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- EMDB-11522: Fully-loaded anthrax lethal toxin in its heptameric pre-pore stat... -

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Basic information

Entry
Database: EMDB / ID: EMD-11522
TitleFully-loaded anthrax lethal toxin in its heptameric pre-pore state, in which the third lethal factor is masked out (PA7LF3-masked)
Map dataPA7LF3 complex, with the third lethal factor (3-LF) masked out.
Sample
  • Complex: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state
    • Complex: Protective antigen
      • Protein or peptide: Protective antigen
    • Complex: Lethal factor
      • Protein or peptide: Lethal factor
Keywordsanthrax lethal toxin / fully-loaded pre-pore state / membrane translocase / cytotoxic substrate / TOXIN
Function / homology
Function and homology information


anthrax lethal factor endopeptidase / positive regulation of apoptotic process in another organism / host cell cytosol / Uptake and function of anthrax toxins / negative regulation of MAPK cascade / host cell endosome membrane / protein homooligomerization / metalloendopeptidase activity / metallopeptidase activity / toxin activity ...anthrax lethal factor endopeptidase / positive regulation of apoptotic process in another organism / host cell cytosol / Uptake and function of anthrax toxins / negative regulation of MAPK cascade / host cell endosome membrane / protein homooligomerization / metalloendopeptidase activity / metallopeptidase activity / toxin activity / host cell plasma membrane / proteolysis / zinc ion binding / extracellular region / identical protein binding / membrane / metal ion binding
Similarity search - Function
Anthrax toxin lethal factor, central domain / Anthrax toxin lethal factor, middle domain / Protective antigen domain 4 / : / Anthrax protective antigen, immunoglobulin-like domain / Anthrax toxin, lethal/endema factor / Anthrax toxin, lethal/endema factor, N-/C-terminal / : / Anthrax toxin lethal factor, N- and C-terminal domain / Anthrax toxin lethal factor (ATLF)-like domain profile. ...Anthrax toxin lethal factor, central domain / Anthrax toxin lethal factor, middle domain / Protective antigen domain 4 / : / Anthrax protective antigen, immunoglobulin-like domain / Anthrax toxin, lethal/endema factor / Anthrax toxin, lethal/endema factor, N-/C-terminal / : / Anthrax toxin lethal factor, N- and C-terminal domain / Anthrax toxin lethal factor (ATLF)-like domain profile. / Bacterial exotoxin B / Protective antigen, heptamerisation domain / Protective antigen, Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA, domain 3 / Protective antigen, heptamerisation domain superfamily / Clostridial binary toxin B/anthrax toxin PA Ca-binding domain / Clostridial binary toxin B/anthrax toxin PA domain 2 / Clostridial binary toxin B/anthrax toxin PA domain 3 / PA14/GLEYA domain / PA14 domain profile. / PA14 domain / PA14 / PA14 domain / Metallopeptidase, catalytic domain superfamily / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Protective antigen / Lethal factor / Protective antigen
Similarity search - Component
Biological speciesBacillus anthracis (anthrax bacterium)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsQuentin D / Antoni C
Funding support Germany, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)615984 Germany
CitationJournal: PLoS Pathog / Year: 2020
Title: Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state.
Authors: Claudia Antoni / Dennis Quentin / Alexander E Lang / Klaus Aktories / Christos Gatsogiannis / Stefan Raunser /
Abstract: Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA) ...Anthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the relative position of the N-terminal α-helices in the three LFs determines which factor is translocated first.
History
DepositionJul 29, 2020-
Header (metadata) releaseSep 2, 2020-
Map releaseSep 2, 2020-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zxj
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11522.png

Downloads & links

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Map

FileDownload / File: emd_11522.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPA7LF3 complex, with the third lethal factor (3-LF) masked out.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 336 pix.
= 359.52 Å		1.07 Å/pix.
x 336 pix.
= 359.52 Å		1.07 Å/pix.
x 336 pix.
= 359.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-1.08765 - 1.9266713
Average (Standard dev.)0.0011153694 (±0.031776376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 359.52002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z336336336
origin x/y/z0.0000.0000.000
length x/y/z359.520359.520359.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS336336336
D min/max/mean-1.0881.9270.001

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Supplemental data

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Sample components

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Entire : Fully-loaded anthrax lethal toxin in its heptameric pre-pore state

EntireName: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state
Components
  • Complex: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state
    • Complex: Protective antigen
      • Protein or peptide: Protective antigen
    • Complex: Lethal factor
      • Protein or peptide: Lethal factor

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Supramolecule #1: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state

SupramoleculeName: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Heptameric pre-pores of proteolytically-acitivated protective antigen were loaded with excess of LFs to create the PA7LF3 complexes. The flexible third LF is masked out in the final ...Details: Heptameric pre-pores of proteolytically-acitivated protective antigen were loaded with excess of LFs to create the PA7LF3 complexes. The flexible third LF is masked out in the final reconstruction to boost the resolution of the rest of the complex.
Molecular weightTheoretical: 93 KDa

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Supramolecule #2: Protective antigen

SupramoleculeName: Protective antigen / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: The trypsin-activated 63 kDa fragments assemble into a hepatameric pre-pore
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)

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Supramolecule #3: Lethal factor

SupramoleculeName: Lethal factor / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Details: Three LF molecules crown the heptameric PA ring. The density corresponding to the third LF is masked out in the reconstruction.
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)

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Macromolecule #1: Protective antigen

MacromoleculeName: Protective antigen / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Molecular weightTheoretical: 85.67993 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGHHHHHHHH HHSSGHIDDD DKHMEVKQEN RLLNESESSS QGLLGYYFSD LNFQAPMVVT SSTTGDLSIP SSELENIPSE NQYFQSAIW SGFIKVKKSD EYTFATSADN HVTMWVDDQE VINKASNSNK IRLEKGRLYQ IKIQYQRENP TEKGLDFKLY W TDSQNKKE ...String:
MGHHHHHHHH HHSSGHIDDD DKHMEVKQEN RLLNESESSS QGLLGYYFSD LNFQAPMVVT SSTTGDLSIP SSELENIPSE NQYFQSAIW SGFIKVKKSD EYTFATSADN HVTMWVDDQE VINKASNSNK IRLEKGRLYQ IKIQYQRENP TEKGLDFKLY W TDSQNKKE VISSDNLQLP ELKQKSSNSR KKRSTSAGPT VPDRDNDGIP DSLEVEGYTV DVKNKRTFLS PWISNIHEKK GL TKYKSSP EKWSTASDPY SDFEKVTGRI DKNVSPEARH PLVAAYPIVH VDMENIILSK NEDQSTQNTD SQTRTISKNT STS RTHTSE VHGNAEVHAS FFDIGGSVSA GFSNSNSSTV AIDHSLSLAG ERTWAETMGL NTADTARLNA NIRYVNTGTA PIYN VLPTT SLVLGKNQTL ATIKAKENQL SQILAPNNYY PSKNLAPIAL NAQDDFSSTP ITMNYNQFLE LEKTKQLRLD TDQVY GNIA TYNFENGRVR VDTGSNWSEV LPQIQETTAR IIFNGKDLNL VERRIAAVNP SDPLETTKPD MTLKEALKIA FGFNEP NGN LQYQGKDITE FDFNFDQQTS QNIKNQLAEL NATNIYTVLD KIKLNAKMNI LIRDKRFHYD RNNIAVGADE SVVKEAH RE VINSSTEGLL LNIDKDIRKI LSGYIVEIED TEGLKEVIND RYDMLNISSL RQDGKTFIDF KKYNDKLPLY ISNPNYKV N VYAVTKENTI INPSENGDTS TNGIKKILIF SKKGYEIG

UniProtKB: Protective antigen

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Macromolecule #2: Lethal factor

MacromoleculeName: Lethal factor / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: anthrax lethal factor endopeptidase
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Molecular weightTheoretical: 93.904211 KDa
Recombinant expressionOrganism: Bacillus anthracis (anthrax bacterium)
SequenceString: MNIKKEFIKV ISMSCLVTAI TLSGPVFIPL VQGAGGHGDV GMHVKEKEKN KDENKRKDEE RNKTQEEHLK EIMKHIVKIE VKGEEAVKK EAAEKLLEKV PSDVLEMYKA IGGKIYIVDG DITKHISLEA LSEDKKKIKD IYGKDALLHE HYVYAKEGYE P VLVIQSSE ...String:
MNIKKEFIKV ISMSCLVTAI TLSGPVFIPL VQGAGGHGDV GMHVKEKEKN KDENKRKDEE RNKTQEEHLK EIMKHIVKIE VKGEEAVKK EAAEKLLEKV PSDVLEMYKA IGGKIYIVDG DITKHISLEA LSEDKKKIKD IYGKDALLHE HYVYAKEGYE P VLVIQSSE DYVENTEKAL NVYYEIGKIL SRDILSKINQ PYQKFLDVLN TIKNASDSDG QDLLFTNQLK EHPTDFSVEF LE QNSNEVQ EVFAKAFAYY IEPQHRDVLQ LYAPEAFNYM DKFNEQEINL SLEELKDQRM LARYEKWEKI KQHYQHWSDS LSE EGRGLL KKLQIPIEPK KDDIIHSLSQ EEKELLKRIQ IDSSDFLSTE EKEFLKKLQI DIRDSLSEEE KELLNRIQVD SSNP LSEKE KEFLKKLKLD IQPYDINQRL QDTGGLIDSP SINLDVRKQY KRDIQNIDAL LHQSIGSTLY NKIYLYENMN INNLT ATLG ADLVDSTDNT KINRGIFNEF KKNFKYSISS NYMIVDINER PALDNERLKW RIQLSPDTRA GYLENGKLIL QRNIGL EIK DVQIIKQSEK EYIRIDAKVV PKSKIDTKIQ EAQLNINQEW NKALGLPKYT KLITFNVHNR YASNIVESAY LILNEWK NN IQSDLIKKVT NYLVDGNGRF VFTDITLPNI AEQYTHQDEI YEQVHSKGLY VPESRSILLH GPSKGVELRN DSEGFIHE F GHAVDDYAGY LLDKNQSDLV TNSKKFIDIF KEEGSNLTSY GRTNEAEFFA EAFRLMHSTD HAERLKVQKN APKTFQFIN DQIKFIINS

UniProtKB: Lethal factor

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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

Concentration0.06 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
20.0 mMTris-HClTris hydrochloride
150.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 286 K / Instrument: GATAN CRYOPLUNGE 3
Details: 4 uL sample was applied to grid (with 2 nm additional carbon layer) and incubated for 45 s prior blotting..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 5238 / Average exposure time: 15.0 sec. / Average electron dose: 74.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.6 µm / Calibrated defocus min: 1.2 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 382000
Startup model#0 - Type of model: PDB ENTRY
#0 - PDB model - PDB ID:

1tzo


#0 - Details: A composite crystal structure was generated, based on 1TZO for the heptameric PA ring, 1J7N (full body of LF) and 3KWV (N-terminal part of LF) for the three LF molecules. This density ...#0 - Details: A composite crystal structure was generated, based on 1TZO for the heptameric PA ring, 1J7N (full body of LF) and 3KWV (N-terminal part of LF) for the three LF molecules. This density was converted into an electron density map, filtered to 30 A and served as initial reference in the 3D refinement.
#1 - Type of model: PDB ENTRY
#1 - PDB model - PDB ID:

1j7n


#1 - Details: A composite crystal structure was generated, based on 1TZO for the heptameric PA ring, 1J7N (full body of LF) and 3KWV (N-terminal part of LF) for the three LF molecules. This density ...#1 - Details: A composite crystal structure was generated, based on 1TZO for the heptameric PA ring, 1J7N (full body of LF) and 3KWV (N-terminal part of LF) for the three LF molecules. This density was converted into an electron density map, filtered to 30 A and served as initial reference in the 3D refinement.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Number images used: 210000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE
Final 3D classificationSoftware - Name: SPHIRE

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Atomic model buiding 1

Initial model
PDB IDChain

3hvd

chain_id: A, source_name: PDB, initial_model_type: experimental model

1j7n

chain_id: A, source_name: PDB, initial_model_type: experimental model
DetailsThe protective antigen (PA) monomer from 3HVD was placed seven times into the density corresponding to the heptameric PA ring using the rigid-body fit in Chimera. Two copies of the monomeric lethal factor (LF) from 1J7N were fitted similarly into the corresponding LF density located atop of the PA7 ring. The resulting model was then flexibly fitted using iMODFIT. The model was further refined in iterative rounds of phenix and coot. Unresolved regions were deleted and side chain information was removed for less well-defined regions.
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6zxj:
Fully-loaded anthrax lethal toxin in its heptameric pre-pore state, in which the third lethal factor is masked out (PA7LF3-masked)

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