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- PDB-6o35: Crystal structure of a de novo designed octameric helical-bundle ... -

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Entry
Database: PDB / ID: 6o35
TitleCrystal structure of a de novo designed octameric helical-bundle protein
Componentsde novo designed WSHC8
KeywordsDE NOVO PROTEIN / Helical bundle / octamer / computational design / pore
Biological speciessynthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 Å
AuthorsBick, M.J. / Xu, C. / Sankaran, B. / Baker, D.
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
DepositionFeb 25, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 18, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Structure summary / Category: audit_author / citation_author
Revision 1.2Sep 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Sep 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_author.identifier_ORCID
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.5Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: de novo designed WSHC8
B: de novo designed WSHC8
C: de novo designed WSHC8
D: de novo designed WSHC8


Theoretical massNumber of molelcules
Total (without water)48,2354
Polymers48,2354
Non-polymers00
Water30617
1
A: de novo designed WSHC8
B: de novo designed WSHC8
C: de novo designed WSHC8
D: de novo designed WSHC8

A: de novo designed WSHC8
B: de novo designed WSHC8
C: de novo designed WSHC8
D: de novo designed WSHC8


Theoretical massNumber of molelcules
Total (without water)96,4698
Polymers96,4698
Non-polymers00
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-x+1,-y,z1
Buried area20890 Å2
ΔGint-241 kcal/mol
Surface area40860 Å2
MethodPISA
Unit cell
Length a, b, c (Å)59.440, 103.684, 72.980
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein
de novo designed WSHC8


Mass: 12058.667 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 17 / 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: 2.34 Å3/Da / Density % sol: 47.39 %
Crystal growTemperature: 290 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 0.3 M Magnesium formate dihydrate 0.1 M BIS-Tris 5.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.1 / Wavelength: 1.00002 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Aug 17, 2018
RadiationMonochromator: Double-crystal Si(111) and multilayer / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.00002 Å / Relative weight: 1
ReflectionResolution: 2.4→34.444 Å / Num. obs: 18231 / % possible obs: 99.57 % / Redundancy: 7 % / Biso Wilson estimate: 40.86 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.1921 / Rpim(I) all: 0.07916 / Rrim(I) all: 0.2081 / Net I/σ(I): 8.28
Reflection shellResolution: 2.4→2.486 Å / Redundancy: 7.2 % / Rmerge(I) obs: 1.698 / Mean I/σ(I) obs: 1.43 / Num. unique obs: 1791 / CC1/2: 0.804 / Rpim(I) all: 0.6821 / Rrim(I) all: 1.823 / % possible all: 99.44

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Processing

Software
NameVersionClassification
PHENIX(dev_3112: ???)refinement
XDSJan 26, 2018data reduction
XDSJan 26, 2018data scaling
PHASER2.8.2.phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Computational model

Resolution: 2.4→34.444 Å / SU ML: 0.4 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 32.47
RfactorNum. reflection% reflection
Rfree0.2984 2172 6.4 %
Rwork0.2609 --
obs0.2634 18190 99.52 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.4→34.444 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2914 0 0 17 2931
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0022936
X-RAY DIFFRACTIONf_angle_d0.3653956
X-RAY DIFFRACTIONf_dihedral_angle_d12.3511844
X-RAY DIFFRACTIONf_chiral_restr0.019480
X-RAY DIFFRACTIONf_plane_restr0.001503
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4-2.45220.43071330.39941993X-RAY DIFFRACTION100
2.4522-2.50920.38191350.34761957X-RAY DIFFRACTION99
2.5092-2.57190.34931390.32781999X-RAY DIFFRACTION100
2.5719-2.64150.2931360.30971985X-RAY DIFFRACTION100
2.6415-2.71920.29261340.31762009X-RAY DIFFRACTION100
2.7192-2.80690.34151350.30611983X-RAY DIFFRACTION100
2.8069-2.90720.34511350.30371979X-RAY DIFFRACTION100
2.9072-3.02350.32061360.2791979X-RAY DIFFRACTION99
3.0235-3.1610.37151370.27282008X-RAY DIFFRACTION100
3.161-3.32750.32881390.27231995X-RAY DIFFRACTION100
3.3275-3.53580.28131340.24991957X-RAY DIFFRACTION100
3.5358-3.80850.2911380.21371989X-RAY DIFFRACTION99
3.8085-4.19120.25861330.20861984X-RAY DIFFRACTION100
4.1912-4.79620.23571390.19531980X-RAY DIFFRACTION99
4.7962-6.03750.29041320.25811983X-RAY DIFFRACTION99
6.0375-34.4480.28791370.28421969X-RAY DIFFRACTION99
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.4228-0.0119-0.19950.32410.04720.10740.12330.0140.0941-0.0736-0.116-0.0622-0.0515-0.039600.43740.0391-0.00020.25350.03160.330724.058617.428836.08
20.3935-0.07780.12271.1779-0.27990.14920.00010.15480.0293-0.13180.001-0.00510.016-0.5656-0.00310.25060.027-0.02260.39890.00770.324312.94348.458335.7695
30.5047-0.20860.00190.77840.18550.166-0.0511-0.05990.02-0.06770.07030.16410.1058-0.036200.2403-0.0727-0.04040.3683-0.01450.339511.902-5.698536.3528
40.4638-0.1783-0.00310.08570.0540.14560.09440.0643-0.0373-0.20960.08870.07230.3757-0.0990.00020.4357-0.0503-0.02670.3455-0.00780.326621.1555-16.516836.2231
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain 'A' and resid 0 through 100)
2X-RAY DIFFRACTION2(chain 'B' and resid 0 through 99)
3X-RAY DIFFRACTION3(chain 'C' and resid 0 through 100)
4X-RAY DIFFRACTION4(chain 'D' and resid 1 through 99)

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