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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
Samplehexameric IgM-C1-C4b2
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 26.7 Å
AuthorsSharp TH
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.
DateDeposition: Apr 17, 2019 / Header (metadata) release: Jun 12, 2019 / Map release: Jun 12, 2019 / Update: Jun 19, 2019

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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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Structure viewerEM map:
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Supplemental images

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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
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/NZ000
NX/NY/NZ858858858
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
Details: Map contains a complex of hexameric IgM, complement component C1 and two molecules of complement component C4b
Number of components: 1

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Component #1: protein, hexameric IgM-C1-C4b2

ProteinName: hexameric IgM-C1-C4b2
Details: Map contains a complex of hexameric IgM, complement component C1 and two molecules of complement component C4b
Recombinant expression: No
MassTheoretical: 2.116 MDa
SourceSpecies: Homo sapiens (human)
Source (natural)Organ or tissue: Human serum

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.4
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.48 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 33000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionDetails: 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

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Image processing

ProcessingMethod: subtomogram averaging / Applied symmetry: C1 (asymmetric) / Number of subtomograms: 259 / Number of class averages: 3
Details: Raw frames were aligned using alignframes and tomograms were reconstructed using weighted back-projection from the software program IMOD.
3D reconstructionSoftware: Dynamo / Resolution: 26.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 1DEE, 4JVU, 4BA8, 4JVW, 1PK6, 6F1C, 1GPZ, 4J1Y, 5JTW

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