[English] 日本語
Yorodumi
- EMDB-8898: Clostridioides difficileC toxinB with DLD-4 darpin -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-8898
TitleClostridioides difficileC toxinB with DLD-4 darpin
Map dataClostridioides difficileC toxinB with DLD-4 darpin
Sample
  • Complex: ternary complex of tcdB and DLD-4 darpin
    • Protein or peptide: Toxin B
    • Protein or peptide: DLD-4 darpin
KeywordsTOXIN-PROTEIN BINDING complex
Function / homology
Function and homology information


glucosyltransferase activity / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis ...glucosyltransferase activity / host cell cytosol / Transferases; Glycosyltransferases; Hexosyltransferases / cysteine-type peptidase activity / host cell endosome membrane / toxin activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / lipid binding / host cell plasma membrane / proteolysis / extracellular region / membrane / metal ion binding
Similarity search - Function
TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / Membrane Localization Domain / TcdA/TcdB catalytic glycosyltransferase domain / TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily ...TcdA/TcdB toxin, N-terminal helical domain / TcdB toxin N-terminal helical domain / Dermonecrotic/RTX toxin, membrane localization domain / TcdA/TcdB toxin, catalytic glycosyltransferase domain / Membrane Localization Domain / TcdA/TcdB catalytic glycosyltransferase domain / TcdA/TcdB toxin, pore forming domain / TcdA/TcdB pore forming domain / CGT/MARTX, cysteine protease (CPD) domain / CGT/MARTX, cysteine protease (CPD) domain superfamily / Peptidase C80 family / CGT/MARTX cysteine protease (CPD) domain profile. / Choline-binding repeat / Putative cell wall binding repeat / Cell wall/choline-binding repeat / Cell wall-binding repeat profile. / Nucleotide-diphospho-sugar transferases
Similarity search - Domain/homology
Biological speciesClostridioides difficile (bacteria) / Drosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsZhang J / Jiang M
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116787 United States
Welch FoundationA-1863 United States
CitationJournal: PLoS Biol / Year: 2019
Title: Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.
Authors: Rudo Simeon / Mengqiu Jiang / Ana M Chamoun-Emanuelli / Hua Yu / Yongrong Zhang / Ran Meng / Zeyu Peng / Joanita Jakana / Junjie Zhang / Hanping Feng / Zhilei Chen /
Abstract: Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted ...Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile-secreted exotoxins-toxin A (TcdA) and toxin B (TcdB)-that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4-1.4E and U3-bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and "kisses" the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the "native" conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti-C. difficile toxin therapeutics.
History
DepositionAug 21, 2017-
Header (metadata) releaseSep 13, 2017-
Map releaseAug 22, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.076
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.076
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6ar6
  • Surface level: 0.076
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8898.png

Downloads & links

-
Map

FileDownload / File: emd_8898.map.gz / Format: CCP4 / Size: 4.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClostridioides difficileC toxinB with DLD-4 darpin
Voxel sizeX=Y=Z: 3 Å
Density
Contour LevelBy AUTHOR: 0.076 / Movie #1: 0.076
Minimum - Maximum-0.2062523 - 0.40994412
Average (Standard dev.)0.001580446 (±0.014951671)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions108108108
Spacing108108108
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z108108108
origin x/y/z0.0000.0000.000
length x/y/z324.000324.000324.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-36
NX/NY/NZ528549
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS108108108
D min/max/mean-0.2060.4100.002

-
Supplemental data

-
Sample components

-
Entire : ternary complex of tcdB and DLD-4 darpin

EntireName: ternary complex of tcdB and DLD-4 darpin
Components
  • Complex: ternary complex of tcdB and DLD-4 darpin
    • Protein or peptide: Toxin B
    • Protein or peptide: DLD-4 darpin

-
Supramolecule #1: ternary complex of tcdB and DLD-4 darpin

SupramoleculeName: ternary complex of tcdB and DLD-4 darpin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridioides difficile (bacteria)

-
Macromolecule #1: Toxin B

MacromoleculeName: Toxin B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases
Source (natural)Organism: Clostridioides difficile (bacteria)
Molecular weightTheoretical: 235.481438 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: VNRKQLEKMA NVRFRTQEDE YVAILDALEE YHNMSENTVV EKYLKLKDIN SLTDIYIDTY KKSGRNKALK KFKEYLVTEV LELKNNNLT PVEKNLHFVW IGGQINDTAI NYINQWKDVN SDYNVNVFYD SNAFLINTLK KTVVESAIND TLESFRENLN D PRFDYNKF ...String:
VNRKQLEKMA NVRFRTQEDE YVAILDALEE YHNMSENTVV EKYLKLKDIN SLTDIYIDTY KKSGRNKALK KFKEYLVTEV LELKNNNLT PVEKNLHFVW IGGQINDTAI NYINQWKDVN SDYNVNVFYD SNAFLINTLK KTVVESAIND TLESFRENLN D PRFDYNKF FRKRMEIIYD KQKNFINYYK AQREENPELI IDDIVKTYLS NEYSKEIDEL NTYIEESLNK ITQNSGNDVR NF EEFKNGE SFNLYEQELV ERWNLAAASD ILRISALKEI GGMYLDVDML PGIQPDLFES IEKPSSVTVD FWEMTKLEAI MKY KEYIPE YTSEHFDMLD EEVQSSFESV LASKSDKSEI FSSLGDMEAS PLEVKIAFNS KGIINQGLIS VKDSYCSNLI VKQI ENRYK ILNNSLNPAI SEDNDFNTTT NTFIDSIMAE ANADNGRFMM ELGKYLRVGF FPDVKTTINL SGPEAYAAAY QDLLM FKEG SMNIHLIEAD LRNFEISKTN ISQSTEQEMA SLWSFDDARA KAQFEEYKRN YFEGSLGEDD NLDFSQNIVV DKEYLL EKI SSLARSSERG YIHYIVQLQG DKISYEAACN LFAKTPYDSV LFQKNIEDSE IAYYYNPGDG EIQEIDKYKI PSIISDR PK IKLTFIGHGK DEFNTDIFAG FDVDSLSTEI EAAIDLAKED ISPKSIEINL LGCNMFSYSI NVEETYPGKL LLKVKDKI S ELMPSISQDS IIVSANQYEV RINSEGRREL LDHSGEWINK EESIIKDISS KEYISFNPKE NKITVKSKNL PELSTLLQE IRNNSNSSDI ELEEKVMLTE CEINVISNID TQIVEERIEE AKNLTSDSIN YIKDEFKLIE SISDALCDLK QQNELEDSHF ISFEDISET DEGFSIRFIN KETGESIFVE TEKTIFSEYA NHITEEISKI KGTIFDTVNG KLVKKVNLDT THEVNTLNAA F FIQSLIEY NSSKESLSNL SVAMKVQVYA QLFSTGLNTI TDAAKVVELV STALDETIDL LPTLSEGLPI IATIIDGVSL GA AIKELSE TSDPLLRQEI EAKIGIMAVN LTTATTAIIT SSLGIASGFS ILLVPLAGIS AGIPSLVNNE LVLRDKATKV VDY FKHVSL VETEGVFTLL DDKIMMPQDD LVISEIDFNN NSIVLGKCEI WRMEGGSGHT VTDDIDHFFS APSITYREPH LSIY DVLEV QKEELDLSKD LMVLPNAPNR VFAWETGWTP GLRSLENDGT KLLDRIRDNY EGEFYWRYFA FIADALITTL KPRYE DTNI RINLDSNTRS FIVPIITTEY IREKLSYSFY GSGGTYALSL SQYNMGINIE LSESDVWIID VDNVVRDVTI ESDKIK KGD LIEGILSTLS IEENKIILNS HEINFSGEVN GSNGFVSLTF SILEGINAII EVDLLSKSYK LLISGELKIL MLNSNHI QQ KIDYIGFNSE LQKNIPYSFV DSEGKENGFI NGSTKEGLFV SELPDVVLIS KVYMDDSKPS FGYYSNNLKD VKVITKDN V NILTGYYLKD DIKISLSLTL QDEKTIKLNS VHLDESGVAE ILKFMNRKGN TNTSDSLMSF LESMNIKSIF VNFLQSNIK FILDANFIIS GTTSIGQFEF ICDENDNIQP YFIKFNTLET NYTLYVGNRQ NMIVEPNYDL DDSGDISSTV INFSQKYLYG IDSCVNKVV ISPNIYTDEI NITPVYETNN TYPEVIVLDA NYINEKINVN INDLSIRYVW SNDGNDFILM STSEENKVSQ V KIRFVNVF KDKTLANKLS FNFSDKQDVP VSEIILSFGL IYINDSLYYF KPPVNNLITG FVTVGDDKYY FNPINGGAAS IG ETIIDDK NYYFNQSGVL QTGVFSTEDG FKYFAPANTL DENLEGEAID FTGKLIIDEN IYYFDDNYRG AVEWKELDGE MHY FSPETG KAFKGLNQIG DYKYYFNSDG VMQKGFVSIN DNKHYFDDSG VMKVGYTEID GKHFYFAENG EMQIGVFNTE DGFK YFAHH NEDLGNEEGE EISYSGILNF NNKIYYFDDS FTAVVGWKDL EDGSKYYFDE DTAEAYILEH HH

UniProtKB: Toxin B, Toxin B

-
Macromolecule #2: DLD-4 darpin

MacromoleculeName: DLD-4 darpin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 34.299195 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDLGKKLLEA ARAGQDDEVR ILMANGADVN ADDRIGMTPL HLAAIGGHLE IVEVLLKNGA DVNADDVHGR TPLHLAAGRG HLEIVEVLH GADVNAPDRW GRTPLHLAAH HGHLEIVEVL LKYGADVNAQ DKFGKTAFDI SIDSGNEDLA EILQSSSEFG G GGSGGGGS ...String:
SDLGKKLLEA ARAGQDDEVR ILMANGADVN ADDRIGMTPL HLAAIGGHLE IVEVLLKNGA DVNADDVHGR TPLHLAAGRG HLEIVEVLH GADVNAPDRW GRTPLHLAAH HGHLEIVEVL LKYGADVNAQ DKFGKTAFDI SIDSGNEDLA EILQSSSEFG G GGSGGGGS GGGGSASDLG KKLLEAARAG QDDEVRILMA NGADVNATDH LGVTPLHLAA VLGHLEIVEV LLKHGADVNA YD ILGRTPL HLAAWRGHLE IVEVLLKYGA DVNADDTSGT TPLHLAAGEG HLEIVEVLLK YGADVNAQDK FGKTAFDISI DNG NEDLAE IL

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

-
Electron microscopy #1

Microscopy ID1
MicroscopeJEOL 3200FSC
Image recordingImage recording ID: 1 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

-
Electron microscopy #1~

Microscopy ID1
MicroscopeFEI TECNAI F20
Image recordingImage recording ID: 2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

+
Image processing

Image recording ID1
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 84420
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: ANGULAR RECONSTITUTION

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbjlvh1.pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more