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- PDB-6u1s: Cryo-EM structure of a de novo designed 16-helix transmembrane na... -

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Entry
Database: PDB / ID: 6u1s
TitleCryo-EM structure of a de novo designed 16-helix transmembrane nanopore, TMHC8_R.
Componentsde novo designed 16-helix transmembrane nanopore, TMHC8_R
KeywordsDE NOVO PROTEIN / Transmembrane / Pore / Rosetta
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsJohnson, M.J. / Reggiano, G. / Xu, C. / Lu, P. / Hsia, Y. / Brunette, T.J. / DiMaio, F. / Baker, D. / Kollman, J.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: 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
DepositionAug 16, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: de novo designed 16-helix transmembrane nanopore, TMHC8_R
B: de novo designed 16-helix transmembrane nanopore, TMHC8_R
D: de novo designed 16-helix transmembrane nanopore, TMHC8_R
F: de novo designed 16-helix transmembrane nanopore, TMHC8_R
H: de novo designed 16-helix transmembrane nanopore, TMHC8_R
J: de novo designed 16-helix transmembrane nanopore, TMHC8_R
L: de novo designed 16-helix transmembrane nanopore, TMHC8_R
N: de novo designed 16-helix transmembrane nanopore, TMHC8_R


Theoretical massNumber of molelcules
Total (without water)280,0138
Polymers280,0138
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
de novo designed 16-helix transmembrane nanopore, TMHC8_R


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

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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 transmembrane nanopore TMHC8_R / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
7Rosettamodel fitting
13Rosettamodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18622 / Symmetry type: POINT

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