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- PDB-7rdr: Circular tandem repeat protein with novel repeat topology and enh... -

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Basic information

Entry
Database: PDB / ID: 7rdr
TitleCircular tandem repeat protein with novel repeat topology and enhanced subunit contact surfaces
ComponentsCircular tendon repeat protein
KeywordsPEPTIDE BINDING PROTEIN / protein display particles
Biological speciesunidentified (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsShen, B.W. / Stoddard, B.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM105691 United States
CitationJournal: Commun Biol / Year: 2021
Title: Design of functionalised circular tandem repeat proteins with longer repeat topologies and enhanced subunit contact surfaces.
Authors: Jazmine P Hallinan / Lindsey A Doyle / Betty W Shen / Mesfin M Gewe / Brittany Takushi / Madison A Kennedy / Della Friend / James M Roberts / Philip Bradley / Barry L Stoddard /
Abstract: Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and ...Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits. In this study, we describe a new generation of cTRPs, with longer repeats and increased interaction surfaces, which enhanced the self-assembly of two significantly different sizes of homotrimeric constructs. Finally, we demonstrated functionalization of these constructs with (1) a hexameric array of peptide-binding SH2 domains, and (2) a trimeric array of anti-SARS CoV-2 VHH domains. The latter proved capable of sub-nanomolar binding affinities towards the viral receptor binding domain and potent viral neutralization function.
History
DepositionJul 10, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 1, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Circular tendon repeat protein
B: Circular tendon repeat protein
C: Circular tendon repeat protein


Theoretical massNumber of molelcules
Total (without water)159,4243
Polymers159,4243
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Circular tendon repeat protein


Mass: 53141.402 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli (E. coli)

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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: trimer of tendon repeat protein / Type: COMPLEX
Details: circular tendon repeats based on de in silico structure design
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: unidentified (others)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: solution was diluted immediate prior to flash freezing
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium ChlorideNaCl1
220 mMHEPESC8H18N2O4S1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was more disperse
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K / Details: blot for 5 seconds before plunging

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 38000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 100 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 420
Image scansWidth: 3710 / Height: 3838 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC2.13particle selectionblob picker
2Leginonimage acquisition
4cryoSPARC2.13CTF correctionpatch CTF
7UCSF Chimeramodel fitting
10Rosettainitial Euler assignment
13cryoSPARC2.133D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 121850
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121400 / Num. of class averages: 4 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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