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- EMDB-27903: Design of Diverse Asymmetric Pockets in de novo Homo-oligomeric P... -

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Basic information

Entry
Database: EMDB / ID: EMD-27903
TitleDesign of Diverse Asymmetric Pockets in de novo Homo-oligomeric Proteins
Map dataSharpened Map
Sample
  • Complex: SG135
    • Protein or peptide: SG135
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsGerben S / Borst AJ / Baker D
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateJan 25, 2023-
Current statusJan 25, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27903.png

Downloads & links

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Map

FileDownload / File: emd_27903.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened Map
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.434
Minimum - Maximum-1.5376469 - 2.2144988
Average (Standard dev.)0.0010285008 (±0.06110269)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 184.79999 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened Map

Fileemd_27903_additional_1.map
AnnotationUnsharpened Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_27903_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_27903_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SG135

EntireName: SG135
Components
  • Complex: SG135
    • Protein or peptide: SG135

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Supramolecule #1: SG135

SupramoleculeName: SG135 / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: SG135

MacromoleculeName: SG135 / type: protein_or_peptide / ID: 1
Details: Deleted loop consisting of residues 173-179 due to lack of confident map density
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.521809 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: DREIKEEARK LIREAIELLQ KGDPRAKEIL RQAILILLAI RLLEEMEENI EKAEKLGNEE LSELAKRAIK LVREALELLK EGDPRAEEI LKLALKIIKA ILLLLEMYEN IKQAEELGDE DLSELAKIAI RLVRQALKLL QEGDPRAEEI LEIALRIIKL I LQLLFLKQ ...String:
DREIKEEARK LIREAIELLQ KGDPRAKEIL RQAILILLAI RLLEEMEENI EKAEKLGNEE LSELAKRAIK LVREALELLK EGDPRAEEI LKLALKIIKA ILLLLEMYEN IKQAEELGDE DLSELAKIAI RLVRQALKLL QEGDPRAEEI LEIALRIIKL I LQLLFLKQ RIEEAKKKGD QQFVFEAEEK IRRIVEELFK LLEG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.5
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 63.56 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2944810
Startup modelType of model: NONE / Details: Ab initio
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Removed large number over over-represented views from initial set of particles.
Number images used: 855664
FSC plot (resolution estimation)

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