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- PDB-8e55: Design of Diverse Asymmetric Pockets in de novo Homo-oligomeric P... -

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Basic information

Entry
Database: PDB / ID: 8.0E+55
TitleDesign of Diverse Asymmetric Pockets in de novo Homo-oligomeric Proteins
ComponentsSG135
KeywordsDE NOVO PROTEIN / tetramer / oligomer / pockets / de novo design / rosetta / cryoEM
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsGerben, S. / Borst, A.J. / 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 19, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: SG135
B: SG135
C: SG135
D: SG135


Theoretical massNumber of molelcules
Total (without water)94,0874
Polymers94,0874
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, negative stain EM, cryoEM
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
SG135


Mass: 23521.809 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Deleted loop consisting of residues 173-179 due to lack of confident map density
Source: (gene. exp.) synthetic construct (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: SG135 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 1700 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 63.56 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
9cryoSPARC3.2initial Euler assignment
10cryoSPARC3.2final Euler assignment
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2944810
3D reconstructionResolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 855664
Details: Removed large number over over-represented views from initial set of particles.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0076092
ELECTRON MICROSCOPYf_angle_d0.658164
ELECTRON MICROSCOPYf_dihedral_angle_d12.3572408
ELECTRON MICROSCOPYf_chiral_restr0.031004
ELECTRON MICROSCOPYf_plane_restr0.0041044

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