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- PDB-6xr2: Computationally designed right-handed alpha/alpha homotrimeric to... -

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Basic information

Entry
Database: PDB / ID: 6xr2
TitleComputationally designed right-handed alpha/alpha homotrimeric toroid with 3 repeats per subunit
ComponentsdTor_3x57R
KeywordsDE NOVO PROTEIN / Computationally designed / de novo / toroid / helix-turn-helix
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.2 Å
AuthorsHallinan, J.P. / Doyle, L. / Bradley, P. / Stoddard, B.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM123378 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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 14, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 10, 2021Group: Database references / Category: citation / citation_author / database_2
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 / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 22, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: dTor_3x57R
B: dTor_3x57R
C: dTor_3x57R
D: dTor_3x57R
E: dTor_3x57R
F: dTor_3x57R


Theoretical massNumber of molelcules
Total (without water)113,7096
Polymers113,7096
Non-polymers00
Water23413
1
A: dTor_3x57R
B: dTor_3x57R
C: dTor_3x57R


Theoretical massNumber of molelcules
Total (without water)56,8553
Polymers56,8553
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5720 Å2
ΔGint-48 kcal/mol
Surface area20960 Å2
MethodPISA
2
D: dTor_3x57R
E: dTor_3x57R
F: dTor_3x57R


Theoretical massNumber of molelcules
Total (without water)56,8553
Polymers56,8553
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5440 Å2
ΔGint-48 kcal/mol
Surface area20540 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.647, 78.930, 112.694
Angle α, β, γ (deg.)90.000, 93.820, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and ((resid 4 and (name N or name...
21(chain B and ((resid 4 and (name N or name...
31(chain C and ((resid 4 and (name N or name...
41(chain D and ((resid 4 and (name O or name...
51(chain E and ((resid 4 and (name O or name...
61(chain F and ((resid 4 and (name O or name...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and ((resid 4 and (name N or name...A0
211(chain B and ((resid 4 and (name N or name...B0
311(chain C and ((resid 4 and (name N or name...C0
411(chain D and ((resid 4 and (name O or name...D0
511(chain E and ((resid 4 and (name O or name...E0
611(chain F and ((resid 4 and (name O or name...F4
621(chain F and ((resid 4 and (name O or name...F2 - 169
631(chain F and ((resid 4 and (name O or name...F2 - 169
641(chain F and ((resid 4 and (name O or name...F2 - 169

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Components

#1: Protein
dTor_3x57R


Mass: 18951.506 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 13 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.25 Å3/Da / Density % sol: 45.33 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 4.5
Details: 0.1 M calcium acetate, 0.1 M sodium acetate, pH 4.5, 25% PEG400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.54 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: Jan 9, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 3.198→50 Å / Num. obs: 14721 / % possible obs: 88 % / Redundancy: 5.5 % / Biso Wilson estimate: 49.76 Å2 / Rmerge(I) obs: 0.221 / Rpim(I) all: 0.097 / Rrim(I) all: 0.243 / Χ2: 1.416 / Net I/σ(I): 5
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
3.2-3.315.50.69814890.7820.3030.7641.49890
3.31-3.455.50.46414550.8310.2020.5091.47587.9
3.45-3.65.50.4714710.8950.2050.5151.44487.2
3.6-3.795.50.2714660.9280.1180.2961.55589.4
3.79-4.035.30.35814620.9520.1580.3931.55187.7
4.03-4.345.30.25114410.9760.1140.2771.44386.6
4.34-4.785.30.22614240.9780.1010.2491.43385.4
4.78-5.475.30.30614260.9650.1370.3371.4185.5
5.47-6.895.30.20514550.9760.0910.2251.35485.4
6.89-5060.07516320.9960.0320.0821.07394.3

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation7.5 Å30.95 Å
Translation7.5 Å30.95 Å

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Processing

Software
NameVersionClassification
PHENIX1.1refinement
HKL-2000data scaling
PHASER2.6.0phasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.2→30.949 Å / SU ML: 0.55 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 36.09 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3292 1472 10.02 %
Rwork0.2426 13218 -
obs0.2514 14690 87.19 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 79.55 Å2 / Biso mean: 30.2152 Å2 / Biso min: 5.46 Å2
Refinement stepCycle: final / Resolution: 3.2→30.949 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7044 0 0 13 7057
Biso mean---20.2 -
Num. residues----1009
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0037060
X-RAY DIFFRACTIONf_angle_d0.7079569
X-RAY DIFFRACTIONf_chiral_restr0.0351193
X-RAY DIFFRACTIONf_plane_restr0.0041291
X-RAY DIFFRACTIONf_dihedral_angle_d18.6534423
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2839X-RAY DIFFRACTION18.079TORSIONAL
12B2839X-RAY DIFFRACTION18.079TORSIONAL
13C2839X-RAY DIFFRACTION18.079TORSIONAL
14D2839X-RAY DIFFRACTION18.079TORSIONAL
15E2839X-RAY DIFFRACTION18.079TORSIONAL
16F2839X-RAY DIFFRACTION18.079TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
3.2-3.30160.37021160.2811110682
3.3016-3.41950.33811460.2309122188
3.4195-3.55620.37971270.2452120689
3.5562-3.71780.33851380.223121889
3.7178-3.91340.39361330.2508119387
3.9134-4.15810.37991250.2451119186
4.1581-4.47820.30131370.2199118487
4.4782-4.92730.33051340.2265116985
4.9273-5.63650.37231300.2901116584
5.6365-7.08740.29851400.2785119787
7.0874-30.9490.26011460.2261136896

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