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- EMDB-15783: 3C9, C5b9-CD59 cryoEM structure; focus refinement map -

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Basic information

Entry
Database: EMDB / ID: EMD-15783
Title3C9, C5b9-CD59 cryoEM structure; focus refinement map
Map data3C9, C5b9-CD59 focus refine map. RELION locally filtered map
Sample
  • Complex: 2C9, CD59 inhibited MAC Complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCouves EC / Gardner S / Bubeck D
Funding supportEuropean Union, United Kingdom, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)No. 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 7, 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_15783.png

Downloads & links

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Map

FileDownload / File: emd_15783.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3C9, C5b9-CD59 focus refine map. RELION locally filtered map
Voxel sizeX=Y=Z: 0.831 Å
Density
Contour LevelBy AUTHOR: 0.0154
Minimum - Maximum-0.03467655 - 0.12288644
Average (Standard dev.)-1.27811045e-05 (±0.0020582057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions540540540
Spacing540540540
CellA=B=C: 448.74 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: 3C9, C5b9-CD59 focus refine map. RELION unfiltered half map

Fileemd_15783_half_map_1.map
Annotation3C9, C5b9-CD59 focus refine map. RELION unfiltered half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3C9, C5b9-CD59 focus refine map. RELION unfiltered half map

Fileemd_15783_half_map_2.map
Annotation3C9, C5b9-CD59 focus refine map. RELION unfiltered half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 2C9, CD59 inhibited MAC Complex

EntireName: 2C9, CD59 inhibited MAC Complex
Components
  • Complex: 2C9, CD59 inhibited MAC Complex

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Supramolecule #1: 2C9, CD59 inhibited MAC Complex

SupramoleculeName: 2C9, CD59 inhibited MAC Complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#9
Details: Solved in a DOPC, MSP2N2 nanodisc with a myristolated cytotopic CD59.
Source (natural)Organism: Homo sapiens (human) / Location in cell: Serum

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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: 7.4
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
120.0 mMNaClSodium chloridesodium chloride

Details: 20 mM HEPES pH 7.4, 120 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK II

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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 / Calibrated defocus max: 4.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.9 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 4 / Number real images: 52838 / Average exposure time: 2.0 sec. / Average electron dose: 50.0 e/Å2 / Details: Collected over 4 separate data collections
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Dataset 1: 737,138 Dataset 2: 1,058,026 Dataset 3: 1,330,232 Dataset 4: 722,870
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generated by CryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 67125
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

7nyd

,

2j8b

)
DetailsInitial fitting was done in ISODLE, final refinements were done in ISOLDE

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