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

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Basic information

Entry
Database: PDB / ID: 8f6q
TitleCryoEM structure of designed modular protein oligomer C8-71
ComponentsC8-71
KeywordsDE NOVO PROTEIN / Synthetic / Self-assembling / Oligomeric / Helical repeats
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 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
CitationJournal: To Be Published
Title: FGF receptor activation using designed cyclic oligomeric assemblies
Authors: Redler, R.L. / Edman, N.I. / Baker, D. / Ekiert, D. / Bhabha, G.
History
DepositionNov 17, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: C8-71
B: C8-71
C: C8-71
D: C8-71
E: C8-71
F: C8-71
G: C8-71
H: C8-71


Theoretical massNumber of molelcules
Total (without water)187,4808
Polymers187,4808
Non-polymers00
Water0
1


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

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Components

#1: Protein
C8-71


Mass: 23435.004 Da / Num. of mol.: 8
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-octamer of de novo designed protein C8-71
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solution
IDSpecimen-IDpH
118
228
Specimen

Experiment-ID: 1 / Conc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample present within the ice layer as both isolated homo-octameric rings and fibrils of variable length

ID
1
2
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid typeDetails
11COPPER300Quantifoil R2/2
22COPPER300Quantifoil R1.2/1.3No pre-treatment
Vitrification

Chamber temperature: 295 K / Cryogen name: ETHANE / Details: blot time = 4s; blot force = 0 / Entry-ID: 8F6Q / Humidity: 100 % / Instrument: FEI VITROBOT MARK IV

IDSpecimen-ID
11
22

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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 FIELDBright-field microscopy / 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: 2 / Num. of real images: 3092

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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
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIX1.16model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132391 / 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: 102.29 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006112528
ELECTRON MICROSCOPYf_angle_d0.711216888
ELECTRON MICROSCOPYf_chiral_restr0.46231936
ELECTRON MICROSCOPYf_plane_restr0.00342152
ELECTRON MICROSCOPYf_dihedral_angle_d10.47927720

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