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- EMDB-28888: CryoEM structure of designed modular protein oligomer C8-71 -

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Basic information

Entry
Database: EMDB / ID: EMD-28888
TitleCryoEM structure of designed modular protein oligomer C8-71
Map dataCryoEM structure of designed modular protein oligomer C8-71
Sample
  • Complex: Self-assembled homo-octamer of de novo designed protein C8-71
    • Protein or peptide: C8-71
KeywordsSynthetic / Self-assembling / Oligomeric / Helical repeats / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsRedler RL / Edman NI / Baker D / Ekiert D / Bhabha G
Funding support United States, 2 items
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: 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.
#1: Journal: Cell(Cambridge,Mass.) / Year: 2024
Title: Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies
Authors: Edman NI / Redler RL / Phal A / Schlichthaerle T / Srivatsan SR / Etemadi A / An SJ / Favor A / Ehnes D / Li Z / Praetorius F / Gordon M / Yang W / Coventry B / Hicks DR / Cao L / Bethel N / ...Authors: Edman NI / Redler RL / Phal A / Schlichthaerle T / Srivatsan SR / Etemadi A / An SJ / Favor A / Ehnes D / Li Z / Praetorius F / Gordon M / Yang W / Coventry B / Hicks DR / Cao L / Bethel N / Heine P / Murray A / Gerben S / Carter L / Miranda M / Negahdari B / Lee S / Trapnell C / Stewart L / Ekiert DC / Schlessinger J / Shendure J / Bhabha G / Ruohola-Baker H / Baker D
History
DepositionNov 17, 2022-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28888.png

Downloads & links

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Map

FileDownload / File: emd_28888.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of designed modular protein oligomer C8-71
Voxel sizeX=Y=Z: 1.069 Å
Density
Contour LevelBy AUTHOR: 0.484
Minimum - Maximum-1.2570164 - 1.977028
Average (Standard dev.)0.00071950746 (±0.084274925)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 213.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_28888_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_28888_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Self-assembled homo-octamer of de novo designed protein C8-71

EntireName: Self-assembled homo-octamer of de novo designed protein C8-71
Components
  • Complex: Self-assembled homo-octamer of de novo designed protein C8-71
    • Protein or peptide: C8-71

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Supramolecule #1: Self-assembled homo-octamer of de novo designed protein C8-71

SupramoleculeName: Self-assembled homo-octamer of de novo designed protein C8-71
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: C8-71

MacromoleculeName: C8-71 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.435004 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MGPEEILERA KESLERAREA SERGDEEEFR KAAEKALELA KRLVEQAKKE GDPELVLEAA RVALWVAELA AKNGDKEVFK KAAESALEV AKRLVEVASK EGDPDLVAWA ALVALWVAFL AFLNGDKEVF KKAAESALEV AKALMEVAMK VGAPWLVELA I AVARAVWL ...String:
MGPEEILERA KESLERAREA SERGDEEEFR KAAEKALELA KRLVEQAKKE GDPELVLEAA RVALWVAELA AKNGDKEVFK KAAESALEV AKRLVEVASK EGDPDLVAWA ALVALWVAFL AFLNGDKEVF KKAAESALEV AKALMEVAMK VGAPWLVELA I AVARAVWL LAELFGDEEV RRRAEAFEII LRIAAIAVKA WLGGGGSLEH HHHHH

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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 #1

Preparation ID1
Concentration0.9 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY ARRAY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Support film - #1 - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: blot time = 4s; blot force = 0.
DetailsSample present within the ice layer as both isolated homo-octameric rings and fibrils of variable length

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Sample preparation #2

Preparation ID2
Concentration0.9 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: HOLEY ARRAY / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: CONTINUOUS / Support film - #1 - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: blot time = 4s; blot force = 0.
DetailsSample present within the ice layer as both isolated homo-octameric rings and fibrils of variable length

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 3092 / Average exposure time: 2.5 sec. / Average electron dose: 61.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab initio model generated in Cryosparc
Final reconstructionApplied symmetry - Point group: C8 (8 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 132391
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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Atomic model buiding 1

DetailsDesigned oligomer was used as initial model and was initially fit into map using UCSF Chimera
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Correlation coefficients
Output model

PDB-8f6q:
CryoEM structure of designed modular protein oligomer C8-71

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