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

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Basic information

Entry
Database: PDB / ID: 9x1h
TitleCryo-EM Structure of human complement C1s CUB domain in complex with RAY121
Components
  • Complement C1s subcomponent heavy chain
  • RAY121 Fab Heavy chain
  • RAY121 Fab Light chain
KeywordsIMMUNE SYSTEM / PH-DEPENDENT / RECYCLING ANTIBODY / 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)
MethodELECTRON MICROSCOPY / single 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 support1items
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, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: RAY121 Fab Light chain
M: RAY121 Fab Light chain
I: RAY121 Fab Heavy chain
B: Complement C1s subcomponent heavy chain
H: RAY121 Fab Heavy chain
A: Complement C1s subcomponent heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)161,43310
Polymers161,2736
Non-polymers1604
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody RAY121 Fab Light chain


Mass: 24034.600 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi293F TM cells / Production host: Homo sapiens (human)
#2: Antibody RAY121 Fab Heavy chain


Mass: 24044.895 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): Expi293F TM cells / Production host: Homo sapiens (human)
#3: Protein Complement C1s subcomponent heavy chain / Complement C1s subcomponent chain A


Mass: 32556.850 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: 278-290: purification tag / Source: (gene. exp.) Homo sapiens (human) / Gene: C1S / Cell line (production host): Expi293F TM cells / Production host: Homo sapiens (human) / References: UniProt: P09871
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN
Has protein modificationY

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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

ComponentName: human complement C1s CUB domain in complex with RAY121
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.16 MDaNO
210.048 MDaNO
310.024 MDaNO
410.032 MDaNO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F TM cells
Buffer solutionpH: 7.5 / Details: 20mM HEPES(7.5), 150mM NaCl, 3mM CaCl2, 0.03% DDM
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5particle selection
4cryoSPARC4.5CTF correction
9PHENIX1.21.2_5419model refinement
10cryoSPARC4.5initial Euler assignment
11cryoSPARC4.5final Euler assignment
12cryoSPARC4.5classification
13cryoSPARC4.53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1101481 / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeInitial refinement model-IDSource nameTypeDetails
14LMF

4lmf

4LMF1PDBexperimental model
2Otherin silico modelModelAngelo (RELION 5.0-beta1)
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 13.43 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00336044
ELECTRON MICROSCOPYf_angle_d0.65158202
ELECTRON MICROSCOPYf_chiral_restr0.0473860
ELECTRON MICROSCOPYf_plane_restr0.00511072
ELECTRON MICROSCOPYf_dihedral_angle_d5.3108810

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