+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-43157 | |||||||||
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Title | A designed tetrahedral protein scaffold - DARP14 | |||||||||
Map data | ||||||||||
Sample |
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Keywords | scaffold / tetrahedral / darpin / symmetrical / DE NOVO PROTEIN | |||||||||
Biological species | synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.7 Å | |||||||||
Authors | Suder DS / Gonen S | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Int J Mol Sci / Year: 2024 Title: Mitigating the Blurring Effect of CryoEM Averaging on a Flexible and Highly Symmetric Protein Complex through Sub-Particle Reconstruction. Authors: Diana S Suder / Shane Gonen / Abstract: Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle CryoEM processing, flexible protein regions can be detrimental to high-resolution ...Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle CryoEM processing, flexible protein regions can be detrimental to high-resolution reconstruction as signals from thousands of particles are averaged together. This "blurring" effect can be difficult to overcome and is possibly more pronounced when averaging highly symmetric complexes. Approaches to mitigating flexibility during CryoEM processing are becoming increasingly critical as the technique advances and is applied to more dynamic proteins and complexes. Here, we detail the use of sub-particle averaging and signal subtraction techniques to precisely target and resolve flexible DARPin protein attachments on a designed tetrahedrally symmetric protein scaffold called DARP14. Particles are first aligned as full complexes, and then the symmetry is reduced by alignment and focused refinement of the constituent subunits. The final reconstructions we obtained were vastly improved over the fully symmetric reconstructions, with observable secondary structure and side-chain placement. Additionally, we were also able to reconstruct the core region of the scaffold to 2.7 Å. The data processing protocol outlined here is applicable to other dynamic and symmetric protein complexes, and our improved maps could allow for new structure-guided variant designs of DARP14. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_43157.map.gz | 9.9 MB | EMDB map data format | |
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Header (meta data) | emd-43157-v30.xml emd-43157.xml | 16.1 KB 16.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_43157_fsc.xml | 10.5 KB | Display | FSC data file |
Images | emd_43157.png | 94.4 KB | ||
Filedesc metadata | emd-43157.cif.gz | 5.3 KB | ||
Others | emd_43157_half_map_1.map.gz emd_43157_half_map_2.map.gz | 77.4 MB 77.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-43157 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-43157 | HTTPS FTP |
-Validation report
Summary document | emd_43157_validation.pdf.gz | 700 KB | Display | EMDB validaton report |
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Full document | emd_43157_full_validation.pdf.gz | 699.5 KB | Display | |
Data in XML | emd_43157_validation.xml.gz | 17.7 KB | Display | |
Data in CIF | emd_43157_validation.cif.gz | 23.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43157 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-43157 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_43157.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 0.9825 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_43157_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_43157_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : A12B12 tetrahedral complex of a designed protein scaffold called ...
Entire | Name: A12B12 tetrahedral complex of a designed protein scaffold called DARP14 |
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Components |
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-Supramolecule #1: A12B12 tetrahedral complex of a designed protein scaffold called ...
Supramolecule | Name: A12B12 tetrahedral complex of a designed protein scaffold called DARP14 type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: synthetic construct (others) |
-Supramolecule #2: Subunit A in a designed protein scaffold called DARP14
Supramolecule | Name: Subunit A in a designed protein scaffold called DARP14 type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: synthetic construct (others) |
-Supramolecule #3: Subunit B in a designed protein scaffold called DARP14
Supramolecule | Name: Subunit B in a designed protein scaffold called DARP14 type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: synthetic construct (others) |
-Macromolecule #1: Subunit A
Macromolecule | Name: Subunit A / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: corrinoid adenosyltransferase |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 15.775438 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: KDSPIIEANG TLDELTSFIG EAKHYVDEEM KGILEEIQND IYKIMGEIGS KGKIEGISEE RIAWLLKLIL RYMEMVNLKS FVLPGGTLE SAKLDVCRTI ARRALRKVLT VTREFGIGAE AAAYLLALSD LLFLLARVIE IE |
-Macromolecule #2: Subunit B
Macromolecule | Name: Subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 12.630332 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: PHLVIEATAN LRLETSPGEL LEQANKALFA SGQFGEADIK SRFVTLEAYR QGTAAVERAY LHACLSILDG RDIATRTLLG ASLCAVLAE AVAGGGEEGV QVSVEVREME RLSYAKRVV |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 8 |
Grid | Material: COPPER / Mesh: 200 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 30.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7000000000000001 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Details | Phenix real space refine |
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Refinement | Space: REAL |
Output model | PDB-8vdz: |