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- EMDB-20613: Cryo-EM structure of a de novo designed 16-helix transmembrane na... -

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Entry
Database: EMDB / ID: EMD-20613
TitleCryo-EM structure of a de novo designed 16-helix transmembrane nanopore, TMHC8_R.
Map data
Sample
  • Organelle or cellular component: de novo designed transmembrane nanopore TMHC8_R
    • Protein or peptide: de novo designed 16-helix transmembrane nanopore, TMHC8_R
KeywordsTransmembrane / Pore / Rosetta / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsJohnson MJ / Reggiano G
Funding support United States, 1 items
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, 2019-
Header (metadata) releaseAug 19, 2020-
Map releaseAug 19, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.009
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  • Surface view with fitted model
  • Atomic models: PDB-6u1s
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6u1s
  • Imaged by Jmol
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20613.png

Downloads & links

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Map

FileDownload / File: emd_20613.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 320 pix.
= 336. Å		1.05 Å/pix.
x 320 pix.
= 336. Å		1.05 Å/pix.
x 320 pix.
= 336. Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.009 / Movie #1: 0.009
Minimum - Maximum-0.0032775076 - 0.016442971
Average (Standard dev.)0.00015542393 (±0.0010988339)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z336.000336.000336.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-205-205-205
NX/NY/NZ411411411
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0030.0160.000

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Supplemental data

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Mask #1

Fileemd_20613_msk_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_20613_half_map_1.map
Projections & Slices
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Histogram

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Half map: #2

Fileemd_20613_half_map_2.map
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Sample components

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Entire : de novo designed transmembrane nanopore TMHC8_R

EntireName: de novo designed transmembrane nanopore TMHC8_R
Components
  • Organelle or cellular component: de novo designed transmembrane nanopore TMHC8_R
    • Protein or peptide: de novo designed 16-helix transmembrane nanopore, TMHC8_R

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Supramolecule #1: de novo designed transmembrane nanopore TMHC8_R

SupramoleculeName: de novo designed transmembrane nanopore TMHC8_R / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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

MacromoleculeName: de novo designed 16-helix transmembrane nanopore, TMHC8_R
type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 35.001648 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EEFMARAISA IAELAKKAIE AIYRLADNHT TDTFMAKAIE AIAELAKEAI KAIADLAKNH TTEEFMARAI SAIAELARKA IDAIYRLAR NHTTDTFMAK AIEAIAELAK EAIKAIADLA KNHTTEDFMD EAISAIAELA RKAIEAILRL ASNLTSETYM R KAQEAIEK ...String:
EEFMARAISA IAELAKKAIE AIYRLADNHT TDTFMAKAIE AIAELAKEAI KAIADLAKNH TTEEFMARAI SAIAELARKA IDAIYRLAR NHTTDTFMAK AIEAIAELAK EAIKAIADLA KNHTTEDFMD EAISAIAELA RKAIEAILRL ASNLTSETYM R KAQEAIEK IARTAEEAIR DLARNLEDQE RRERAKSARD EIKRFAEDAR KKIEVLALLK RSREYLKKVA LIQLVIAFVF LI LLILLSW RSEELIRELE EKGAASEAEL ARMKQQHMTA YLQAALTAWE IISKSVIALL LLQQNQLNLE LRH

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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

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: CisTEM ab initio EM map
Final reconstructionApplied symmetry - Point group: C8 (8 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 18622
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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