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- PDB-8utm: CryoEM Structure of Allosterically Switchable De Novo Protein sr3... -

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Basic information

Entry
Database: PDB / ID: 8utm
TitleCryoEM Structure of Allosterically Switchable De Novo Protein sr322, In Closed State without Effector Peptide, off Target Multimeric State
Componentsde novo protein sr322
KeywordsDE NOVO PROTEIN / Allosterically Switchable Protein / sr322 / closed state
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.32 Å
AuthorsWeidle, C. / Borst, A.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2024
Title: De novo design of allosterically switchable protein assemblies.
Authors: Arvind Pillai / Abbas Idris / Annika Philomin / Connor Weidle / Rebecca Skotheim / Philip J Y Leung / Adam Broerman / Cullen Demakis / Andrew J Borst / Florian Praetorius / David Baker /
Abstract: Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of ...Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such 'action at a distance' modulation and to create synthetic proteins whose functions can be regulated by effectors. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge. Here, inspired by the classic Monod-Wyman-Changeux model of cooperativity, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules to protein interfaces that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain-side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.
History
DepositionOct 31, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Sep 4, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.3Sep 18, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: de novo protein sr322
B: de novo protein sr322
C: de novo protein sr322
D: de novo protein sr322
E: de novo protein sr322
F: de novo protein sr322
G: de novo protein sr322
H: de novo protein sr322


Theoretical massNumber of molelcules
Total (without water)320,7148
Polymers320,7148
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
de novo protein sr322


Mass: 40089.242 Da / Num. of mol.: 8
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: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: sr322 / Type: COMPLEX
Details: Complex is made up of 4 monomeric proteins, and is purified as a 4 sided multimer sr322. In this structure two copies of sr322 interact in an off target way.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.326592 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 8 / Details: 150 mM NaCl, 40 mM Tris pH 8.0
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
240 mMTris hydrochloric acidTris-HCL1
SpecimenConc.: 0.971 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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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: 1800 nm / Nominal defocus min: 800 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
Image recordingAverage exposure time: 5 sec. / Electron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4213

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Processing

EM software
IDNameCategory
7UCSF ChimeraXmodel fitting
8ISOLDEmodel fitting
10Cootmodel refinement
11PyMOLmodel refinement
12PHENIXmodel refinement
15cryoSPARCclassification
16cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 699357
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144551 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Initial fit was done using Chimera using Rigid body from In silico model. Then Isolde and Namdinator were used. Coot and Phenix were also used.
Atomic model buildingType: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 176.36 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009114168
ELECTRON MICROSCOPYf_angle_d1.548719760
ELECTRON MICROSCOPYf_chiral_restr0.0872752
ELECTRON MICROSCOPYf_plane_restr0.00982848
ELECTRON MICROSCOPYf_dihedral_angle_d7.59322848

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