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- EMDB-24425: Circular tandem repeat protein with novel repeat topology and enh... -

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Basic information

Entry
Database: EMDB / ID: EMD-24425
TitleCircular tandem repeat protein with novel repeat topology and enhanced subunit contact surfaces
Map datalow resolution cryoEM map of cTRP trimers
Sample
  • Complex: trimer of tendon repeat protein
    • Protein or peptide: Circular tendon repeat protein
Biological speciesunidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsShen BW / Stoddard BL
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseSep 1, 2021-
Map releaseSep 1, 2021-
UpdateNov 17, 2021-
Current statusNov 17, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.26
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.26
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rdr
  • Surface level: 0.26
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7rdr
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24425.png

Downloads & links

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Map

FileDownload / File: emd_24425.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlow resolution cryoEM map of cTRP trimers
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.26 / Movie #1: 0.26
Minimum - Maximum-0.18786378 - 0.7552734
Average (Standard dev.)-0.0014736541 (±0.033771)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 296.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.161.161.16
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z296.960296.960296.960
α/β/γ90.00090.00090.000
start NX/NY/NZ127131139
NX/NY/NZ1079278
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1880.755-0.001

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Supplemental data

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Sample components

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Entire : trimer of tendon repeat protein

EntireName: trimer of tendon repeat protein
Components
  • Complex: trimer of tendon repeat protein
    • Protein or peptide: Circular tendon repeat protein

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Supramolecule #1: trimer of tendon repeat protein

SupramoleculeName: trimer of tendon repeat protein / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: circular tendon repeats based on de in silico structure design
Source (natural)Organism: unidentified (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: Circular tendon repeat protein

MacromoleculeName: Circular tendon repeat protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 53.141402 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SELAARCLII LFQQLVELAR LAIESGDEEL LRRVSEWLEE VIKDMRRVVE QALREGNSEL AARILIILFQ QLVELARLAI ESGDEELLR RVSEWLEEVI KDMRRVVEQA LREGNSELAA RILIILFQQL VELARLAIES GDEELLRRVS EWLEEVIKDM R RVVEQALR ...String:
SELAARCLII LFQQLVELAR LAIESGDEEL LRRVSEWLEE VIKDMRRVVE QALREGNSEL AARILIILFQ QLVELARLAI ESGDEELLR RVSEWLEEVI KDMRRVVEQA LREGNSELAA RILIILFQQL VELARLAIES GDEELLRRVS EWLEEVIKDM R RVVEQALR EGNSELAARI LIILFQQLVE LARLAIESGD EELLRRVSEW LEEVIKDMRR VVEQALREGN SELAARILII LF QQLVELA RLAIESGDEE LLRRVSEWLE EVIKDMRRVV EQALREGNSE LAARILIILF QQLVELARLA IESGDEELLR RVS EWLEEV IKDMRRVVEQ ALREGNSELA ARILIILFQQ LVELARLAIE SGDEELLRRV SEWLEEVIKD MRRVVEQALR EGNS ELACR ILIILFQQLV ELARLAIESG DEELLRRVSE WLEEVIKDMR RVVEQALREG N

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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

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium Chloride
20.0 mMC8H18N2O4SHEPES

Details: solution was diluted immediate prior to flash freezing
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5 seconds before plunging.
DetailsThis sample was more disperse

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Electron microscopy

MicroscopeFEI TECNAI 20
TemperatureMin: 100.0 K / Max: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 420 / Average exposure time: 10.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus min: 1.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 38000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 121850
CTF correctionSoftware - Name: cryoSPARC (ver. 2.13) / Software - details: patch CTF
Startup modelType of model: INSILICO MODEL / In silico model: based on helix-turn-helix units.
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.13) / Number images used: 121400
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: Rosetta
Final angle assignmentType: ANGULAR RECONSTITUTION

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7rdr:
Circular tandem repeat protein with novel repeat topology and enhanced subunit contact surfaces

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