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Entry
Database: PDB / ID: 6tms
TitleCrystal structure of a de novo designed hexameric helical-bundle protein
Components
  • (a novel designed pore protein) x 2
  • affinity 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: 2.7 Å
AuthorsXu, C. / Pei, X.Y. / Luisi, B.F. / Baker, D.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute 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
DepositionDec 5, 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 / diffrn_radiation / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _diffrn_radiation.pdbx_diffrn_protocol / _diffrn_radiation.pdbx_monochromatic_or_laue_m_l

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: a novel designed pore protein
B: a novel designed pore protein
C: a novel designed pore protein
E: a novel designed pore protein
F: a novel designed pore protein
G: a novel designed pore protein
D: a novel designed pore protein
H: a novel designed pore protein
I: a novel designed pore protein
J: a novel designed pore protein
K: a novel designed pore protein
L: a novel designed pore protein
Q: affinity purification tag
R: affinity purification tag
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,28119
Polymers98,80014
Non-polymers4805
Water19,4021077
1
A: a novel designed pore protein
B: a novel designed pore protein
C: a novel designed pore protein
E: a novel designed pore protein
F: a novel designed pore protein
G: a novel designed pore protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,7218
Polymers47,5296
Non-polymers1922
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
D: a novel designed pore protein
H: a novel designed pore protein
I: a novel designed pore protein
J: a novel designed pore protein
K: a novel designed pore protein
L: a novel designed pore protein
Q: affinity purification tag
R: affinity purification tag
hetero molecules


Theoretical massNumber of molelcules
Total (without water)51,55911
Polymers51,2718
Non-polymers2883
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)45.880, 54.330, 79.333
Angle α, β, γ (deg.)89.800, 90.080, 89.770
Int Tables number1
Space group name H-MP1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 1 through 8 or resid 10...
21(chain B and (resid 1 through 8 or resid 10...
31(chain C and (resid 1 through 8 or resid 10...
41(chain D and (resid 1 through 8 or resid 10...
51(chain E and (resid 1 through 8 or resid 10...
61(chain F and (resid 1 through 8 or resid 10...
71(chain G and (resid 1 through 8 or resid 10...
81(chain H and (resid 1 through 8 or resid 10...
91(chain I and (resid 1 through 8 or resid 10...
101(chain J and (resid 1 through 8 or resid 10...
111(chain K and (resid 1 through 8 or resid 10 through 69))
121(chain L and (resid 1 through 8 or resid 10...

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resid 1 through 8 or resid 10...A1 - 8
121(chain A and (resid 1 through 8 or resid 10...A10 - 48
131(chain A and (resid 1 through 8 or resid 10...A1 - 69
141(chain A and (resid 1 through 8 or resid 10...A1 - 69
151(chain A and (resid 1 through 8 or resid 10...A1 - 69
161(chain A and (resid 1 through 8 or resid 10...A1 - 69
171(chain A and (resid 1 through 8 or resid 10...A1 - 69
181(chain A and (resid 1 through 8 or resid 10...A1 - 69
211(chain B and (resid 1 through 8 or resid 10...B1 - 8
221(chain B and (resid 1 through 8 or resid 10...B10 - 48
231(chain B and (resid 1 through 8 or resid 10...B49
241(chain B and (resid 1 through 8 or resid 10...B1 - 69
251(chain B and (resid 1 through 8 or resid 10...B1 - 69
261(chain B and (resid 1 through 8 or resid 10...B1 - 69
271(chain B and (resid 1 through 8 or resid 10...B1 - 69
311(chain C and (resid 1 through 8 or resid 10...C1 - 8
321(chain C and (resid 1 through 8 or resid 10...C10 - 48
331(chain C and (resid 1 through 8 or resid 10...C49
341(chain C and (resid 1 through 8 or resid 10...C1 - 69
351(chain C and (resid 1 through 8 or resid 10...C1 - 69
361(chain C and (resid 1 through 8 or resid 10...C1 - 69
371(chain C and (resid 1 through 8 or resid 10...C1 - 69
411(chain D and (resid 1 through 8 or resid 10...D1 - 8
421(chain D and (resid 1 through 8 or resid 10...D10 - 48
431(chain D and (resid 1 through 8 or resid 10...D49
441(chain D and (resid 1 through 8 or resid 10...D1 - 69
451(chain D and (resid 1 through 8 or resid 10...D1 - 69
461(chain D and (resid 1 through 8 or resid 10...D1 - 69
471(chain D and (resid 1 through 8 or resid 10...D1 - 69
511(chain E and (resid 1 through 8 or resid 10...E1 - 8
521(chain E and (resid 1 through 8 or resid 10...E10 - 48
531(chain E and (resid 1 through 8 or resid 10...E49
541(chain E and (resid 1 through 8 or resid 10...E1 - 69
551(chain E and (resid 1 through 8 or resid 10...E1 - 69
561(chain E and (resid 1 through 8 or resid 10...E1 - 69
571(chain E and (resid 1 through 8 or resid 10...E1 - 69
611(chain F and (resid 1 through 8 or resid 10...F1 - 8
621(chain F and (resid 1 through 8 or resid 10...F10 - 48
631(chain F and (resid 1 through 8 or resid 10...F49
641(chain F and (resid 1 through 8 or resid 10...F1 - 69
651(chain F and (resid 1 through 8 or resid 10...F1 - 69
661(chain F and (resid 1 through 8 or resid 10...F1 - 69
671(chain F and (resid 1 through 8 or resid 10...F1 - 69
711(chain G and (resid 1 through 8 or resid 10...G1 - 8
721(chain G and (resid 1 through 8 or resid 10...G10 - 48
731(chain G and (resid 1 through 8 or resid 10...G1 - 69
741(chain G and (resid 1 through 8 or resid 10...G1 - 69
751(chain G and (resid 1 through 8 or resid 10...G1 - 69
761(chain G and (resid 1 through 8 or resid 10...G1 - 69
771(chain G and (resid 1 through 8 or resid 10...G1 - 69
781(chain G and (resid 1 through 8 or resid 10...G1 - 69
811(chain H and (resid 1 through 8 or resid 10...H1 - 8
821(chain H and (resid 1 through 8 or resid 10...H10 - 48
831(chain H and (resid 1 through 8 or resid 10...H49
841(chain H and (resid 1 through 8 or resid 10...H1 - 69
851(chain H and (resid 1 through 8 or resid 10...H1 - 69
861(chain H and (resid 1 through 8 or resid 10...H1 - 69
871(chain H and (resid 1 through 8 or resid 10...H1 - 69
911(chain I and (resid 1 through 8 or resid 10...I1 - 8
921(chain I and (resid 1 through 8 or resid 10...I10 - 48
931(chain I and (resid 1 through 8 or resid 10...I49
941(chain I and (resid 1 through 8 or resid 10...I1 - 69
951(chain I and (resid 1 through 8 or resid 10...I1 - 69
961(chain I and (resid 1 through 8 or resid 10...I1 - 69
971(chain I and (resid 1 through 8 or resid 10...I1 - 69
1011(chain J and (resid 1 through 8 or resid 10...J1 - 8
1021(chain J and (resid 1 through 8 or resid 10...J10 - 48
1031(chain J and (resid 1 through 8 or resid 10...J1 - 69
1041(chain J and (resid 1 through 8 or resid 10...J1 - 69
1051(chain J and (resid 1 through 8 or resid 10...J1 - 69
1061(chain J and (resid 1 through 8 or resid 10...J1 - 69
1071(chain J and (resid 1 through 8 or resid 10...J1 - 69
1081(chain J and (resid 1 through 8 or resid 10...J1 - 69
1111(chain K and (resid 1 through 8 or resid 10 through 69))K0
1211(chain L and (resid 1 through 8 or resid 10...L1 - 8
1221(chain L and (resid 1 through 8 or resid 10...L10 - 48
1231(chain L and (resid 1 through 8 or resid 10...L49
1241(chain L and (resid 1 through 8 or resid 10...L1 - 69
1251(chain L and (resid 1 through 8 or resid 10...L1 - 69
1261(chain L and (resid 1 through 8 or resid 10...L1 - 69
1271(chain L and (resid 1 through 8 or resid 10...L1 - 69

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Components

#1: Protein
a novel designed pore protein


Mass: 7926.179 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein a novel designed pore protein


Mass: 7898.166 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Protein/peptide affinity purification tag


Mass: 1871.095 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) synthetic construct (others)
#4: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1077 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 1.96 Å3/Da / Density % sol: 36.6 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.6 / Details: 0.1 M MES pH 6.6, 17.5% PEG550MME

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Type: OXFORD DIFFRACTION NOVA / Wavelength: 0.98 Å
DetectorType: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Sep 24, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
Reflection twinOperator: h,-k,-l / Fraction: 0.5
ReflectionResolution: 2.5→54 Å / Num. obs: 77817 / % possible obs: 98.7 % / Observed criterion σ(F): 3 / Observed criterion σ(I): 3 / Redundancy: 3 % / Biso Wilson estimate: 58 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.145 / Rpim(I) all: 0.134 / Rrim(I) all: 0.198 / Χ2: 0.76 / Net I/av σ(I): 4.5 / Net I/σ(I): 4.5
Reflection shellResolution: 2.5→2.6 Å / Redundancy: 3 % / Rmerge(I) obs: 1.047 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 2858 / CC1/2: 0.424 / Rpim(I) all: 0.954 / Rrim(I) all: 1.421 / Χ2: 0.73 / % possible all: 94.4

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Processing

Software
NameVersionClassification
PHENIX1.11.1_2575refinement
Aimlessdata scaling
PDB_EXTRACT3.25data extraction
iMOSFLMdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Rossetta model

Resolution: 2.7→15.991 Å / Cross valid method: THROUGHOUT / σ(F): 0.01 / Phase error: 29.5 / Stereochemistry target values: TWIN_LSQ_F
RfactorNum. reflection% reflection
Rfree0.2994 2084 5.2 %
Rwork0.2879 38025 -
obs0.2958 40063 95.74 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 166.7 Å2 / Biso mean: 47.9781 Å2 / Biso min: 4.2 Å2
Refinement stepCycle: final / Resolution: 2.7→15.991 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6663 0 25 1077 7765
Biso mean--41.56 56.99 -
Num. residues----835
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0196693
X-RAY DIFFRACTIONf_angle_d2.5798942
X-RAY DIFFRACTIONf_chiral_restr0.1091123
X-RAY DIFFRACTIONf_plane_restr0.0141094
X-RAY DIFFRACTIONf_dihedral_angle_d27.0584406
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4860X-RAY DIFFRACTION39.083TORSIONAL
12B4860X-RAY DIFFRACTION39.083TORSIONAL
13C4860X-RAY DIFFRACTION39.083TORSIONAL
14D4860X-RAY DIFFRACTION39.083TORSIONAL
15E4860X-RAY DIFFRACTION39.083TORSIONAL
16F4860X-RAY DIFFRACTION39.083TORSIONAL
17G4860X-RAY DIFFRACTION39.083TORSIONAL
18H4860X-RAY DIFFRACTION39.083TORSIONAL
19I4860X-RAY DIFFRACTION39.083TORSIONAL
110J4860X-RAY DIFFRACTION39.083TORSIONAL
111K4860X-RAY DIFFRACTION39.083TORSIONAL
112L4860X-RAY DIFFRACTION39.083TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.7003-2.76740.33661800.31832562274289
2.7674-2.84180.31221410.33642817295891
2.8418-2.92480.2774700.32552749281994
2.9248-3.01850.26151130.29872836294993
3.0185-3.12550.25251270.28542674280191
3.1255-3.24950.27681310.27792798292992
3.2495-3.39580.26891640.27852734289891
3.3958-3.57260.29941450.27772748289392
3.5726-3.79310.31640.2472754291891
3.7931-4.08070.24011450.26482684282991
4.0807-4.48170.25711200.25162701282192
4.4817-5.10830.26642120.24472691290388
5.1083-6.35590.62951650.47672572273787
6.3559-15.15140.25011220.33482705282790

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