex region
Map data
Sample
  • Complex: Diffocin
    • Protein or peptide: TRI-2 (CD1371)
    • Protein or peptide: TRI-1 (CD1372)
    • Protein or peptide: Spike (CD1369)
    • Protein or peptide: Tape measure protein (CD1366)
    • Protein or peptide: Tube tail (CD1367)
    • Protein or peptide: Sheath initiator (CD1370)
    • Protein or peptide: Sheath (CD1363)
    • Protein or peptide: Tube (CD1364)
    • Protein or peptide: Hub-Hydrolase (CD1368)
KeywordsPhage tail-like / bacteriocin / baseplate / pre-contraction / VIRUS LIKE PARTICLE
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases) / cysteine-type peptidase activity
Similarity search - Function
Protein of unknown function, DUF2577 / Protein of unknown function (DUF2577) / : / YQBQ-like / Bacteriophage Mu-like, Gp48 / : / Bacteriophage Mu-like, Gp48 / : / Protein of unknown function DUF2634 / Contractile injection system sheath initiator ...Protein of unknown function, DUF2577 / Protein of unknown function (DUF2577) / : / YQBQ-like / Bacteriophage Mu-like, Gp48 / : / Bacteriophage Mu-like, Gp48 / : / Protein of unknown function DUF2634 / Contractile injection system sheath initiator / Phage tail tube protein / XkdM-like superfamily / Phage tail tube protein / Baseplate protein J-like / Baseplate J-like protein barrel domain / NlpC/P60 family / NlpC/P60 domain profile. / Endopeptidase, NLPC/P60 domain / Tail sheath protein, subtilisin-like domain / Phage tail sheath protein subtilisin-like domain / Tail sheath protein, C-terminal domain / Phage tail sheath C-terminal domain / Papain-like cysteine peptidase superfamily
Similarity search - Domain/homology
DUF2577 family protein / Phage tail tube protein / XkdP-like protein / DUF2634 domain-containing protein / Base plate protein / Base plate protein / C40 family peptidase / Phage tail sheath protein / Phage-related protein
Similarity search - Component
Biological speciesClostridioides difficile (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsCai XY / He Y / Zhou ZH
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM071940 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI094386 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI085318 United States
CitationJournal: Nat Commun / Year: 2024
Title: Atomic structures of a bacteriocin targeting Gram-positive bacteria.
Authors: Xiaoying Cai / Yao He / Iris Yu / Anthony Imani / Dean Scholl / Jeff F Miller / Z Hong Zhou /
Abstract: Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; ...Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics.
History
DepositionNov 28, 2023-
Header (metadata) releaseAug 28, 2024-
Map releaseAug 28, 2024-
UpdateAug 28, 2024-
Current statusAug 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42956.png

Downloads & links

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Map

FileDownload / File: emd_42956.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å		1.1 Å/pix.
x 300 pix.
= 330. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.10730729 - 0.19504143
Average (Standard dev.)0.0010434823 (±0.01174838)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42956_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_42956_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_42956_half_map_2.map
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Sample components

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Entire : Diffocin

EntireName: Diffocin
Components
  • Complex: Diffocin
    • Protein or peptide: TRI-2 (CD1371)
    • Protein or peptide: TRI-1 (CD1372)
    • Protein or peptide: Spike (CD1369)
    • Protein or peptide: Tape measure protein (CD1366)
    • Protein or peptide: Tube tail (CD1367)
    • Protein or peptide: Sheath initiator (CD1370)
    • Protein or peptide: Sheath (CD1363)
    • Protein or peptide: Tube (CD1364)
    • Protein or peptide: Hub-Hydrolase (CD1368)

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Supramolecule #1: Diffocin

SupramoleculeName: Diffocin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridioides difficile (bacteria)

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Macromolecule #1: TRI-2 (CD1371)

MacromoleculeName: TRI-2 (CD1371) / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 39.603281 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MYSDQTYEVI KNRTLENINL DIYKGEGSFL NNMVSGNNLE LSKIYLELSK MHKMAFIQDT YNQFLDKRVN EFGVYRKLGT ESNGEVEFI GEKGTVINNG TIISYRDLLF VVIKDVTIGS EEGDNSPVQA LEVGKKYNLP TNCEFKLVDN ISGVTKITNT R SFEGGTDI ...String:
MYSDQTYEVI KNRTLENINL DIYKGEGSFL NNMVSGNNLE LSKIYLELSK MHKMAFIQDT YNQFLDKRVN EFGVYRKLGT ESNGEVEFI GEKGTVINNG TIISYRDLLF VVIKDVTIGS EEGDNSPVQA LEVGKKYNLP TNCEFKLVDN ISGVTKITNT R SFEGGTDI ETDEELKERF YKIQRNQATS GNKAHYEEWA LEVDGVYNVK VYPRWDGPGT VKVLIFGKNN QAVDTETIER CQ QHIDEEK PIGPTITVVT PLPIEISISA VMKLEDGYTL DNVKESFLES INTYFRDIRG EIIYTKVMGI LINTTGVHDL SNL LINGST DNITINEDKI PSVTTVNFSE VENQ

UniProtKB: Base plate protein

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Macromolecule #2: TRI-1 (CD1372)

MacromoleculeName: TRI-1 (CD1372) / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 26.473488 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MKLIDKLPSF DRNYIVEEIQ GAYDTELNIL KEDIDDTFNQ LFVDTATWGL DMWEDILCIE KKELDFDTRR SNIKAKMRSR GTSTIEVIK SICEAYTKSE TDIKVYSDEF TFVLSFIANN CDYKTLLDCS DMIERVKPAH LLHYLEPIIL DKSMVYCGGG M VCSEEVKV ...String:
MKLIDKLPSF DRNYIVEEIQ GAYDTELNIL KEDIDDTFNQ LFVDTATWGL DMWEDILCIE KKELDFDTRR SNIKAKMRSR GTSTIEVIK SICEAYTKSE TDIKVYSDEF TFVLSFIANN CDYKTLLDCS DMIERVKPAH LLHYLEPIIL DKSMVYCGGG M VCSEEVKV HPYFEPIIKC SAVVNCGAGM ISREEIKVYP LSIKCIENNC KINIAIANDT GVENVVVYPK SEVV

UniProtKB: Base plate protein

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Macromolecule #3: Spike (CD1369)

MacromoleculeName: Spike (CD1369) / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 12.626413 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString:
MANPINEFIG IIREEGKYHN QPSFFIGKIK SKLPDLKIET NNIILEKEDI LIDSWMIDRQ LETFDTETNQ EHQHEVKNPF IDNFESGDM VIMFRIGEKF AVVSKLVSL

UniProtKB: DUF2577 family protein

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Macromolecule #4: Tape measure protein (CD1366)

MacromoleculeName: Tape measure protein (CD1366) / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 89.157289 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MGNVREEGIN MYLTDNYTPK MNQIISVTDN FRRATVAVSL STNVMASSIK NSIGSASNRV NSLNSSLRKV QTTASSVSST MTKLSSSIN AVSGVIGSLN GSIMRLAITI AMIIDYFNKL IQKKNEFNSN IMIILIFKAK SDEVEKTKNK LLGNLKKIGG K IWNIVIKA ...String:
MGNVREEGIN MYLTDNYTPK MNQIISVTDN FRRATVAVSL STNVMASSIK NSIGSASNRV NSLNSSLRKV QTTASSVSST MTKLSSSIN AVSGVIGSLN GSIMRLAITI AMIIDYFNKL IQKKNEFNSN IMIILIFKAK SDEVEKTKNK LLGNLKKIGG K IWNIVIKA KDMTKRVISS ILGKLKQVEK RPYQGSINLK DMVSSAMGRI LPKLMLFKNT FWSGVIAIKD MASGIISKVF PK LRLFAGK VWSGAIAVKD MASGILGSIK GKISDLTNGA TIGVAVKKGV DLLGQEQNQK VVLESVMKRN TGKVNQIDVD DYY GSLVRM ANDTPFDPED VVAMGTKAKM ISNITGGKKE KDITQAMVDV RALNMNTSSE QDVSAAFLSA AKGNMESLNT LVGE NYKTF DEALEGISVK QMGLAKEMSN TIPGIISGAQ TSINNGLKSI VKPFDDILGQ GLKKIKTFIE SGLGNLAGLS EKMAG KIGN VMNGKIIIGN KYDQMQSRSV KNGKEFSDST QYRISNEAEK RKMMVENKQE RFENHAATMI GNAPKAIVNA GSTLLQ NID FTALIDSLLP VVNLVNNLLD SINNKSPIAQ GLISIFGTIV TTAFQLIGPV VEAVSPIITR IFTFLGEYAP QINNFIE TL GVIWKTVWET LGPLLETGWK IIEPILGAFF NILDKVCKIV KDICKWWQTM INKIKNGSIT GTVLNLVEKS KKNYKDNP Y AGTKAGDSGK AYSGKKGNNA FGLNYVPYND YQTRLHEGEM VLTKQEANQY RSRKNGGNIN IAKLADTIVI REEADIEKI TSKLVASIQL AQLGGVL

UniProtKB: Phage-related protein

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Macromolecule #5: Tube tail (CD1367)

MacromoleculeName: Tube tail (CD1367) / type: protein_or_peptide / ID: 5 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 16.234616 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString:
MEMWLRQAED RFRFPVFPSS FSINGKAAVN SSSILKIGEI ATFGGVALKS ISISSFFPNK DYTFCDYTGF PSPYDCVNKI EKWMKEGFI LRFTITETNI NMEVIIEGFS YEERDGTRDV YFTLDLKEYK RIKIPKVTPK Q

UniProtKB: XkdP-like protein

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Macromolecule #6: Sheath initiator (CD1370)

MacromoleculeName: Sheath initiator (CD1370) / type: protein_or_peptide / ID: 6 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 16.549959 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString:
MSTIFPFIGV PEDYILPKTE ELPIFREVAW DFEKDEPILE KGDFKIIEKK EALKVWIYKC IKTNRYEHEI YSLEYGTELS ELIGQKYTK GLTESEASRF IKEALLINPY ILEVNVKSAN FNRDILSANV KVSTIYGEVE INV

UniProtKB: DUF2634 domain-containing protein

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Macromolecule #7: Sheath (CD1363)

MacromoleculeName: Sheath (CD1363) / type: protein_or_peptide / ID: 7 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 39.26843 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MAIGLPSINI SFKELATTVK ERSARGIIAM VLKDAKALGL NEIHEKEDIP VDLSAENKEY INLALMGNVN TPNKLLVYVI EGEADIQTA LDFLETKEFN YLCMPKAVEA DKTAIKNWII KLRDIDKVKV KAVLGKVVGN HEGIINFTTE DVLVGEKKYS V DEFTSRVA ...String:
MAIGLPSINI SFKELATTVK ERSARGIIAM VLKDAKALGL NEIHEKEDIP VDLSAENKEY INLALMGNVN TPNKLLVYVI EGEADIQTA LDFLETKEFN YLCMPKAVEA DKTAIKNWII KLRDIDKVKV KAVLGKVVGN HEGIINFTTE DVLVGEKKYS V DEFTSRVA GLIAGTPLSQ SVTYTKLSDV VDIPKMTKVD AESRVNKGEL ILIKEAGAIR IARGVNSLTE LTAEKGEMFQ KI KIVDTLD IIHSDIRKVI IDDYIGKVTN SYDNKCLLIV AIKSYLEELE KSALIESDST VEIDFEAQKS YLKSKGVDLS YMT LQEIKE ANTGSKVFLK AKIKVLDAME DIDLSIEI

UniProtKB: Phage tail sheath protein

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Macromolecule #8: Tube (CD1364)

MacromoleculeName: Tube (CD1364) / type: protein_or_peptide / ID: 8 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 16.028353 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString:
MANMEARNVM SGTWGELWLD GNKVAEVKKF QAKMEFTKED IIIAGQMGTD TKYMGYKGKG SITLYHVSSR MHKLIGEKIK RGSEPRFVA ISKLNDPDSY GAERIAVKNI AFDDLTLADW EVGVKGEIEA PFTFTEYDFL DII

UniProtKB: Phage tail tube protein

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Macromolecule #9: Hub-Hydrolase (CD1368)

MacromoleculeName: Hub-Hydrolase (CD1368) / type: protein_or_peptide / ID: 9 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 65.426082 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MIKIWVHIKN GSIYDITDIV DKVSWSGDYK SPSRTLEFSI IQSSFDNVFQ QIDIPIASTV CFYVDEKELF RGMIINRSKD SSSNEISFV SKDMGFLLTQ SEVSYNFKDK LVEDIAKQVF AENRLSVGII AKTNVKYTKM FIGVNGYDTI MSAYTEASKK T KKKYMIEA ...String:
MIKIWVHIKN GSIYDITDIV DKVSWSGDYK SPSRTLEFSI IQSSFDNVFQ QIDIPIASTV CFYVDEKELF RGMIINRSKD SSSNEISFV SKDMGFLLTQ SEVSYNFKDK LVEDIAKQVF AENRLSVGII AKTNVKYTKM FIGVNGYDTI MSAYTEASKK T KKKYMIEA NLDKFNVIEK GTVTLSVMFE EGFNIINTTF SESMENVKNK VIVVDQYGSK ISEKIDNEIF KEVNVIMQKV IQ QQENQDV DIDSEFNGIE KSCSLKGYGD VSCITGRGVK VKDSYTKLVG LFYIDTDKHT WQNGEYQIEL ELNFQNLMDE KSA GQDEPK EESNLGGEDY AGGKEFTAEF TAYCPRKEEG GDTDCRKKKL DPSKKTCAAP MVGKYEQTYY TKEFLNKHPL LNYG DEIQV ITGVSGRDGV YKVNDVGPAI TIEKNGTYHI DILFGNVEEA SKFGRRKGKI IIGGYSGNVS DKAKIVISEA KKHLG KPYK WGGNGPSSFD CSGLMVYCFK KVNVSLPRTS NQQSKKGKKV EQKNLQAGDL VFFHNPVSHV GLYIGNGEFL HAPQKG DVV KISKLSSRRD FNTARRVL

UniProtKB: C40 family peptidase

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 116539
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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