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- EMDB-11525: Fully-loaded anthrax lethal toxin in its heptameric pre-pore stat... -

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Basic information

Entry
Database: EMDB / ID: EMD-11525
TitleFully-loaded anthrax lethal toxin in its heptameric pre-pore state and PA7LF(2+1A)' arrangement
Map dataPA7LF(2 1A)'
Sample
  • Complex: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state and PA7LF(2+1A)' arrangement
    • Complex: Protective antigen
      • Protein or peptide: Protective antigen
    • Complex: Lethal factor
      • Protein or peptide: Lethal factor
Biological speciesBacillus anthracis (anthrax bacterium)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsQuentin D / Antoni C / Gatsogiannis C / Raunser S
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-
UpdateSep 2, 2020-
Current statusSep 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11525.png

Downloads & links

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Map

FileDownload / File: emd_11525.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPA7LF(2 1A)'
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: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.66913307 - 1.0001136
Average (Standard dev.)0.0012471832 (±0.023108173)
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-0.6691.0000.001

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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 stat...

EntireName: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state and PA7LF(2+1A)' arrangement
Components
  • Complex: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state and PA7LF(2+1A)' arrangement
    • 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 stat...

SupramoleculeName: Fully-loaded anthrax lethal toxin in its heptameric pre-pore state and PA7LF(2+1A)' arrangement
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Three LF molecules crown the heptameric PA ring. The LFs form a continuous chain of head-to-tail interactions. In the PA7LF(2+1A)' arrangement, the third LF binds with its N-terminal domain ...Details: Three LF molecules crown the heptameric PA ring. The LFs form a continuous chain of head-to-tail interactions. In the PA7LF(2+1A)' arrangement, the third LF binds with its N-terminal domain to the C-terminal region of the 2nd LF. In addition, two LFs likely interact with each other via their C-terminal regions close to the central axis 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)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3)

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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 LFs form a continuous chain of head-to-tail interactions. In the PA7LF(2+1A) arrangement, the third LF binds with its N-terminal domain ...Details: Three LF molecules crown the heptameric PA ring. The LFs form a continuous chain of head-to-tail interactions. In the PA7LF(2+1A) arrangement, the third LF binds with its N-terminal domain to the C-terminus of the 2nd LF. In addition, two LFs likely interact with each other via their C-terminal regions close to the central axis of the complex.
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Recombinant expressionOrganism: Bacillus anthracis (anthrax bacterium)

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Macromolecule #1: Protective antigen

MacromoleculeName: Protective antigen / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGHHHHHHHH HHSSGHIDDD DKHMEVKQEN RLLNESESSS QGLLGYYFSD LNFQAPMVVT SSTTGDLSIP SSELENIPSE NQYFQSAIWS GFIKVKKSDE YTFATSADNH VTMWVDDQEV INKASNSNKI RLEKGRLYQI KIQYQRENPT EKGLDFKLYW TDSQNKKEVI ...String:
MGHHHHHHHH HHSSGHIDDD DKHMEVKQEN RLLNESESSS QGLLGYYFSD LNFQAPMVVT SSTTGDLSIP SSELENIPSE NQYFQSAIWS GFIKVKKSDE YTFATSADNH VTMWVDDQEV INKASNSNKI RLEKGRLYQI KIQYQRENPT EKGLDFKLYW TDSQNKKEVI SSDNLQLPEL KQKSSNSRKK RSTSAGPTVP DRDNDGIPDS LEVEGYTVDV KNKRTFLSPW ISNIHEKKGL TKYKSSPEKW STASDPYSDF EKVTGRIDKN VSPEARHPLV AAYPIVHVDM ENIILSKNED QSTQNTDSQT RTISKNTSTS RTHTSEVHGN AEVHASFFDI GGSVSAGFSN SNSSTVAIDH SLSLAGERTW AETMGLNTAD TARLNANIRY VNTGTAPIYN VLPTTSLVLG KNQTLATIKA KENQLSQILA PNNYYPSKNL APIALNAQDD FSSTPITMNY NQFLELEKTK QLRLDTDQVY GNIATYNFEN GRVRVDTGSN WSEVLPQIQE TTARIIFNGK DLNLVERRIA AVNPSDPLET TKPDMTLKEA LKIAFGFNEP NGNLQYQGKD ITEFDFNFDQ QTSQNIKNQL AELNATNIYT VLDKIKLNAK MNILIRDKRF HYDRNNIAVG ADESVVKEAH REVINSSTEG LLLNIDKDIR KILSGYIVEI EDTEGLKEVI NDRYDMLNIS SLRQDGKTFI DFKKYNDKLP LYISNPNYKV NVYAVTKENT IINPSENGDT STNGIKKILI FSKKGYEIG

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Macromolecule #2: Lethal factor

MacromoleculeName: Lethal factor / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus anthracis (anthrax bacterium)
Recombinant expressionOrganism: Bacillus anthracis (anthrax bacterium)
SequenceString: MNIKKEFIKV ISMSCLVTAI TLSGPVFIPL VQGAGGHGDV GMHVKEKEKN KDENKRKDEE RNKTQEEHL KEIMKHIVKI EVKGEEAVKK EAAEKLLEKV PSDVLEMYKA IGGKIYIVDG D ITKHISLE ALSEDKKKIK DIYGKDALLH EHYVYAKEGY EPVLVIQSSE ...String:
MNIKKEFIKV ISMSCLVTAI TLSGPVFIPL VQGAGGHGDV GMHVKEKEKN KDENKRKDEE RNKTQEEHL KEIMKHIVKI EVKGEEAVKK EAAEKLLEKV PSDVLEMYKA IGGKIYIVDG D ITKHISLE ALSEDKKKIK DIYGKDALLH EHYVYAKEGY EPVLVIQSSE DYVENTEKAL NV YYEIGKI LSRDILSKIN QPYQKFLDVL NTIKNASDSD GQDLLFTNQL KEHPTDFSVE FLE QNSNEV QEVFAKAFAY YIEPQHRDVL QLYAPEAFNY MDKFNEQEIN LSLEELKDQR MLAR YEKWE KIKQHYQHWS DSLSEEGRGL LKKLQIPIEP KKDDIIHSLS QEEKELLKRI QIDSS DFLS TEEKEFLKKL QIDIRDSLSE EEKELLNRIQ VDSSNPLSEK EKEFLKKLKL DIQPYD INQ RLQDTGGLID SPSINLDVRK QYKRDIQNID ALLHQSIGST LYNKIYLYEN MNINNLT AT LGADLVDSTD NTKINRGIFN EFKKNFKYSI SSNYMIVDIN ERPALDNERL KWRIQLSP D TRAGYLENGK LILQRNIGLE IKDVQIIKQS EKEYIRIDAK VVPKSKIDTK IQEAQLNIN QEWNKALGLP KYTKLITFNV HNRYASNIVE SAYLILNEWK NNIQSDLIKK VTNYLVDGNG RFVFTDITL PNIAEQYTHQ DEIYEQVHSK GLYVPESRSI LLHGPSKGVE LRNDSEGFIH E FGHAVDDY AGYLLDKNQS DLVTNSKKFI DIFKEEGSNL TSYGRTNEAE FFAEAFRLMH ST DHAERLK VQKNAPKTFQ FINDQIKFII NS

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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: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE / 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
CTF correctionSoftware - Name: SPHIRE
Startup model#0 - Type of model: PDB ENTRY
#0 - PDB model - PDB ID:

1tzo


#0 - Details: The corresponding 3D class of the 3D classification step served as initial model for the refinement
#1 - Type of model: PDB ENTRY
#1 - PDB model - PDB ID:

1j7n


#1 - Details: The corresponding 3D class of the 3D classification step served as initial model for the refinement
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Number images used: 30000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE
Final 3D classificationSoftware - Name: SPHIRE

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