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- EMDB-30126: A de novo designed transmembrane nanopore, TMH4C4 -

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Basic information

Entry
Database: EMDB / ID: EMD-30126
TitleA de novo designed transmembrane nanopore, TMH4C4
Map data
Sample
  • Complex: A de novo designed transmembrane nanopore
    • Protein or peptide: TMH4C4
Keywordsnanopore / de novo design / MEMBRANE PROTEIN / DE NOVO PROTEIN
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsLu P / Xu C
Funding support China, United States, 2 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31901054 China
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
DepositionMar 16, 2020-
Header (metadata) releaseJun 24, 2020-
Map releaseJun 24, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6m6z
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30126.png

Downloads & links

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Map

FileDownload / File: emd_30126.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.087 Å
Density
Contour LevelBy AUTHOR: 0.026 / Movie #1: 0.026
Minimum - Maximum-0.048209827 - 0.09325138
Average (Standard dev.)-0.0001038213 (±0.0032721732)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 217.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0871.0871.087
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z217.400217.400217.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0480.093-0.000

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Supplemental data

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Mask #1

Fileemd_30126_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : A de novo designed transmembrane nanopore

EntireName: A de novo designed transmembrane nanopore
Components
  • Complex: A de novo designed transmembrane nanopore
    • Protein or peptide: TMH4C4

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Supramolecule #1: A de novo designed transmembrane nanopore

SupramoleculeName: A de novo designed transmembrane nanopore / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: TMH4C4

MacromoleculeName: TMH4C4 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 23.455572 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SAEELLRRSR EYLKKVALIQ LVIAFVFLIL LILLSWRSEE LIRELEEKGA ASEAELARMK QQHMTAYLQA ALTAWEIISK SVIALLLLQ QNQLNLELNT DTDKNVAEEL LRRSREYLKK VALIQLVIAF VFLILLILLS WRSEELIREL EEKGAASEAE L ARMKQQHM ...String:
SAEELLRRSR EYLKKVALIQ LVIAFVFLIL LILLSWRSEE LIRELEEKGA ASEAELARMK QQHMTAYLQA ALTAWEIISK SVIALLLLQ QNQLNLELNT DTDKNVAEEL LRRSREYLKK VALIQLVIAF VFLILLILLS WRSEELIREL EEKGAASEAE L ARMKQQHM TAYLQAALTA WEIISKSVIA LLLLQQNQLN LELRH

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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

Concentration6 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE-PROPANE

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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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 64739
FSC plot (resolution estimation)

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