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- EMDB-66460: Cryo-EM Structure of human complement C1s CUB domain in complex w... -

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Basic information

Entry
Database: EMDB / ID: EMD-66460
TitleCryo-EM Structure of human complement C1s CUB domain in complex with RAY121
Map data
Sample
  • Complex: human complement C1s CUB domain in complex with RAY121
    • Protein or peptide: RAY121 Fab Light chain
    • Protein or peptide: RAY121 Fab Heavy chain
    • Protein or peptide: Complement C1s subcomponent heavy chain
  • Ligand: CALCIUM ION
KeywordsPH-DEPENDENT / RECYCLING ANTIBODY / IMMUNE SYSTEM / COMPLEMENT C1s / FAB / COMPLEX / CUB DOMAIN / EGF-LIKE DOMAIN
Function / homology
Function and homology information


complement subcomponent C_overbar_1s_ / Classical antibody-mediated complement activation / Initial triggering of complement / complement activation, classical pathway / Regulation of Complement cascade / blood microparticle / innate immune response / serine-type endopeptidase activity / calcium ion binding / proteolysis ...complement subcomponent C_overbar_1s_ / Classical antibody-mediated complement activation / Initial triggering of complement / complement activation, classical pathway / Regulation of Complement cascade / blood microparticle / innate immune response / serine-type endopeptidase activity / calcium ion binding / proteolysis / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Peptidase S1A, complement C1r/C1S/mannan-binding / CUB domain / Domain first found in C1r, C1s, uEGF, and bone morphogenetic protein. / CUB domain / CUB domain profile. / Spermadhesin, CUB domain superfamily / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/CCP/SCR domain profile. ...Peptidase S1A, complement C1r/C1S/mannan-binding / CUB domain / Domain first found in C1r, C1s, uEGF, and bone morphogenetic protein. / CUB domain / CUB domain profile. / Spermadhesin, CUB domain superfamily / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/CCP/SCR domain profile. / Sushi/SCR/CCP superfamily / Coagulation Factor Xa inhibitory site / EGF-type aspartate/asparagine hydroxylation site / EGF-like calcium-binding, conserved site / Calcium-binding EGF-like domain signature. / Aspartic acid and asparagine hydroxylation site. / EGF-like calcium-binding domain / Calcium-binding EGF-like domain / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan
Similarity search - Domain/homology
Complement C1s subcomponent
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsKawauchi H / Adrian H / Gupta G / Koga H / Fujii T / Fukumura T / Ishino S / Irie M / Torizawa T
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: EBioMedicine / Year: 2025
Title: Long lasting complement neutralisation by RAY121, an engineered anti-C1s antibody with C1q displacement function.
Authors: Adrian W S Ho / Taku Fukuzawa / Hikaru Koga / Ken Ohmine / Hiroki Kawauchi / Masaru Muraoka / Yuri Ikuta / Garvita Gupta / Momoko Okuda / Ichio Onami / Miho Ayabe / Akira Takeiri / Hideyuki ...Authors: Adrian W S Ho / Taku Fukuzawa / Hikaru Koga / Ken Ohmine / Hiroki Kawauchi / Masaru Muraoka / Yuri Ikuta / Garvita Gupta / Momoko Okuda / Ichio Onami / Miho Ayabe / Akira Takeiri / Hideyuki Konishi / Yui Sugawara / Shoko Usami / Eriko Ito / Norihito Shibahara / Kazuhisa Ozeki / Yukiko Wada / Ayano Hirako / Noriyuki Takahashi / Zenjiro Sampei / Kenta Haraya / Naoaki Murao / Takashi Fujii / Takuya Torizawa / Hideaki Shimada / Tomoyuki Igawa /
Abstract: BACKGROUND: Antibody drugs blocking the complement classical pathway (CP) often require frequent intravenous administration to overcome the high abundance or rapid turnover of complement proteins. ...BACKGROUND: Antibody drugs blocking the complement classical pathway (CP) often require frequent intravenous administration to overcome the high abundance or rapid turnover of complement proteins. This makes treatment compliance burdensome for patients.
METHODS: Screening was performed to identify anti-C1s antibodies specific for the CUB domain of C1s with CP neutralising function. Antibody engineering was performed on the anti-C1s antibody to ...METHODS: Screening was performed to identify anti-C1s antibodies specific for the CUB domain of C1s with CP neutralising function. Antibody engineering was performed on the anti-C1s antibody to prolong its half-life in circulation. The variable region was mutated to confer pH-dependent binding to C1s, and the antibody Fc was modified to lower its isoelectric point and to have enhanced binding to the neonatal Fc receptor.
FINDINGS: A combination of pH-dependent binding to C1s and optimisation of charge characteristics was required for the final engineered antibody, RAY121, to achieve a long half-life in circulation ...FINDINGS: A combination of pH-dependent binding to C1s and optimisation of charge characteristics was required for the final engineered antibody, RAY121, to achieve a long half-life in circulation and long-lasting neutralisation of the CP. In male cynomolgus monkeys, a single subcutaneous injection suppressed CP activity for more than a month. RAY121 neutralises the CP by using a unique mechanism of displacing C1q from the C1 complex and blocking its reassembly. Structure analysis by cryo-electron microscopy revealed that the RAY121 epitope on C1s is distinct from the C1q binding site, and that C1q displacement is mediated by the Fab arm sterically obstructing C1q binding to C1s.
INTERPRETATION: RAY121 is able to neutralise the CP for an extended duration and is effective at doses that can be administered subcutaneously for convenient treatment. These data present RAY121 as a ...INTERPRETATION: RAY121 is able to neutralise the CP for an extended duration and is effective at doses that can be administered subcutaneously for convenient treatment. These data present RAY121 as a promising agent for the treatment of CP-driven autoimmune disorders.
FUNDING: Chugai Pharmaceutical Co. Ltd.
History
DepositionOct 2, 2025-
Header (metadata) releaseNov 19, 2025-
Map releaseNov 19, 2025-
UpdateNov 19, 2025-
Current statusNov 19, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_66460.png

Downloads & links

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Map

FileDownload / File: emd_66460.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.2993127 - 1.6727394
Average (Standard dev.)0.00021435609 (±0.020404603)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_66460_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_66460_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_66460_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : human complement C1s CUB domain in complex with RAY121

EntireName: human complement C1s CUB domain in complex with RAY121
Components
  • Complex: human complement C1s CUB domain in complex with RAY121
    • Protein or peptide: RAY121 Fab Light chain
    • Protein or peptide: RAY121 Fab Heavy chain
    • Protein or peptide: Complement C1s subcomponent heavy chain
  • Ligand: CALCIUM ION

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Supramolecule #1: human complement C1s CUB domain in complex with RAY121

SupramoleculeName: human complement C1s CUB domain in complex with RAY121
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32 KDa

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Macromolecule #1: RAY121 Fab Light chain

MacromoleculeName: RAY121 Fab Light chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.0346 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCQAHEILH DKKNLAWYQQ KPGQPPKLLI YSASILESGV PSRFSGSGSG TDFTLTISSL QPEDFATYY CHGEFSHGSG DLNHFGQGTK VEIKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA L QSGNSQES ...String:
DIQMTQSPSS LSASVGDRVT ITCQAHEILH DKKNLAWYQQ KPGQPPKLLI YSASILESGV PSRFSGSGSG TDFTLTISSL QPEDFATYY CHGEFSHGSG DLNHFGQGTK VEIKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA L QSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC

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Macromolecule #2: RAY121 Fab Heavy chain

MacromoleculeName: RAY121 Fab Heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.044895 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVESGGG VVQPGRSLRL SCAASGFTFS AYAMNWIRQP PGKGLEWIGL IYGSGHEFYA SWAEERVTIS VDTSKNQFSL KLSSVTAAD TAVYYCARGR SKNYNSDFHL WGQGTLVTVS SASTKGPSVF PLAPSSRSTS ESTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
QVQLVESGGG VVQPGRSLRL SCAASGFTFS AYAMNWIRQP PGKGLEWIGL IYGSGHEFYA SWAEERVTIS VDTSKNQFSL KLSSVTAAD TAVYYCARGR SKNYNSDFHL WGQGTLVTVS SASTKGPSVF PLAPSSRSTS ESTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDK

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Macromolecule #3: Complement C1s subcomponent heavy chain

MacromoleculeName: Complement C1s subcomponent heavy chain / type: protein_or_peptide / ID: 3 / Details: 278-290: purification tag / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.55685 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EPTMYGEILS PNYPQAYPSE VEKSWDIEVP EGYGIHLYFT HLDIELSENC AYDSVQIISG DTEEGRLCGQ RSSNNPHSPI VEEFQVPYN KLQVIFKSDF SNEERFTGFA AYYVATDINE CTDFVDVPCS HFCNNFIGGY FCSCPPEYFL HDDMKNCGVN C SGDVFTAL ...String:
EPTMYGEILS PNYPQAYPSE VEKSWDIEVP EGYGIHLYFT HLDIELSENC AYDSVQIISG DTEEGRLCGQ RSSNNPHSPI VEEFQVPYN KLQVIFKSDF SNEERFTGFA AYYVATDINE CTDFVDVPCS HFCNNFIGGY FCSCPPEYFL HDDMKNCGVN C SGDVFTAL IGEIASPNYP KPYPENSRCE YQIRLEKGFQ VVVTLRREDF DVEAADSAGN CLDSLVFVAG DRQFGPYCGH GF PGPLNIE TKSNALDIIF QTDLTGQKKG WKLRYHGDPM GGGGSDYKDD DDK

UniProtKB: Complement C1s subcomponent

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration5 mg/mL
BufferpH: 7.5 / Details: 20mM HEPES(7.5), 150mM NaCl, 3mM CaCl2, 0.03% DDM
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.5) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5) / Number images used: 1101481
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

4lmf

source_name: PDB, initial_model_type: experimental model
source_name: Other, initial_model_type: in silico modelModelAngelo (RELION 5.0-beta1)
Output model

PDB-9x1h:
Cryo-EM Structure of human complement C1s CUB domain in complex with RAY121

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