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Yorodumi- PDB-6tj1: Crystal structure of a de novo designed hexameric helical-bundle ... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6tj1 | ||||||
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Title | Crystal structure of a de novo designed hexameric helical-bundle protein | ||||||
Components |
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Keywords | BIOSYNTHETIC PROTEIN / Helical bundle / hexamer / computational protein design / pore / de novo protein | ||||||
Biological species | synthetic construct (others) | ||||||
Method | X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å | ||||||
Authors | Xu, C. / Pei, X.Y. / Luisi, B.F. / Baker, D. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nature / Year: 2020 Title: Computational design of transmembrane pores. Authors: Chunfu Xu / Peilong Lu / Tamer M Gamal El-Din / Xue Y Pei / Matthew C Johnson / Atsuko Uyeda / Matthew J Bick / Qi Xu / Daohua Jiang / Hua Bai / Gabriella Reggiano / Yang Hsia / T J Brunette ...Authors: Chunfu Xu / Peilong Lu / Tamer M Gamal El-Din / Xue Y Pei / Matthew C Johnson / Atsuko Uyeda / Matthew J Bick / Qi Xu / Daohua Jiang / Hua Bai / Gabriella Reggiano / Yang Hsia / T J Brunette / Jiayi Dou / Dan Ma / Eric M Lynch / Scott E Boyken / Po-Ssu Huang / Lance Stewart / Frank DiMaio / Justin M Kollman / Ben F Luisi / Tomoaki Matsuura / William A Catterall / David Baker / Abstract: Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in considerable interest in the ...Transmembrane channels and pores have key roles in fundamental biological processes and in biotechnological applications such as DNA nanopore sequencing, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels, and there have been recent advances in de novo membrane protein design and in redesigning naturally occurring channel-containing proteins. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 6tj1.cif.gz | 55.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6tj1.ent.gz | 38.9 KB | Display | PDB format |
PDBx/mmJSON format | 6tj1.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6tj1_validation.pdf.gz | 453.6 KB | Display | wwPDB validaton report |
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Full document | 6tj1_full_validation.pdf.gz | 457.3 KB | Display | |
Data in XML | 6tj1_validation.xml.gz | 10.2 KB | Display | |
Data in CIF | 6tj1_validation.cif.gz | 13.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/tj/6tj1 ftp://data.pdbj.org/pub/pdb/validation_reports/tj/6tj1 | HTTPS FTP |
-Related structure data
-Links
-Assembly
Deposited unit |
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Unit cell |
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Components on special symmetry positions |
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-Components
#1: Protein | Mass: 10557.086 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria) #2: Protein/peptide | | Mass: 486.562 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: A protein purification cleavage tag with a disordered electronic map density Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli) #3: Water | ChemComp-HOH / | |
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-Experimental details
-Experiment
Experiment | Method: X-RAY DIFFRACTION / Number of used crystals: 1 |
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-Sample preparation
Crystal | Density Matthews: 2.04 Å3/Da / Density % sol: 39.79 % |
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Crystal grow | Temperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.6 / Details: 0.1 M MES, pH 6.6, 17.5% PEG 550 MME / PH range: 6.6 |
-Data collection
Diffraction | Mean temperature: 100 K / Ambient temp details: 100 / Serial crystal experiment: Y |
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Diffraction source | Source: SYNCHROTRON / Site: Diamond / Beamline: I02 / Wavelength: 0.9801 Å |
Detector | Type: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jul 29, 2015 / Details: Double Crystal |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Wavelength: 0.9801 Å / Relative weight: 1 |
Reflection | Resolution: 2.35→54.14 Å / Num. obs: 8149 / % possible obs: 95.5 % / Observed criterion σ(I): 3 / Redundancy: 2.9 % / Biso Wilson estimate: 34.41 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.135 / Rpim(I) all: 0.089 / Net I/σ(I): 8.8 |
Reflection shell | Resolution: 2.457622→2.52 Å / Redundancy: 2.9 % / Rmerge(I) obs: 0.08 / Mean I/σ(I) obs: 1 / Num. unique obs: 7210 / CC1/2: 0.812 / % possible all: 95.3 |
Serial crystallography sample delivery | Method: fixed target |
-Processing
Software |
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Refinement | Method to determine structure: MOLECULAR REPLACEMENT Starting model: Rosetta design model Resolution: 2.4→39.5 Å / Cross valid method: THROUGHOUT
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Displacement parameters | Biso max: 158.82 Å2 / Biso mean: 68.1204 Å2 / Biso min: 9.87 Å2 | ||||||||||||||||||||
Refinement step | Cycle: LAST / Resolution: 2.4→39.5 Å
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