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- PDB-8f6r: CryoEM structure of designed modular protein oligomer C6-79 -

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Entry
Database: PDB / ID: 8f6r
TitleCryoEM structure of designed modular protein oligomer C6-79
ComponentsDe novo designed oligomeric protein C6-79
KeywordsDE NOVO PROTEIN / Synthetic / Self-assembling / Oligomeric / Helical repeats
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsRedler, R.L. / Edman, N.I. / Baker, D. / Ekiert, D. / Bhabha, G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128777 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)R01AG063845 United States
Citation
Journal: Cell / Year: 2024
Title: Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies.
Authors: Natasha I Edman / Ashish Phal / Rachel L Redler / Thomas Schlichthaerle / Sanjay R Srivatsan / Devon Duron Ehnes / Ali Etemadi / Seong J An / Andrew Favor / Zhe Li / Florian Praetorius / Max ...Authors: Natasha I Edman / Ashish Phal / Rachel L Redler / Thomas Schlichthaerle / Sanjay R Srivatsan / Devon Duron Ehnes / Ali Etemadi / Seong J An / Andrew Favor / Zhe Li / Florian Praetorius / Max Gordon / Thomas Vincent / Silvia Marchiano / Leslie Blakely / Chuwei Lin / Wei Yang / Brian Coventry / Derrick R Hicks / Longxing Cao / Neville Bethel / Piper Heine / Analisa Murray / Stacey Gerben / Lauren Carter / Marcos Miranda / Babak Negahdari / Sangwon Lee / Cole Trapnell / Ying Zheng / Charles E Murry / Devin K Schweppe / Benjamin S Freedman / Lance Stewart / Damian C Ekiert / Joseph Schlessinger / Jay Shendure / Gira Bhabha / Hannele Ruohola-Baker / David Baker /
Abstract: Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase ...Many growth factors and cytokines signal by binding to the extracellular domains of their receptors and driving association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affect signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo-designed fibroblast growth factor receptor (FGFR)-binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca release and mitogen-activated protein kinase (MAPK) pathway activation. The high specificity of the designed agonists reveals distinct roles for two FGFR splice variants in driving arterial endothelium and perivascular cell fates during early vascular development. Our designed modular assemblies should be broadly useful for unraveling the complexities of signaling in key developmental transitions and for developing future therapeutic applications.
#1: Journal: bioRxiv / Year: 2023
Title: Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies.
Authors: Natasha I Edman / Rachel L Redler / Ashish Phal / Thomas Schlichthaerle / Sanjay R Srivatsan / Ali Etemadi / Seong J An / Andrew Favor / Devon Ehnes / Zhe Li / Florian Praetorius / Max ...Authors: Natasha I Edman / Rachel L Redler / Ashish Phal / Thomas Schlichthaerle / Sanjay R Srivatsan / Ali Etemadi / Seong J An / Andrew Favor / Devon Ehnes / Zhe Li / Florian Praetorius / Max Gordon / Wei Yang / Brian Coventry / Derrick R Hicks / Longxing Cao / Neville Bethel / Piper Heine / Analisa Murray / Stacey Gerben / Lauren Carter / Marcos Miranda / Babak Negahdari / Sangwon Lee / Cole Trapnell / Lance Stewart / Damian C Ekiert / Joseph Schlessinger / Jay Shendure / Gira Bhabha / Hannele Ruohola-Baker / David Baker /
Abstract: Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, ...Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways.
#2: Journal: Cell(Cambridge,Mass.) / Year: 2024
Title: Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies
Authors: Edman, N.I. / Redler, R.L. / Phal, A. / Schlichthaerle, T. / Srivatsan, S.R. / Etemadi, A. / An, S.J. / Favor, A. / Ehnes, D. / Li, Z. / Praetorius, F. / Gordon, M. / Yang, W. / Coventry, B. ...Authors: Edman, N.I. / Redler, R.L. / Phal, A. / Schlichthaerle, T. / Srivatsan, S.R. / Etemadi, A. / An, S.J. / Favor, A. / Ehnes, D. / Li, Z. / Praetorius, F. / Gordon, M. / Yang, W. / Coventry, B. / Hicks, D.R. / Cao, L. / Bethel, N. / Heine, P. / Murray, A. / Gerben, S. / Carter, L. / Miranda, M. / Negahdari, B. / Lee, S. / Trapnell, C. / Stewart, L. / Ekiert, D.C. / Schlessinger, J. / Shendure, J. / Bhabha, G. / Ruohola-Baker, H. / Baker, D.
History
DepositionNov 17, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jun 19, 2024Group: Database references / Category: citation / citation_author
Revision 1.3Jun 26, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: De novo designed oligomeric protein C6-79
B: De novo designed oligomeric protein C6-79
C: De novo designed oligomeric protein C6-79
D: De novo designed oligomeric protein C6-79
E: De novo designed oligomeric protein C6-79
F: De novo designed oligomeric protein C6-79


Theoretical massNumber of molelcules
Total (without water)161,3056
Polymers161,3056
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, gel filtration, light scattering, multi-angle light scattering (MALS)
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
De novo designed oligomeric protein C6-79


Mass: 26884.201 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21 (bacteria)

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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: Self-assembled homo-hexamer of de novo designed protein C6-79
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: blot time = 6s; blot force = 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 61.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6434

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correctionCTFFIND
7UCSF Chimeramodel fitting
9PHENIX1.16model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 864566 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficients
Details: Designed oligomer was used as initial model and was initially fit into map using UCSF Chimera
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 111.52 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00510368
ELECTRON MICROSCOPYf_angle_d0.728514028
ELECTRON MICROSCOPYf_chiral_restr0.31741662
ELECTRON MICROSCOPYf_plane_restr0.00461884
ELECTRON MICROSCOPYf_dihedral_angle_d14.82746642

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