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

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Basic information

Entry
Database: PDB / ID: 8ure
TitleCryoEM Structure of Allosterically Switchable De Novo Protein sr312, in Open State with Effector Peptide
Components
  • Effector peptide cs221B
  • sr312
KeywordsDE NOVO PROTEIN / de novo / allosterically switchable de novo protein / sr312 / effector peptide
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsWeidle, C. / Skotheim, R.
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 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 4, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Sep 11, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: sr312
B: Effector peptide cs221B
C: sr312
D: Effector peptide cs221B
E: sr312
F: Effector peptide cs221B
G: sr312
H: Effector peptide cs221B


Theoretical massNumber of molelcules
Total (without water)201,5578
Polymers201,5578
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, CryoEM and Negative Stain, light scattering, Mass Photometry, gel filtration, Size-exclusion chromatography
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
sr312


Mass: 47297.691 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Protein/peptide
Effector peptide cs221B


Mass: 3091.635 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Complex is comprised of 4 sr312 proteins, each sr312 protein is bound to an effector peptide, putting the protein in an open state. Complex is formed with C4 symmetry.COMPLEXall0MULTIPLE SOURCES
2sr312 proteinCOMPLEX#11RECOMBINANT
3effector peptideCOMPLEX#21SYNTHETIC
Molecular weightValue: 0.2061 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22synthetic construct (others)32630
33synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 150 mM NaCl, 40 mM Tris, pH 8.0
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
240 mMTris - Hydrochloric Acid1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/2
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 recordingElectron dose: 43 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3795

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 971294
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58251 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 207.87 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00858888
ELECTRON MICROSCOPYf_angle_d1.550312392
ELECTRON MICROSCOPYf_chiral_restr0.06531728
ELECTRON MICROSCOPYf_plane_restr0.00991784
ELECTRON MICROSCOPYf_dihedral_angle_d6.57921784

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