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- EMDB-4878: In situ structure of a hexameric IgM complex bound to complement ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4878
TitleIn situ structure of a hexameric IgM complex bound to complement components C1 and two molecules of C4b
Map data
Sample
  • Complex: hexameric IgM-C1-C4b2
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 26.7 Å
AuthorsSharp TH
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
European Research Council759517 Netherlands
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b.
Authors: Thomas H Sharp / Aimee L Boyle / Christoph A Diebolder / Alexander Kros / Abraham J Koster / Piet Gros /
Abstract: Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates ...Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates complement-immune responses remains unclear. We present cryoelectron tomography structures of IgM, C1, and C4b complexes formed on antigen-bearing lipid membranes by normal human serum at 4 °C. The IgM-C1-C4b complexes revealed C4b product release as the temperature-limiting step in complement activation. Both IgM hexamers and pentamers adopted hexagonal, dome-shaped structures with Fab pairs, dimerized by hinge domains, bound to surface antigens that support a platform of Fc regions. C1 binds IgM through widely spread C1q-collagen helices, with C1r proteases pointing outward and C1s bending downward and interacting with surface-attached C4b, which further interacts with the adjacent IgM-Fab and globular C1q-recognition unit. Based on these data, we present mechanistic models for antibody-mediated, C1q-transmitted activation of C1 and for C4b deposition, while further conformational rearrangements are required to form C3 convertases.
History
DepositionApr 17, 2019-
Header (metadata) releaseJun 12, 2019-
Map releaseJun 12, 2019-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4878.png

Downloads & links

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Map

FileDownload / File: emd_4878.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.3 Å/pix.
x 160 pix.
= 688. Å		4.3 Å/pix.
x 160 pix.
= 688. Å		4.3 Å/pix.
x 160 pix.
= 688. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-1.5185909 - 2.9356854
Average (Standard dev.)0.016613847 (±0.15100342)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-1
Dimensions160160160
Spacing160160160
CellA=B=C: 688.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.34.34.3
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z688.000688.000688.000
α/β/γ90.00090.00090.000
start NX/NY/NZ434333
NX/NY/NZ116118137
MAP C/R/S123
start NC/NR/NS00-1
NC/NR/NS160160160
D min/max/mean-1.5192.9360.017

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Supplemental data

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Sample components

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Entire : hexameric IgM-C1-C4b2

EntireName: hexameric IgM-C1-C4b2
Components
  • Complex: hexameric IgM-C1-C4b2

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Supramolecule #1: hexameric IgM-C1-C4b2

SupramoleculeName: hexameric IgM-C1-C4b2 / type: complex / ID: 1 / Parent: 0
Details: Map contains a complex of hexameric IgM, complement component C1 and two molecules of complement component C4b
Source (natural)Organism: Homo sapiens (human) / Organ: Human serum
Molecular weightTheoretical: 2.116 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Component - Formula: PBS / Component - Name: PBS
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber temperature: 277.15 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 2.4 sec. / Average electron dose: 1.48 e/Å2
Details: Exposures of 2.4 sec were dose-fractionated into 6 movie frames per tilt angle. The total dose for each tilt series was 80 e-/A^2. Focusing to -300 nm was performed before each image ...Details: Exposures of 2.4 sec were dose-fractionated into 6 movie frames per tilt angle. The total dose for each tilt series was 80 e-/A^2. Focusing to -300 nm was performed before each image acquisition using a low-dose routine
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 33000
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

DetailsRaw frames were aligned using alignframes and tomograms were reconstructed using weighted back-projection from the software program IMOD.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 26.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo / Number subtomograms used: 259
ExtractionNumber tomograms: 54 / Number images used: 1522 / Software - Name: EMAN2 (ver. 2.2)
Final 3D classificationNumber classes: 3 / Software - Name: Dynamo
Final angle assignmentType: NOT APPLICABLE / Software - Name: Dynamo
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

1dee

,

4jvu

,

4ba8

,

4jvw

,

1pk6

,

6f1c

,

1gpz

,

4j1y

,

5jtw

)
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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