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Yorodumi- EMDB-11988: Cryo-EM structure of complement C4b in complex with nanobody E3 -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-11988 | |||||||||
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Title | Cryo-EM structure of complement C4b in complex with nanobody E3 | |||||||||
Map data | DeepEMhancer-postprocessed, locally sharpened cryo-EM density map of complement C4b in complex with nanobody E3. | |||||||||
Sample |
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Function / homology | Function and homology information complement component C1q complex binding / positive regulation of apoptotic cell clearance / Activation of C3 and C5 / complement activation / endopeptidase inhibitor activity / Initial triggering of complement / complement activation, classical pathway / Regulation of Complement cascade / Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) ...complement component C1q complex binding / positive regulation of apoptotic cell clearance / Activation of C3 and C5 / complement activation / endopeptidase inhibitor activity / Initial triggering of complement / complement activation, classical pathway / Regulation of Complement cascade / Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / blood microparticle / inflammatory response / endoplasmic reticulum lumen / axon / innate immune response / neuronal cell body / dendrite / synapse / extracellular space / extracellular exosome / extracellular region / plasma membrane Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / Lama glama (llama) / Human (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.39 Å | |||||||||
Authors | Oosterheert W / De la O Becerra KI / van den Bos RM / Gros P | |||||||||
Funding support | Mexico, 1 items
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Citation | Journal: J Immunol / Year: 2022 Title: Multifaceted Activities of Seven Nanobodies against Complement C4b. Authors: Karla I De la O Becerra / Wout Oosterheert / Ramon M van den Bos / Katerina T Xenaki / Joseph H Lorent / Maartje Ruyken / Arie Schouten / Suzan H M Rooijakkers / Paul M P van Bergen En Henegouwen / Piet Gros / Abstract: Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. ...Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama () immunization, that bind to human C4b () with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined single-particle cryo-electron microscopy structures in complex with C4b at 3.4-4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_11988.map.gz | 91.3 MB | EMDB map data format | |
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Header (meta data) | emd-11988-v30.xml emd-11988.xml | 31.8 KB 31.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_11988_fsc.xml | 10.7 KB | Display | FSC data file |
Images | emd_11988.png | 92.9 KB | ||
Masks | emd_11988_msk_1.map | 103 MB | Mask map | |
Others | emd_11988_additional_1.map.gz emd_11988_additional_2.map.gz emd_11988_half_map_1.map.gz emd_11988_half_map_2.map.gz | 80.8 MB 63 MB 80.9 MB 80.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-11988 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-11988 | HTTPS FTP |
-Validation report
Summary document | emd_11988_validation.pdf.gz | 721.1 KB | Display | EMDB validaton report |
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Full document | emd_11988_full_validation.pdf.gz | 720.6 KB | Display | |
Data in XML | emd_11988_validation.xml.gz | 18 KB | Display | |
Data in CIF | emd_11988_validation.cif.gz | 23.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-11988 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-11988 | HTTPS FTP |
-Related structure data
Related structure data | 7b2mMC 7b2pC 7b2qC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_11988.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | DeepEMhancer-postprocessed, locally sharpened cryo-EM density map of complement C4b in complex with nanobody E3. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.0285 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_11988_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Refined, unsharpened density map of complement C4b in...
File | emd_11988_additional_1.map | ||||||||||||
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Annotation | Refined, unsharpened density map of complement C4b in complex with nanobody E3. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Local-resolution filtered, sharpenend (B = -72 A2) density...
File | emd_11988_additional_2.map | ||||||||||||
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Annotation | Local-resolution filtered, sharpenend (B = -72 A2) density map of complement C4b in complex with nanobody E3. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 1 of the refinement of complement...
File | emd_11988_half_map_1.map | ||||||||||||
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Annotation | Half map 1 of the refinement of complement C4b in complex with nanobody E3. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2 of the refinement of complement...
File | emd_11988_half_map_2.map | ||||||||||||
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Annotation | Half map 2 of the refinement of complement C4b in complex with nanobody E3. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Complement C4b bound to nanobody E3
+Supramolecule #1: Complement C4b bound to nanobody E3
+Supramolecule #2: Complement C4 beta chain
+Supramolecule #3: Complement C4 alpha chain
+Supramolecule #4: Complement C4 gamma chain
+Supramolecule #5: Nanobody E3
+Macromolecule #1: Complement C4 beta chain
+Macromolecule #2: Complement C4 alpha chain
+Macromolecule #3: Complement C4 gamma chain
+Macromolecule #4: Anti-C4b nanobody E3
+Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.25 mg/mL | |||||||||
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Buffer | pH: 7.3 Component:
Details: 1x PBS. | |||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot 4.5 seconds, force 1. | |||||||||
Details | Purified components were mixed before vitrification. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-32 / Number grids imaged: 1 / Number real images: 1396 / Average exposure time: 6.4 sec. / Average electron dose: 51.7 e/Å2 / Details: 32 frames of 0.2 seconds. |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Details | Model was refined in the local-resolution filtered Relion map using Phenix real space refine. | ||||||||
Refinement | Space: REAL / Target criteria: Map to model FSC | ||||||||
Output model | PDB-7b2m: |