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- EMDB-0149: PTC3 holotoxin complex from Photorhabdus luminecens in prepore st... -

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Basic information

Entry
Database: EMDB / ID: EMD-0149
TitlePTC3 holotoxin complex from Photorhabdus luminecens in prepore state (TcdA1, TcdB2, TccC3)
Map dataPhotorhabdus luminescens ABC holotoxin Volume is filtered according to its local resolution
Sample
  • Complex: PTC3 holotoxin complex in prepore state (5 x TcdA1, 1 x TcdB2, 1 x TccC3)
    • Complex: TcdA1
      • Protein or peptide: TcdA1
    • Complex: TcdB2/TccC3
      • Protein or peptide: TcdB2,TccC3
Function / homology
Function and homology information


extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / ABC toxin, N-terminal domain / ABC toxin N-terminal region ...Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / ABC toxin, N-terminal domain / ABC toxin N-terminal region / TcA receptor binding domain / TcA receptor binding domain / Insecticidal toxin complex/plasmid virulence protein / Tc toxin complex TcA, C-terminal TcB-binding domain / Neuraminidase-like domain / Salmonella virulence plasmid 28.1kDa A protein / Tc toxin complex TcA C-terminal TcB-binding domain / Neuraminidase-like domain / Rhs repeat-associated core / Integrin alpha, N-terminal
Similarity search - Domain/homology
TccC3 / TcdB2 / TcdA1
Similarity search - Component
Biological speciesPhotorhabdus luminescens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsGatsogiannis C / Merino F / Roderer D / Balchin D / Schubert E / Kuhlee A / Hayer-Hartl M / Raunser S
CitationJournal: Nature / Year: 2018
Title: Tc toxin activation requires unfolding and refolding of a β-propeller.
Authors: Christos Gatsogiannis / Felipe Merino / Daniel Roderer / David Balchin / Evelyn Schubert / Anne Kuhlee / Manajit Hayer-Hartl / Stefan Raunser /
Abstract: Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB- ...Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB-TcC heterodimer functions as a cocoon that shields the toxic enzyme. Binding of the cocoon to the channel triggers opening of the cocoon and translocation of the toxic enzyme into the channel. Here we show in atomic detail how the assembly of the three components activates the toxin. We find that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding. The presence of the toxic enzyme inside the cocoon is essential for its subnanomolar binding affinity for the TcA subunit. The enzyme passes through a narrow negatively charged constriction site inside the cocoon, probably acting as an extruder that releases the unfolded protein with its C terminus first into the translocation channel.
History
DepositionJul 27, 2018-
Header (metadata) releaseSep 5, 2018-
Map releaseOct 3, 2018-
UpdateNov 14, 2018-
Current statusNov 14, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6h6e
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0149.png

Downloads & links

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Map

FileDownload / File: emd_0149.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhotorhabdus luminescens ABC holotoxin Volume is filtered according to its local resolution
Voxel sizeX=Y=Z: 1.14 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.13690102 - 0.12409625
Average (Standard dev.)0.00017070811 (±0.0030184903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 547.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z547.200547.200547.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.1370.1240.000

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Supplemental data

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Sample components

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Entire : PTC3 holotoxin complex in prepore state (5 x TcdA1, 1 x TcdB2, 1 ...

EntireName: PTC3 holotoxin complex in prepore state (5 x TcdA1, 1 x TcdB2, 1 x TccC3)
Components
  • Complex: PTC3 holotoxin complex in prepore state (5 x TcdA1, 1 x TcdB2, 1 x TccC3)
    • Complex: TcdA1
      • Protein or peptide: TcdA1
    • Complex: TcdB2/TccC3
      • Protein or peptide: TcdB2,TccC3

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Supramolecule #1: PTC3 holotoxin complex in prepore state (5 x TcdA1, 1 x TcdB2, 1 ...

SupramoleculeName: PTC3 holotoxin complex in prepore state (5 x TcdA1, 1 x TcdB2, 1 x TccC3)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #2: TcdA1

SupramoleculeName: TcdA1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: 5 TcdA1 subunits in the holotoxin complex
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: TcdB2/TccC3

SupramoleculeName: TcdB2/TccC3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 / Details: 1 TcdB2/TccC3 monomer in the holotoxin complex
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: TcdA1

MacromoleculeName: TcdA1 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightTheoretical: 283.229406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNESVKEIPD VLKSQCGFNC LTDISHSSFN EFRQQVSEHL SWSETHDLYH DAQQAQKDNR LYEARILKRA NPQLQNAVHL AILAPNAEL IGYNNQFSGR ASQYVAPGTV SSMFSPAAYL TELYREARNL HASDSVYYLD TRRPDLKSMA LSQQNMDIEL S TLSLSNEL ...String:
MNESVKEIPD VLKSQCGFNC LTDISHSSFN EFRQQVSEHL SWSETHDLYH DAQQAQKDNR LYEARILKRA NPQLQNAVHL AILAPNAEL IGYNNQFSGR ASQYVAPGTV SSMFSPAAYL TELYREARNL HASDSVYYLD TRRPDLKSMA LSQQNMDIEL S TLSLSNEL LLESIKTESK LENYTKVMEM LSTFRPSGAT PYHDAYENVR EVIQLQDPGL EQLNASPAIA GLMHQASLLG IN ASISPEL FNILTEEITE GNAEELYKKN FGNIEPASLA MPEYLKRYYN LSDEELSQFI GKASNFGQQE YSNNQLITPV VNS SDGTVK VYRITREYTT NAYQMDVELF PFGGENYRLD YKFKNFYNAS YLSIKLNDKR ELVRTEGAPQ VNIEYSANIT LNTA DISQP FEIGLTRVLP SGSWAYAAAK FTVEEYNQYS FLLKLNKAIR LSRATELSPT ILEGIVRSVN LQLDINTDVL GKVFL TKYY MQRYAIHAET ALILCNAPIS QRSYDNQPSQ FDRLFNTPLL NGQYFSTGDE EIDLNSGSTG DWRKTILKRA FNIDDV SLF RLLKITDHDN KDGKIKNNLK NLSNLYIGKL LADIHQLTID ELDLLLIAVG EGKTNLSAIS DKQLATLIRK LNTITSW LH TQKWSVFQLF IMTSTSYNKT LTPEIKNLLD TVYHGLQGFD KDKADLLHVM APYIAATLQL SSENVAHSVL LWADKLQP G DGAMTAEKFW DWLNTKYTPG SSEAVETQEH IVQYCQALAQ LEMVYHSTGI NENAFRLFVT KPEMFGAATG AAPAHDALS LIMLTRFADW VNALGEKASS VLAAFEANSL TAEQLADAMN LDANLLLQAS IQAQNHQHLP PVTPENAFSC WTSINTILQW VNVAQQLNV APQGVSALVG LDYIQSMKET PTYAQWENAA GVLTAGLNSQ QANTLHAFLD ESRSAALSTY YIRQVAKAAA A IKSRDDLY QYLLIDNQVS AAIKTTRIAE AIASIQLYVN RALENVEENA NSGVISRQFF IDWDKYNKRY STWAGVSQLV YY PENYIDP TMRIGQTKMM DALLQSVSQS QLNADTVEDA FMSYLTSFEQ VANLKVISAY HDNINNDQGL TYFIGLSETD AGE YYWRSV DHSKFNDGKF AANAWSEWHK IDCPINPYKS TIRPVIYKSR LYLLWLEQKE ITKQTGNSKD GYQTETDYRY ELKL AHIRY DGTWNTPITF DVNKKISELK LEKNRAPGLY CAGYQGEDTL LVMFYNQQDT LDSYKNASMQ GLYIFADMAS KDMTP EQSN VYRDNSYQQF DTNNVRRVNN RYAEDYEIPS SVSSRKDYGW GDYYLSMVYN GDIPTINYKA ASSDLKIYIS PKLRII HNG YEGQKRNQCN LMNKYGKLGD KFIVYTSLGV NPNNSSNKLM FYPVYQYSGN TSGLNQGRLL FHRDTTYPSK VEAWIPG AK RSLTNQNAAI GDDYATDSLN KPDDLKQYIF MTDSKGTATD VSGPVEINTA ISPAKVQIIV KAGGKEQTFT ADKDVSIQ P SPSFDEMNYQ FNALEIDGSG LNFINNSASI DVTFTAFAED GRKLGYESFS IPVTLKVSTD NALTLHHNEN GAQYMQWQS YRTRLNTLFA RQLVARATTG IDTILSMETQ NIQEPQLGKG FYATFVIPPY NLSTHGDERW FKLYIKHVVD NNSHIIYSGQ LTDTNINIT LFIPLDDVPL NQDYHAKVYM TFKKSPSDGT WWGPHFVRDD KGIVTINPKS ILTHFESVNV LNNISSEPMD F SGANSLYF WELFYYTPML VAQRLLHEQN FDEANRWLKY VWSPSGYIVH GQIQNYQWNV RPLLEDTSWN SDPLDSVDPD AV AQHDPMH YKVSTFMRTL DLLIARGDHA YRQLERDTLN EAKMWYMQAL HLLGDKPYLP LSTTWSDPRL DRAADITTQN AHD SAIVAL RQNIPTPAPL SLRSANTLTD LFLPQINEVM MNYWQTLAQR VYNLRHNLSI DGQPLYLPIY ATPADPKALL SAAV ATSQG GGKLPESFMS LWRFPHMLEN ARGMVSQLTQ FGSTLQNIIE RQDAEALNAL LQNQAAELIL TNLSIQDKTI EELDA EKTV LEKSKAGAQS RFDSYGKLYD ENINAGENQA MTLRASAAGL TTAVQASRLA GAAADLVPNI FGFAGGGSRW GAIAEA TGY VMEFSANVMN TEADKISQSE TYRRRRQEWE IQRNNAEAEL KQIDAQLKSL AVRREAAVLQ KTSLKTQQEQ TQSQLAF LQ RKFSNQALYN WLRGRLAAIY FQFYDLAVAR CLMAEQAYRW ELNDDSARFI KPGAWQGTYA GLLAGETLML SLAQMEDA H LKRDKRALEV ERTVSLAEVY AGLPKDNGPF SLAQEIDKLV SQGSGSAGSG NNNLAFGAGT DTKTSLQASV SFADLKIRE DYPASLGKIR RIKQISVTLP ALLGPYQDVQ AILSYGDKAG LANGCEALAV SHGMNDSGQF QLDFNDGKFL PFEGIAIDQG TLTLSFPNA SMPEKGKQAT MLKTLNDIIL HIRYTIK

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Macromolecule #2: TcdB2,TccC3

MacromoleculeName: TcdB2,TccC3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightTheoretical: 273.678656 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQNSQDFSIT ELSLPKGGGA ITGMGEALTP TGPDGMAALS LPLPISAGRG YAPAFTLNYN SGAGNSPFGL GWDCNVMTIR RRTHFGVPH YDETDTFLGP EGEVLVVADQ PRDESTLQGI NLGATFTVTG YRSRLESHFS RLEYWQPKTT GKTDFWLIYS P DGQVHLLG ...String:
MQNSQDFSIT ELSLPKGGGA ITGMGEALTP TGPDGMAALS LPLPISAGRG YAPAFTLNYN SGAGNSPFGL GWDCNVMTIR RRTHFGVPH YDETDTFLGP EGEVLVVADQ PRDESTLQGI NLGATFTVTG YRSRLESHFS RLEYWQPKTT GKTDFWLIYS P DGQVHLLG KSPQARISNP SQTTQTAQWL LEASVSSRGE QIYYQYRAED DTGCEADEIT HHLQATAQRY LHIVYYGNRT AS ETLPGLD GSAPSQADWL FYLVFDYGER SNNLKTPPAF STTGSWLCRQ DRFSRYEYGF EIRTRRLCRQ VLMYHHLQAL DSK ITEHNG PTLVSRLILN YDESAIASTL VFVRRVGHEQ DGNVVTLPPL ELAYQDFSPR HHAHWQPMDV LANFNAIQRW QLVD LKGEG LPGLLYQDKG AWWYRSAQRL GEIGSDAVTW EKMQPLSVIP SLQSNASLVD INGDGQLDWV ITGPGLRGYH SQRPD GSWT RFTPLNALPV EYTHPRAQLA DLMGAGLSDL VLIGPKSVRL YANTRDGFAK GKDVVQSGDI TLPVPGADPR KLVAFS DVL GSGQAHLVEV SATKVTCWPN LGRGRFGQPI TLPGFSQPAT EFNPAQVYLA DLDGSGPTDL IYVHTNRLDI FLNKSGN GF AEPVTLRFPE GLRFDHTCQL QMADVQGLGV ASLILSVPHM SPHHWRCDLT NMKPWLLNEM NNNMGVHHTL RYRSSSQF W LDEKAAALTT GQTPVCYLPF PIHTLWQTET EDEISGNKLV TTLRYARGAW DGREREFRGF GYVEQTDSHQ LAQGNAPER TPPALTKNWY ATGLPVIDNA LSTEYWRDDQ AFAGFSPRFT TWQDNKDVPL TPEDDNSRYW FNRALKGQLL RSELYGLDDS TNKHVPYTV TEFRSQVRRL QHTDSRYPVL WSSVVESRNY HYERIASDPQ CSQNITLSSD RFGQPLKQLS VQYPRRQQPA I NLYPDTLP DKLLANSYDD QQRQLRLTYQ QSSWHHLTNN TVRVLGLPDS TRSDIFTYGA ENVPAGGLNL ELLSDKNSLI AD DKPREYL GQQKTAYTDG QNTTPLQTPT RQALIAFTET TVFNQSTLSA FNGSIPSDKL STTLEQAGYQ QTNYLFPRTG EDK VWVAHH GYTDYGTAAQ FWRPQKQSNT QLTGKITLIW DANYCVVVQT RDAAGLTTSA KYDWRFLTPV QLTDINDNQH LITL DALGR PITLRFWGTE NGKMTGYSSP EKASFSPPSD VNAAIELKKP LPVAQCQVYA PESWMPVLSQ KTFNRLAEQD WQKLY NARI ITEDGRICTL AYRRWVQSQK AIPQLISLLN NGPRLPPHSL TLTTDRYDHD PEQQIRQQVV FSDGFGRLLQ AAARHE AGM ARQRNEDGSL IINVQHTENR WAVTGRTEYD NKGQPIRTYQ PYFLNDWRYV SNDSARQEKE AYADTHVYDP IGREIKV IT AKGWFRRTLF TPWFTVNEDE NDTAAEVKKV KMMKNIDPKL YQKTPTVSVY DNRGLIIRNI DFHRTTANGD PDTRITRH Q YDIHGHLNQS IDPRLYEAKQ TNNTIKPNFL WQYDLTGNPL CTESIDAGRT VTLNDIEGRP LLTVTATGVI QTRQYETSS LPGRLLSVAE QTPEEKTSRI TERLIWAGNT EAEKDHNLAG QCVRHYDTAG VTRLESLSLT GTVLSQSSQL LIDTQEANWT GDNETVWQN MLADDIYTTL STFDATGALL TQTDAKGNIQ RLAYDVAGQL NGSWLTLKGQ TEQVIIKSLT YSAAGQKLRE E HGNDVITE YSYEPETQRL IGIKTRRPSD TKVLQDLRYE YDPVGNVISI RNDAEATRFW HNQKVMPENT YTYDSLYQLI SA TGREMAN IGQQSHQFPS PALPSDNNTY TNYTRTYTYD RGGNLTKIQH SSPATQNNYT TNITVSNRSN RAVLSTLTED PAQ VDALFD AGGHQNTLIS GQNLNWNTRG ELQQVTLVKR DKGANDDREW YRYSGDGRRM LKINEQQASN NAQTQRVTYL PNLE LRLTQ NSTATTEDLQ VITVGEAGRA QVRVLHWESG KPEDIDNNQL RYSYDNLIGS SQLELDSEGQ IISEEEYYPY GGTAL WAAR NQTEASYKTI RYSGKERDAT GLYYYGYRYY QPWIGRWLSS DPAGTIDGLN LYRMVRNNPV TLLDPDGLMP TIAERI AAL KKNKVTDSAP SPANATNVAI NIRPPVAPKP SLPKASTSSQ PTTHPIGAAN IKPTTSGSSI VAPLSPVGNK STSEISL PE SAQSSSSSTT STNLQKKSFT LYRADNRSFE EMQSKFPEGF KAWTPLDTKM ARQFASIFIG QKDTSNLPKE TVKNISTW G AKPKLKDLSN YIKYTKDKST VWVSTAINTE AGGQSSGAPL HKIDMDLYEF AIDGQKLNPL PEGRTKNMVP SLLLDTPQI ETSSIIALNH GPVNDAEISF LTTIPLKNVK PHKR

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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: 8
Component:
ConcentrationFormula
20.0 mMTris
150.0 mMNaClSodium chloride
0.02 %Tween20
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 94 % / Instrument: GATAN CRYOPLUNGE 3 / Details: blot 3 sec before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number real images: 3068 / Average electron dose: 2.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 205336
CTF correctionSoftware - Name: SPHIRE
Startup modelType of model: EMDB MAP
EMDB ID:

2551

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPHIRE
Final 3D classificationSoftware - Name: SPHIRE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Number images used: 89148

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Atomic model buiding 1

Initial model
PDB IDChain

1vw1
PDB Unreleased entry

chain_id: A

4o9x

chain_id: A

1vw1
PDB Unreleased entry

chain_id: B

1vw1
PDB Unreleased entry

chain_id: C

1vw1
PDB Unreleased entry

chain_id: D

1vw1
PDB Unreleased entry

chain_id: E
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 68.03 / Target criteria: Model to Map FSC
Output model

PDB-6h6e:
PTC3 holotoxin complex from Photorhabdus luminecens in prepore state (TcdA1, TcdB2, TccC3)

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