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- EMDB-15800: CryoEM reconstruction of C5b8-CD59, density subtracted -

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Basic information

Entry
Database: EMDB / ID: EMD-15800
TitleCryoEM reconstruction of C5b8-CD59, density subtracted
Map dataCryoEM reconstruction of C5b8-CD59.
Sample
  • Complex: C5b8-CD59
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBubeck D / Couves EC / Gardner S
Funding supportEuropean Union, United Kingdom, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)864751European Union
Cancer Research UKC24523/A26234 United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for membrane attack complex inhibition by CD59.
Authors: Emma C Couves / Scott Gardner / Tomas B Voisin / Jasmine K Bickel / Phillip J Stansfeld / Edward W Tate / Doryen Bubeck /
Abstract: CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to ...CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.
History
DepositionSep 8, 2022-
Header (metadata) releaseFeb 22, 2023-
Map releaseFeb 22, 2023-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15800.png

Downloads & links

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Map

FileDownload / File: emd_15800.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM reconstruction of C5b8-CD59.
Voxel sizeX=Y=Z: 1.171 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-4.923125 - 10.287653
Average (Standard dev.)0.002511437 (±0.08988831)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 524.60803 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15800_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoEM reconstruction of C5b8-CD59.

Fileemd_15800_half_map_1.map
AnnotationCryoEM reconstruction of C5b8-CD59.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoEM reconstruction of C5b8-CD59.

Fileemd_15800_half_map_2.map
AnnotationCryoEM reconstruction of C5b8-CD59.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : C5b8-CD59

EntireName: C5b8-CD59
Components
  • Complex: C5b8-CD59

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Supramolecule #1: C5b8-CD59

SupramoleculeName: C5b8-CD59 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#7 / Details: Inhibited complement membrane attack complex
Source (natural)Organism: Homo sapiens (human)

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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: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 294 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.9 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number real images: 12805 / Average exposure time: 9.8 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1138825
Details: CrYOLO neural network based particle picking, standard model, low threshold (0.01).
Startup modelType of model: INSILICO MODEL
In silico model: An initial model was generated using ab-inito reconstruction in cryoSPARC.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 4 / Avg.num./class: 51965 / Software - Name: RELION (ver. 3.1)
Details: 3D Classification was used to remove junk particles and particles with graphene oxide edges.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 206782
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial rigid body fits were performed using UCSF Chimera and UCSF ChimeraX. Carbohydrates were added using Coot. Models were refined iteratively using Isolde and Phenix Real Space Refine.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 87

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