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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
Title: De novo design of allosterically switchable protein assemblies
Authors: Pillai, A. / Idris, A. / Philomin, A. / Weidle, C. / Skotheim, R. / Leung, P.J.Y. / Broerman, A. / Demakis, C. / Borst, A.J. / Praetorius, F. / Baker, D.
History
DepositionOct 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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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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