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- PDB-6tj1: Crystal structure of a de novo designed hexameric helical-bundle ... -

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Basic information

Entry
Database: PDB / ID: 6tj1
TitleCrystal structure of a de novo designed hexameric helical-bundle protein
Components
  • De novo designed WSHC6
  • purification tag
KeywordsBIOSYNTHETIC PROTEIN / Helical bundle / hexamer / computational protein design / pore / de novo protein
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å
AuthorsXu, C. / Pei, X.Y. / Luisi, B.F. / Baker, D.
Funding support United States, 1items
OrganizationGrant numberCountry
United States Department of Agriculture (USDA)FA9550-18-1-0297 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
DepositionNov 23, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 29, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 16, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: De novo designed WSHC6
B: De novo designed WSHC6
C: De novo designed WSHC6
D: purification tag


Theoretical massNumber of molelcules
Total (without water)32,1584
Polymers32,1584
Non-polymers00
Water66737
1
A: De novo designed WSHC6
B: De novo designed WSHC6
C: De novo designed WSHC6
D: purification tag

A: De novo designed WSHC6
B: De novo designed WSHC6
C: De novo designed WSHC6
D: purification tag


Theoretical massNumber of molelcules
Total (without water)64,3168
Polymers64,3168
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area16880 Å2
ΔGint-162 kcal/mol
Surface area19970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)45.720, 54.130, 79.170
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21221
Components on special symmetry positions
IDModelComponents
11A-111-

HOH

21A-112-

HOH

31A-113-

HOH

41A-114-

HOH

51A-115-

HOH

61B-105-

HOH

71B-106-

HOH

81B-107-

HOH

91B-108-

HOH

101B-109-

HOH

111B-110-

HOH

121B-111-

HOH

131B-112-

HOH

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Components

#1: Protein De novo designed WSHC6


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 purification tag


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 / water / Water


Mass: 18.015 Da / Num. of mol.: 37 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.04 Å3/Da / Density % sol: 39.79 %
Crystal growTemperature: 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

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: 100 / Serial crystal experiment: Y
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I02 / Wavelength: 0.9801 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jul 29, 2015 / Details: Double Crystal
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9801 Å / Relative weight: 1
ReflectionResolution: 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 shellResolution: 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 deliveryMethod: fixed target

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Processing

Software
NameVersionClassification
PHENIXv1.11refinement
Aimlessccp4-7.0data scaling
PDB_EXTRACT3.25data extraction
MOSFLMccp4-7.0data reduction
AutoSolphenix phaser-MRphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Rosetta design model

Resolution: 2.4→39.5 Å / Cross valid method: THROUGHOUT
RfactorNum. reflection% reflectionSelection details
Rfree0.2802 -0.05 %random selection
Rwork0.2539 ---
obs0.2547 7622 95.3 %-
Displacement parametersBiso max: 158.82 Å2 / Biso mean: 68.1204 Å2 / Biso min: 9.87 Å2
Refinement stepCycle: LAST / Resolution: 2.4→39.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1598 0 0 37 1635

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