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- EMDB-11991: Cryo-EM density map of complement C4b in complex with nanobody G3 -

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Basic information

Entry
Database: EMDB / ID: EMD-11991
TitleCryo-EM density map of complement C4b in complex with nanobody G3
Map dataDeepEMhancer-postprocessed, local-sharpened cryo-EM density map of complement C4b in complex with nanobody G3.
Sample
  • Complex: Complement C4b bound to nanobody G3
    • Complex: Complement C4b
      • Protein or peptide: Complement C4b
    • Complex: Nanobody G3
      • Protein or peptide: Nanobody G3
Function / homology
Function and homology information


detection of molecule of bacterial origin / classical-complement-pathway C3/C5 convertase complex / positive regulation of opsonization / opsonization / symbiont cell surface / complement binding / positive regulation of apoptotic cell clearance / Activation of C3 and C5 / complement activation / endopeptidase inhibitor activity ...detection of molecule of bacterial origin / classical-complement-pathway C3/C5 convertase complex / positive regulation of opsonization / opsonization / symbiont cell surface / complement 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 / positive regulation of immune response / carbohydrate binding / blood microparticle / inflammatory response / axon / innate immune response / dendrite / synapse / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Alpha-2-macroglobulin, conserved site / Alpha-2-macroglobulin family thiolester region signature. / Anaphylatoxin, complement system domain / Anaphylatoxin domain signature. / Anaphylatoxin/fibulin / Anaphylatoxin, complement system / Anaphylotoxin-like domain / Anaphylatoxin domain profile. / Anaphylatoxin homologous domain / Netrin C-terminal Domain ...Alpha-2-macroglobulin, conserved site / Alpha-2-macroglobulin family thiolester region signature. / Anaphylatoxin, complement system domain / Anaphylatoxin domain signature. / Anaphylatoxin/fibulin / Anaphylatoxin, complement system / 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. / Tissue inhibitor of metalloproteinases-like, OB-fold / 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 / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.96 Å
AuthorsOosterheert W / De la O Becerra KI / Gros P
Funding support Mexico, 1 items
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, 2020-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateMay 11, 2022-
Current statusMay 11, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11991.png

Downloads & links

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Map

FileDownload / File: emd_11991.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer-postprocessed, local-sharpened cryo-EM density map of complement C4b in complex with nanobody G3.
Voxel sizeX=Y=Z: 1.0285 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.0017775423 - 2.2719932
Average (Standard dev.)0.0010497267 (±0.022660503)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 308.55 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.02851.02851.0285
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z308.550308.550308.550
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0022.2720.001

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Supplemental data

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Mask #1

Fileemd_11991_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Refined, unsharpened cryo-EM density map of complement C4b...

Fileemd_11991_additional_1.map
AnnotationRefined, unsharpened cryo-EM density map of complement C4b in complex with nanobody G3.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local-resolution filtered, sharpened cryo-EM density map of complement...

Fileemd_11991_additional_2.map
AnnotationLocal-resolution filtered, sharpened cryo-EM density map of complement C4b in complex with nanobody G3.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of the refinement of the...

Fileemd_11991_half_map_1.map
AnnotationHalf map 2 of the refinement of the cryo-EM density map of complement C4b in complex with nanobody G3.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of the refinement of the...

Fileemd_11991_half_map_2.map
AnnotationHalf map 1 of the refinement of the cryo-EM density map of complement C4b in complex with nanobody G3.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complement C4b bound to nanobody G3

EntireName: Complement C4b bound to nanobody G3
Components
  • Complex: Complement C4b bound to nanobody G3
    • Complex: Complement C4b
      • Protein or peptide: Complement C4b
    • Complex: Nanobody G3
      • Protein or peptide: Nanobody G3

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Supramolecule #1: Complement C4b bound to nanobody G3

SupramoleculeName: Complement C4b bound to nanobody G3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 14.4 KDa

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Supramolecule #2: Complement C4b

SupramoleculeName: Complement C4b / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: The purchased C4b is generated from a mixture of C4A and C4B.
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Nanobody G3

SupramoleculeName: Nanobody G3 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 Codon Plus (DE3)-RIL / Recombinant plasmid: pHEN6-Thrombin-his

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Macromolecule #1: Complement C4b

MacromoleculeName: Complement C4b / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MRLLWGLIWA SSFFTLSLQK PRLLLFSPSV VHLGVPLSVG VQLQDVPRGQ VVKGSVFLRN PSRNNVPCS PKVDFTLSSE RDFALLSLQV PLKDAKSCGL HQLLRGPEVQ LVAHSPWLKD S LSRTTNIQ GINLLFSSRR GHLFLQTDQP IYNPGQRVRY RVFALDQKMR ...String:
MRLLWGLIWA SSFFTLSLQK PRLLLFSPSV VHLGVPLSVG VQLQDVPRGQ VVKGSVFLRN PSRNNVPCS PKVDFTLSSE RDFALLSLQV PLKDAKSCGL HQLLRGPEVQ LVAHSPWLKD S LSRTTNIQ GINLLFSSRR GHLFLQTDQP IYNPGQRVRY RVFALDQKMR PSTDTITVMV EN SHGLRVR KKEVYMPSSI FQDDFVIPDI SEPGTWKISA RFSDGLESNS STQFEVKKYV LPN FEVKIT PGKPYILTVP GHLDEMQLDI QARYIYGKPV QGVAYVRFGL LDEDGKKTFF RGLE SQTKL VNGQSHISLS KAEFQDALEK LNMGITDLQG LRLYVAAAII ESPGGEMEEA ELTSW YFVS SPFSLDLSKT KRHLVPGAPF LLQALVREMS GSPASGIPVK VSATVSSPGS VPEVQD IQQ NTDGSGQVSI PIIIPQTISE LQLSVSAGSP HPAIARLTVA APPSGGPGFL SIERPDS RP PRVGDTLNLN LRAVGSGATF SHYYYMILSR GQIVFMNREP KRTLTSVSVF VDHHLAPS F YFVAFYYHGD HPVANSLRVD VQAGACEGKL ELSVDGAKQY RNGESVKLHL ETDSLALVA LGALDTALYA AGSKSHKPLN MGKVFEAMNS YDLGCGPGGG DSALQVFQAA GLAFSDGDQW TLSRKRLSC PKEKTTRKKR NVNFQKAINE KLGQYASPTA KRCCQDGVTR LPMMRSCEQR A ARVQQPDC REPFLSCCQF AESLRKKSRD KGQAGLQRAL EILQEEDLID EDDIPVRSFF PE NWLWRVE TVDRFQILTL WLPDSLTTWE IHGLSLSKTK GLCVATPVQL RVFREFHLHL RLP MSVRRF EQLELRPVLY NYLDKNLTVS VHVSPVEGLC LAGGGGLAQQ VLVPAGSARP VAFS VVPTA AAAVSLKVVA RGSFEFPVGD AVSKVLQIEK EGAIHREELV YELNPLDHRG RTLEI PGNS DPNMIPDGDF NSYVRVTASD PLDTLGSEGA LSPGGVASLL RLPRGCGEQT MIYLAP TLA ASRYLDKTEQ WSTLPPETKD HAVDLIQKGY MRIQQFRKAD GSYAAWLSRD SSTWLTA FV LKVLSLAQEQ VGGSPEKLQE TSNWLLSQQQ ADGSFQDPCP VLDRSMQGGL VGNDETVA L TAFVTIALHH GLAVFQDEGA EPLKQRVEAS ISKANSFLGE KASAGLLGAH AAAITAYAL SLTKAPVDLL GVAHNNLMAM AQETGDNLYW GSVTGSQSNA VSPTPAPRNP SDPMPQAPAL WIETTAYAL LHLLLHEGKA EMADQASAWL TRQGSFQGGF RSTQDTVIAL DALSAYWIAS H TTEERGLN VTLSSTGRNG FKSHALQLNN RQIRGLEEEL QFSLGSKINV KVGGNSKGTL KV LRTYNVL DMKNTTCQDL QIEVTVKGHV EYTMEANEDY EDYEYDELPA KDDPDAPLQP VTP LQLFEG RRNRRRREAP KVVEEQESRV HYTVCIWRNG KVGLSGMAIA DVTLLSGFHA LRAD LEKLT SLSDRYVSHF ETEGPHVLLY FDSVPTSREC VGFEAVQEVP VGLVQPASAT LYDYY NPER RCSVFYGAPS KSRLLATLCS AEVCQCAEGK CPRQRRALER GLQDEDGYRM KFACYY PRV EYGFQVKVLR EDSRAAFRLF ETKITQVLHF TKDVKAAANQ MRNFLVRASC RLRLEPG KE YLIMGLDGAT YDLEGHPQYL LDSNSWIEEM PSERLCRSTR QRAACAQLND FLQEYGTQ G CQV

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Macromolecule #2: Nanobody G3

MacromoleculeName: Nanobody G3 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
EVQLVESGGG LVQAGGSLRL SCAASESIFI NNMGWFRQAP GKERELVATI ARDYGPNYAD SAKGRFTITR DNAKNMYLQM NNLKTEDTGV YYCRVVVAGG YYYWGQGTQV TVSSHGSGLV PRGSGGGHHH HHH

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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

Concentration0.25 mg/mL
BufferpH: 7.3
Component:
ConcentrationFormulaName
10.0 mMNaPO4sodium phosphate
145.0 mMNaClSodium chloridesodium chloride

Details: 1x PBS.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot 4.5 seconds, force 1.
DetailsPurified components were mixed before vitrification.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm 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: 1658 / Average exposure time: 6.4 sec. / Average electron dose: 50.5 e/Å2 / Details: 32 frames of 0.2 seconds.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 845087
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4xam
PDB Unreleased entry


Details: First crystal structure of C4b.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Performed in RELION
Final 3D classificationNumber classes: 3 / Avg.num./class: 258848 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Details: Performed in RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 207769
FSC plot (resolution estimation)

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