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- EMDB-4068: Cryo-EM structure of the Tc toxin TcdA1 in its pore state -

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Basic information

Entry
Database: EMDB / ID: EMD-4068
TitleCryo-EM structure of the Tc toxin TcdA1 in its pore state
Map dataStructure of TcdA1 in pore state, determined in lipid nano disc (processed volume)
Sample
  • Complex: Structure of TcdA1 in pore state, determined in lipid nanodisc
    • Protein or peptide: TcdA1
Function / homology
Function and homology information


identical protein binding
Similarity search - Function
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
Similarity search - Domain/homology
Biological speciesPhotorhabdus luminescens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsGatsogiannis C / Merino F / Prumbaum D / Roderer D / Leidreiter F / Meusch D / Raunser S
Funding support Germany, 2 items
OrganizationGrant numberCountry
European Research Council615984 Germany
Max Planck Society Germany
CitationJournal: Nat Struct Mol Biol / Year: 2016
Title: Membrane insertion of a Tc toxin in near-atomic detail.
Authors: Christos Gatsogiannis / Felipe Merino / Daniel Prumbaum / Daniel Roderer / Franziska Leidreiter / Dominic Meusch / Stefan Raunser /
Abstract: Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual ...Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual injection system is poorly understood. Using electron cryomicroscopy, we determined the structure of TcdA1 from Photorhabdus luminescens embedded in lipid nanodiscs. In our structure, compared with the previous structure of TcdA1 in the prepore state, the transmembrane helices rearrange in the membrane and open the initially closed pore. However, the helices do not span the complete membrane; instead, the loops connecting the helices form the rim of the funnel. Lipid head groups reach into the space between the loops and consequently stabilize the pore conformation. The linker domain is folded and packed into a pocket formed by the other domains of the toxin, thereby considerably contributing to stabilization of the pore state.
History
DepositionJul 22, 2016-
Header (metadata) releaseAug 31, 2016-
Map releaseAug 31, 2016-
UpdateOct 23, 2019-
Current statusOct 23, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 6.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5lkh, PDB-5lki
  • Surface level: 6.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5lkh
  • Surface level: 6.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5lki
  • Surface level: 6.75
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5lkh
  • Imaged by Jmol
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5lki
  • Imaged by Jmol
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4068.png

Downloads & links

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Map

FileDownload / File: emd_4068.map.gz / Format: CCP4 / Size: 85.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of TcdA1 in pore state, determined in lipid nano disc (processed volume)
Voxel sizeX=Y=Z: 1.57 Å
Density
Contour LevelBy AUTHOR: 6.75 / Movie #1: 6.75
Minimum - Maximum-15.199429500000001 - 29.238513999999999
Average (Standard dev.)0.000000002739475 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions282282282
Spacing282282282
CellA=B=C: 442.74002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.571.571.57
M x/y/z282282282
origin x/y/z0.0000.0000.000
length x/y/z442.740442.740442.740
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS282282282
D min/max/mean-15.19929.2390.000

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Supplemental data

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Additional map: Structure of TcdA1 in pore state, determined in...

Fileemd_4068_additional.map
AnnotationStructure of TcdA1 in pore state, determined in lipid nano disc (unsharpened, unfiltered raw volume)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of TcdA1 in pore state, determined in lipid nanodisc

EntireName: Structure of TcdA1 in pore state, determined in lipid nanodisc
Components
  • Complex: Structure of TcdA1 in pore state, determined in lipid nanodisc
    • Protein or peptide: TcdA1

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Supramolecule #1: Structure of TcdA1 in pore state, determined in lipid nanodisc

SupramoleculeName: Structure of TcdA1 in pore state, determined in lipid nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET-19b
Molecular weightTheoretical: 1.4 MDa

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 11
Component:
ConcentrationFormula
20.0 mMCAPS
250.0 mMNaClSodium chloride
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: GATAN CRYOPLUNGE 3

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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 / Nominal magnification: 59000
Sample stageCooling holder cryogen: NITROGEN
DetailsCs corrected microscope
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number real images: 1957 / Average electron dose: 15.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 30061 / Details: particles were picked manually
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: OTHER
Details: initial model was obtained using VIPER (SPARX) from negative stain class averages
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION
Details: The density was filtered to its local resolution, using localfilt (SPARX) and RESMAP
Number images used: 13000

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-5lkh:
Cryo-EM structure of the Tc toxin TcdA1 in its pore state (obtained by flexible fitting)

PDB-5lki:
Cryo-EM structure of the Tc toxin TcdA1 in its pore state

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