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- PDB-7b2p: Cryo-EM structure of complement C4b in complex with nanobody B5 -

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Basic information

Entry
Database: PDB / ID: 7b2p
TitleCryo-EM structure of complement C4b in complex with nanobody B5
Components
  • (Complement C4 ...) x 3
  • Nanobody B5
KeywordsIMMUNE SYSTEM / Complement / nanobody / C4b / inhibitor / complement classical pathway / nanobody-antigen complex
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
: / Complement C4, MG1 domain / Complement C4A/B CUB C-terminal domain / Alpha-2-macroglobulin, conserved site / Alpha-2-macroglobulin family thiolester region signature. / : / Alpha-macro-globulin thiol-ester bond-forming region / Anaphylatoxin, complement system domain / Anaphylatoxin domain signature. / Anaphylatoxin, complement system ...: / Complement C4, MG1 domain / Complement C4A/B CUB C-terminal domain / Alpha-2-macroglobulin, conserved site / Alpha-2-macroglobulin family thiolester region signature. / : / Alpha-macro-globulin thiol-ester bond-forming region / Anaphylatoxin, complement system domain / Anaphylatoxin domain signature. / Anaphylatoxin, complement system / Anaphylatoxin/fibulin / Anaphylotoxin-like domain / Anaphylatoxin domain profile. / Anaphylatoxin homologous domain / Netrin C-terminal Domain / Netrin module, non-TIMP type / UNC-6/NTR/C345C module / : / Alpha-macroglobulin, receptor-binding / Alpha-macroglobulin, receptor-binding domain superfamily / Macroglobulin domain MG4 / Macroglobulin domain MG3 / A-macroglobulin receptor binding domain / Macroglobulin domain MG4 / Macroglobulin domain MG3 / A-macroglobulin receptor / Netrin domain / NTR domain profile. / Alpha-2-macroglobulin / Macroglobulin domain / Alpha-2-macroglobulin, bait region domain / Alpha-macroglobulin-like, TED domain / Alpha-2-macroglobulin family / MG2 domain / A-macroglobulin TED domain / Alpha-2-macroglobulin bait region domain / Alpha-2-Macroglobulin / Alpha-2-macroglobulin family / Tissue inhibitor of metalloproteinases-like, OB-fold / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesLama glama (llama)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsOosterheert, W. / De la O Becerra, K.I. / Gros, P.
Funding support Mexico, 1items
OrganizationGrant numberCountry
Consejo Nacional de Ciencia y Tecnologia (CONACYT)604718 Mexico
CitationJournal: 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
DepositionNov 27, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 2, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 4, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Complement C4 beta chain
B: Complement C4 alpha chain
C: Complement C4 gamma chain
D: Nanobody B5
hetero molecules


Theoretical massNumber of molelcules
Total (without water)205,2868
Polymers204,1984
Non-polymers1,0884
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area18740 Å2
ΔGint-75 kcal/mol
Surface area63990 Å2
MethodPISA

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Components

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Complement C4 ... , 3 types, 3 molecules ABC

#1: Protein Complement C4 beta chain


Mass: 71761.688 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
Plasmid details: Purchased from Complement Technology Inc., purified from normal human serum
References: UniProt: P0C0L4
#2: Protein Complement C4 alpha chain


Mass: 84270.055 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P0C0L4
#3: Protein Complement C4 gamma chain


Mass: 33115.629 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P0C0L4

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Antibody , 1 types, 1 molecules D

#4: Antibody Nanobody B5


Mass: 15050.476 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 Codon Plus (DE3)-RIL

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Sugars , 2 types, 4 molecules

#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complement C4b bound to nanobody B5COMPLEXThe purchased C4b is generated from a mixture of C4A and C4B. C4b is modelled with the sequence of C4A based on pdb 5jtw.#1-#40MULTIPLE SOURCES
2Complement C4 beta chainCOMPLEX#11NATURAL
3Complement C4 alpha chainCOMPLEX#21NATURAL
4Complement C4 gamma chainCOMPLEX#31NATURAL
5Nanobody B5COMPLEX#41RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.204 MDaNO
220.072 MDaNO
330.084 MDaNO
440.033 MDaNO
550.015 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Homo sapiens (human)9606
34Homo sapiens (human)9606
45Lama glama (llama)9844
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21 Codon Plus (DE3)-RIL / Plasmid: pHEN6-Thrombin-his
Buffer solutionpH: 7.3 / Details: 1x PBS.
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMsodium phosphateNaPO41
2145 mMsodium chlorideNaCl1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified components were mixed before vitrification.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K / Details: blot 4.5 seconds, force 1

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7.2 sec. / Electron dose: 51.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1032 / Details: 36 frames of 0.2 seconds.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 36 / Used frames/image: 1-36

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.18CTF correction
7UCSF Chimera1.12model fitting
9PHENIX1.18.2-3874model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 522079
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 274925 / Details: Performed in RELION. / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL / Target criteria: Map to model FSC
Details: Model was refined in the local-resolution filtered Relion map using Phenix real space refine.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
15JTW

5jtw

A1
25JTW

5jtw

B1
35JTW

5jtw

C1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311942
ELECTRON MICROSCOPYf_angle_d0.48916218
ELECTRON MICROSCOPYf_dihedral_angle_d20.0311654
ELECTRON MICROSCOPYf_chiral_restr0.0421849
ELECTRON MICROSCOPYf_plane_restr0.0032096

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