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- PDB-8e1e: Scaffolding protein functional sites using deep learning -

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Basic information

Entry
Database: PDB / ID: 8e1e
TitleScaffolding protein functional sites using deep learning
ComponentsSG122_C3
KeywordsDE NOVO PROTEIN / DE NOVO DESIGN / Scaffolding protein / ligand binding / pocket
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 4.27 Å
AuthorsBera, A.K. / Gerben, S. / Baker, D.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Biochemistry / Year: 2023
Title: Design of Diverse Asymmetric Pockets in Homo-oligomeric Proteins.
Authors: Stacey R Gerben / Andrew J Borst / Derrick R Hicks / Isabelle Moczygemba / David Feldman / Brian Coventry / Wei Yang / Asim K Bera / Marcos Miranda / Alex Kang / Hannah Nguyen / David Baker /
Abstract: A challenge for design of protein-small-molecule recognition is that incorporation of cavities with size, shape, and composition suitable for specific recognition can considerably destabilize protein ...A challenge for design of protein-small-molecule recognition is that incorporation of cavities with size, shape, and composition suitable for specific recognition can considerably destabilize protein monomers. This challenge can be overcome through binding pockets formed at homo-oligomeric interfaces between folded monomers. Interfaces surrounding the central homo-oligomer symmetry axes necessarily have the same symmetry and so may not be well suited to binding asymmetric molecules. To enable general recognition of arbitrary asymmetric substrates and small molecules, we developed an approach to designing asymmetric interfaces at off-axis sites on homo-oligomers, analogous to those found in native homo-oligomeric proteins such as glutamine synthetase. We symmetrically dock curved helical repeat proteins such that they form pockets at the asymmetric interface of the oligomer with sizes ranging from several angstroms, appropriate for binding a single ion, to up to more than 20 Å across. Of the 133 proteins tested, 84 had soluble expression in , 47 had correct oligomeric states in solution, 35 had small-angle X-ray scattering (SAXS) data largely consistent with design models, and 8 had negative-stain electron microscopy (nsEM) 2D class averages showing the structures coming together as designed. Both an X-ray crystal structure and a cryogenic electron microscopy (cryoEM) structure are close to the computational design models. The nature of these proteins as homo-oligomers allows them to be readily built into higher-order structures such as nanocages, and the asymmetric pockets of these structures open rich possibilities for small-molecule binder design free from the constraints associated with monomer destabilization.
History
DepositionAug 10, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 16, 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: SG122_C3
B: SG122_C3
C: SG122_C3


Theoretical massNumber of molelcules
Total (without water)83,4763
Polymers83,4763
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)234.092, 234.092, 234.092
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number197
Space group name H-MI23
Space group name HallI223
Symmetry operation#1: x,y,z
#2: z,x,y
#3: y,z,x
#4: -y,-z,x
#5: z,-x,-y
#6: -y,z,-x
#7: -z,-x,y
#8: -z,x,-y
#9: y,-z,-x
#10: x,-y,-z
#11: -x,y,-z
#12: -x,-y,z
#13: x+1/2,y+1/2,z+1/2
#14: z+1/2,x+1/2,y+1/2
#15: y+1/2,z+1/2,x+1/2
#16: -y+1/2,-z+1/2,x+1/2
#17: z+1/2,-x+1/2,-y+1/2
#18: -y+1/2,z+1/2,-x+1/2
#19: -z+1/2,-x+1/2,y+1/2
#20: -z+1/2,x+1/2,-y+1/2
#21: y+1/2,-z+1/2,-x+1/2
#22: x+1/2,-y+1/2,-z+1/2
#23: -x+1/2,y+1/2,-z+1/2
#24: -x+1/2,-y+1/2,z+1/2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1SERLYSA1 - 232
d_21ens_1SERLYSB1 - 232
d_31ens_1SERLYSC1 - 232

NCS oper:
IDCodeMatrixVector
1given(-0.168559394801, -0.738593306307, -0.652738583433), (-0.505139351846, 0.633372937459, -0.58623626407), (0.846417134539, 0.230908315078, -0.479853503047)-48.2978766606, -10.6324169042, 100.483568008
2given(-0.188427840534, -0.456296611438, 0.869648406715), (-0.73363920145, 0.654087800205, 0.184235370432), (-0.652892388536, -0.603293089644, -0.458005433352)-99.6919666687, -49.6434899269, 9.40321322715

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Components

#1: Protein SG122_C3


Mass: 27825.336 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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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: 6.4 Å3/Da / Density % sol: 80.79 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: morphous c9

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER2 S 16M / Detector: PIXEL / Date: Mar 18, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 4.26→117.05 Å / Num. obs: 28776 / % possible obs: 99.9 % / Redundancy: 40.9 % / Biso Wilson estimate: 281.65 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.143 / Net I/σ(I): 22.1
Reflection shellResolution: 4.26→4.76 Å / Rmerge(I) obs: 4.145 / Mean I/σ(I) obs: 1.2 / Num. unique obs: 4200 / CC1/2: 0.507

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Designed model

Resolution: 4.27→95.57 Å / SU ML: 0.9466 / Cross valid method: FREE R-VALUE / σ(F): 0.37 / Phase error: 54.1727
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.4269 1432 4.98 %
Rwork0.405 27344 -
obs0.4061 28776 99.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 249.98 Å2
Refinement stepCycle: LAST / Resolution: 4.27→95.57 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5622 0 0 0 5622
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00315661
X-RAY DIFFRACTIONf_angle_d0.70227557
X-RAY DIFFRACTIONf_chiral_restr0.039867
X-RAY DIFFRACTIONf_plane_restr0.0036969
X-RAY DIFFRACTIONf_dihedral_angle_d4.8852738
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AX-RAY DIFFRACTIONTorsion NCS2.49695553443
ens_1d_3AX-RAY DIFFRACTIONTorsion NCS2.07690491042
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
4.27-4.420.44871960.39622652X-RAY DIFFRACTION97.33
4.42-4.60.40791100.40592792X-RAY DIFFRACTION99.93
4.6-4.80.43381630.4082697X-RAY DIFFRACTION99.97
4.81-5.060.39171460.41922734X-RAY DIFFRACTION99.97
5.06-5.370.42571780.43662673X-RAY DIFFRACTION99.96
5.38-5.790.43351040.43022785X-RAY DIFFRACTION100
5.79-6.370.37411260.46152791X-RAY DIFFRACTION100
6.38-7.290.59071700.46982697X-RAY DIFFRACTION100
7.3-9.180.41621070.40162783X-RAY DIFFRACTION100
9.2-95.570.39751320.38042740X-RAY DIFFRACTION99.38

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