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- PDB-8gel: Cryo-EM structure of synthetic tetrameric building block sC4 -

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Basic information

Entry
Database: PDB / ID: 8gel
TitleCryo-EM structure of synthetic tetrameric building block sC4
ComponentssC4
KeywordsDE NOVO PROTEIN / synthetic / tetramer / self-assembling
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsRedler, R.L. / Huddy, T.F. / Hsia, Y. / Baker, D. / Ekiert, D. / Bhabha, G.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nature / Year: 2024
Title: Blueprinting extendable nanomaterials with standardized protein blocks.
Authors: Timothy F Huddy / Yang Hsia / Ryan D Kibler / Jinwei Xu / Neville Bethel / Deepesh Nagarajan / Rachel Redler / Philip J Y Leung / Connor Weidle / Alexis Courbet / Erin C Yang / Asim K Bera / ...Authors: Timothy F Huddy / Yang Hsia / Ryan D Kibler / Jinwei Xu / Neville Bethel / Deepesh Nagarajan / Rachel Redler / Philip J Y Leung / Connor Weidle / Alexis Courbet / Erin C Yang / Asim K Bera / Nicolas Coudray / S John Calise / Fatima A Davila-Hernandez / Hannah L Han / Kenneth D Carr / Zhe Li / Ryan McHugh / Gabriella Reggiano / Alex Kang / Banumathi Sankaran / Miles S Dickinson / Brian Coventry / T J Brunette / Yulai Liu / Justas Dauparas / Andrew J Borst / Damian Ekiert / Justin M Kollman / Gira Bhabha / David Baker /
Abstract: A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The ...A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.
History
DepositionMar 7, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 10, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: sC4
B: sC4
C: sC4
D: sC4


Theoretical massNumber of molelcules
Total (without water)231,3044
Polymers231,3044
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
sC4


Mass: 57826.109 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)

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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: De novo designed tetramer sC4 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: blot time = 4s

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.6 sec. / Electron dose: 47.07 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3850
EM imaging opticsEnergyfilter slit width: 30 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC2particle selection
2Leginon3image acquisition
4cryoSPARC2CTF correction
5CTFFINDCTF correction
8UCSF Chimera1.13model fitting
10cryoSPARCinitial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
14PHENIX1.16model refinement
15Coot0.8.9.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3781336
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 378829 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 224.3 / Protocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00715600
ELECTRON MICROSCOPYf_angle_d0.86120904
ELECTRON MICROSCOPYf_dihedral_angle_d14.38210080
ELECTRON MICROSCOPYf_chiral_restr0.0442588
ELECTRON MICROSCOPYf_plane_restr0.0032668

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