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Yorodumi- PDB-6u1s: Cryo-EM structure of a de novo designed 16-helix transmembrane na... -
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-Basic information
Entry | Database: PDB / ID: 6u1s | ||||||
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Title | Cryo-EM structure of a de novo designed 16-helix transmembrane nanopore, TMHC8_R. | ||||||
Components | de novo designed 16-helix transmembrane nanopore, TMHC8_R | ||||||
Keywords | DE NOVO PROTEIN / Transmembrane / Pore / Rosetta | ||||||
Biological species | synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.6 Å | ||||||
Authors | Johnson, M.J. / Reggiano, G. / Xu, C. / Lu, P. / Hsia, Y. / Brunette, T.J. / DiMaio, F. / Baker, D. / Kollman, J. | ||||||
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
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Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 6u1s.cif.gz | 789.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6u1s.ent.gz | 658 KB | Display | PDB format |
PDBx/mmJSON format | 6u1s.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6u1s_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 6u1s_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 6u1s_validation.xml.gz | 70.5 KB | Display | |
Data in CIF | 6u1s_validation.cif.gz | 99.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/u1/6u1s ftp://data.pdbj.org/pub/pdb/validation_reports/u1/6u1s | HTTPS FTP |
-Related structure data
Related structure data | 20613MC 6m6zC 6o35C 6tj1C 6tmsC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 35001.648 Da / Num. of mol.: 8 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
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: de novo designed transmembrane nanopore TMHC8_R / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT |
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Source (natural) | Organism: synthetic construct (others) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 70 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||
Symmetry | Point symmetry: C8 (8 fold cyclic) | |||||||||
3D reconstruction | Resolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18622 / Symmetry type: POINT |