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Yorodumi- EMDB-16730: Cryo-EM structure of complement C5 in complex with nanobodies UNb... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-16730 | |||||||||||||||
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Title | Cryo-EM structure of complement C5 in complex with nanobodies UNbC5-1 and UNbC5-2 | |||||||||||||||
Map data | map | |||||||||||||||
Sample |
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Keywords | nanobody / inhibitor / complement / C5 / inhibition / convertase / classical pathway / nanobody-antigen complex / alternative pathway / IMMUNE SYSTEM | |||||||||||||||
Function / homology | Function and homology information Terminal pathway of complement / membrane attack complex / Activation of C3 and C5 / negative regulation of macrophage chemotaxis / complement activation, alternative pathway / chemokine activity / endopeptidase inhibitor activity / positive regulation of vascular endothelial growth factor production / positive regulation of chemokine production / complement activation, classical pathway ...Terminal pathway of complement / membrane attack complex / Activation of C3 and C5 / negative regulation of macrophage chemotaxis / complement activation, alternative pathway / chemokine activity / endopeptidase inhibitor activity / positive regulation of vascular endothelial growth factor production / positive regulation of chemokine production / complement activation, classical pathway / Peptide ligand-binding receptors / Regulation of Complement cascade / chemotaxis / G alpha (i) signalling events / killing of cells of another organism / cell surface receptor signaling pathway / inflammatory response / G protein-coupled receptor signaling pathway / signaling receptor binding / extracellular space / extracellular exosome / extracellular region Similarity search - Function | |||||||||||||||
Biological species | Homo sapiens (human) / Lama glama (llama) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||||||||
Authors | De la O Becerra KI / Gros P | |||||||||||||||
Funding support | Netherlands, European Union, Mexico, 4 items
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Citation | Journal: J Biol Chem / Year: 2023 Title: Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies. Authors: Eva M Struijf / Karla I De la O Becerra / Maartje Ruyken / Carla J C de Haas / Fleur van Oosterom / Danique Y Siere / Joanne E van Keulen / Dani A C Heesterbeek / Edward Dolk / Raimond ...Authors: Eva M Struijf / Karla I De la O Becerra / Maartje Ruyken / Carla J C de Haas / Fleur van Oosterom / Danique Y Siere / Joanne E van Keulen / Dani A C Heesterbeek / Edward Dolk / Raimond Heukers / Bart W Bardoel / Piet Gros / Suzan H M Rooijakkers / Abstract: The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector ...The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5. | |||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_16730.map.gz | 118.1 MB | EMDB map data format | |
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Header (meta data) | emd-16730-v30.xml emd-16730.xml | 24.7 KB 24.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_16730_fsc.xml | 12 KB | Display | FSC data file |
Images | emd_16730.png | 52.6 KB | ||
Masks | emd_16730_msk_1.map | 125 MB | Mask map | |
Others | emd_16730_half_map_1.map.gz emd_16730_half_map_2.map.gz | 116.1 MB 116.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-16730 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-16730 | HTTPS FTP |
-Validation report
Summary document | emd_16730_validation.pdf.gz | 919.5 KB | Display | EMDB validaton report |
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Full document | emd_16730_full_validation.pdf.gz | 919.1 KB | Display | |
Data in XML | emd_16730_validation.xml.gz | 18.6 KB | Display | |
Data in CIF | emd_16730_validation.cif.gz | 23.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16730 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16730 | HTTPS FTP |
-Related structure data
Related structure data | 8cmlMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_16730.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.04 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_16730_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: half map b
File | emd_16730_half_map_1.map | ||||||||||||
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Annotation | half map b | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map a
File | emd_16730_half_map_2.map | ||||||||||||
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Annotation | half map a | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2
Entire | Name: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2 |
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Components |
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-Supramolecule #1: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2
Supramolecule | Name: Complement C5 bound to nanobodies UNbC5-1 and UNbC5-2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1, #4 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 218 KDa |
-Macromolecule #1: Nanobody UNbC5-2
Macromolecule | Name: Nanobody UNbC5-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 16.095776 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MEVQLVESGG GLVQPGGSLR LSCAASGRTF STNTMGWFRQ APGQEREFVA LISGNGRILD YSDSAKGRFT ISRDNAKNTV YLQMNSLKP EDTGVYFCAA EFRGRTLASY WGQGTQVTVS SAAASGSLEQ KLISEEDLNG AAHHHHHHGA A |
-Macromolecule #2: Complement C5 beta chain
Macromolecule | Name: Complement C5 beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 73.361453 KDa |
Sequence | String: QEQTYVISAP KIFRVGASEN IVIQVYGYTE AFDATISIKS YPDKKFSYSS GHVHLSSENK FQNSAILTIQ PKQLPGGQNP VSYVYLEVV SKHFSKSKRM PITYDNGFLF IHTDKPVYTP DQSVKVRVYS LNDDLKPAKR ETVLTFIDPE GSEVDMVEEI D HIGIISFP ...String: QEQTYVISAP KIFRVGASEN IVIQVYGYTE AFDATISIKS YPDKKFSYSS GHVHLSSENK FQNSAILTIQ PKQLPGGQNP VSYVYLEVV SKHFSKSKRM PITYDNGFLF IHTDKPVYTP DQSVKVRVYS LNDDLKPAKR ETVLTFIDPE GSEVDMVEEI D HIGIISFP DFKIPSNPRY GMWTIKAKYK EDFSTTGTAY FEVKEYVLPH FSVSIEPEYN FIGYKNFKNF EITIKARYFY NK VVTEADV YITFGIREDL KDDQKEMMQT AMQNTMLING IAQVTFDSET AVKELSYYSL EDLNNKYLYI AVTVIESTGG FSE EAEIPG IKYVLSPYKL NLVATPLFLK PGIPYPIKVQ VKDSLDQLVG GVPVTLNAQT IDVNQETSDL DPSKSVTRVD DGVA SFVLN LPSGVTVLEF NVKTDAPDLP EENQAREGYR AIAYSSLSQS YLYIDWTDNH KALLVGEHLN IIVTPKSPYI DKITH YNYL ILSKGKIIHF GTREKFSDAS YQSINIPVTQ NMVPSSRLLV YYIVTGEQTA ELVSDSVWLN IEEKCGNQLQ VHLSPD ADA YSPGQTVSLN MATGMDSWVA LAAVDSAVYG VQRGAKKPLE RVFQFLEKSD LGCGAGGGLN NANVFHLAGL TFLTNAN AD DSQENDEPCK EIL UniProtKB: Complement C5 |
-Macromolecule #3: Complement C5 alpha chain
Macromolecule | Name: Complement C5 alpha chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 112.635008 KDa |
Sequence | String: TLQKKIEEIA AKYKHSVVKK CCYDGACVNN DETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGRLHMKTL LPVSKPEIR SYFPESWLWE VHLVPRRKQL QFALPDSLTT WEIQGVGISN TGICVADTVK AKVFKDVFLE MNIPYSVVRG E QIQLKGTV ...String: TLQKKIEEIA AKYKHSVVKK CCYDGACVNN DETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGRLHMKTL LPVSKPEIR SYFPESWLWE VHLVPRRKQL QFALPDSLTT WEIQGVGISN TGICVADTVK AKVFKDVFLE MNIPYSVVRG E QIQLKGTV YNYRTSGMQF CVKMSAVEGI CTSESPVIDH QGTKSSKCVR QKVEGSSSHL VTFTVLPLEI GLHNINFSLE TW FGKEILV KTLRVVPEGV KRESYSGVTL DPRGIYGTIS RRKEFPYRIP LDLVPKTEIK RILSVKGLLV GEILSAVLSQ EGI NILTHL PKGSAEAELM SVVPVFYVFH YLETGNHWNI FHSDPLIEKQ KLKKKLKEGM LSIMSYRNAD YSYSVWKGGS ASTW LTAFA LRVLGQVNKY VEQNQNSICN SLLWLVENYQ LDNGSFKENS QYQPIKLQGT LPVEARENSL YLTAFTVIGI RKAFD ICPL VKIDTALIKA DNFLLENTLP AQSTFTLAIS AYALSLGDKT HPQFRSIVSA LKREALVKGN PPIYRFWKDN LQHKDS SVP NTGTARMVET TAYALLTSLN LKDINYVNPV IKWLSEEQRY GGGFYSTQDT INAIEGLTEY SLLVKQLRLS MDIDVSY KH KGALHNYKMT DKNFLGRPVE VLLNDDLIVS TGFGSGLATV HVTTVVHKTS TSEEVCSFYL KIDTQDIEAS HYRGYGNS D YKRIVACASY KPSREESSSG SSHAVMDISL PTGISANEED LKALVEGVDQ LFTDYQIKDG HVILQLNSIP SSDFLCVRF RIFELFEVGF LSPATFTVYE YHRPDKQCTM FYSTSNIKIQ KVCEGAACKC VEADCGQMQE ELDLTISAET RKQTACKPEI AYAYKVSIT SITVENVFVK YKATLLDIYK TGEAVAEKDS EITFIKKVTC TNAELVKGRQ YLIMGKEALQ IKYNFSFRYI Y PLDSLTWI EYWPRDTTCS SCQAFLANLD EFAEDIFLNG C UniProtKB: Complement C5 |
-Macromolecule #4: Nanobody UNbC5-1
Macromolecule | Name: Nanobody UNbC5-1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 16.757295 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MEVQLVESGG GLVQAGGSLR LSCAASGFTF DDYAIGWFRQ APGKEREGVS CISTSDGSTY YADSVKGRFT ISSDNAKNTV YLQMNSLKP EDTAVYYCAA DPYLPIRGRG IESTDFGSWG QGTQVTVSSA AASGSLEQKL ISEEDLNGAA HHHHHHGAA |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.26 mg/mL | |||||||||
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Buffer | pH: 7.4 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 / Pretreatment - Time: 30 sec. | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot 4 seconds at blot force 1. | |||||||||
Details | Purified protein were mixed before vitrification in |
-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: 1-40 / Number grids imaged: 2 / Number real images: 3810 / Average electron dose: 55.0 e/Å2 |
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: 2.6 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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Refinement | Space: REAL / Protocol: RIGID BODY FIT | ||||||||||||
Output model | PDB-8cml: |