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

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基本情報

登録情報
データベース: PDB / ID: 6tms
タイトルCrystal structure of a de novo designed hexameric helical-bundle protein
要素
  • (a novel designed pore protein) x 2
  • affinity purification tag
キーワードBIOSYNTHETIC PROTEIN / Helical bundle / hexamer / computational protein design / pore / de novo protein
生物種synthetic construct (人工物)
手法X線回折 / シンクロトロン / 分子置換 / 解像度: 2.7 Å
データ登録者Xu, C. / Pei, X.Y. / Luisi, B.F. / Baker, D.
資金援助 米国, 1件
組織認可番号国
Howard Hughes Medical Institute 米国
引用ジャーナル: Nature / 年: 2020
タイトル: Computational design of transmembrane pores.
著者: 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 ...著者: 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 /
要旨: 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.
履歴
登録2019年12月5日登録サイト: PDBE / 処理サイト: PDBE
改定 1.02020年4月29日Provider: repository / タイプ: Initial release
改定 1.12020年9月16日Group: Database references / カテゴリ: 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
改定 1.22024年5月1日Group: Data collection / Database references / Refinement description
カテゴリ: 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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構造の表示

構造ビューア分子:
MolmilJmol/JSmol

ダウンロードとリンク

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集合体

登録構造単位
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
ヘテロ分子


分子量 (理論値)分子数
合計 (水以外)99,28119
ポリマ－98,80014
非ポリマー4805
水19,4021077
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
ヘテロ分子


分子量 (理論値)分子数
合計 (水以外)47,7218
ポリマ－47,5296
非ポリマー1922
水1086
タイプ名称対称操作数
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
ヘテロ分子


分子量 (理論値)分子数
合計 (水以外)51,55911
ポリマ－51,2718
非ポリマー2883
水1448
タイプ名称対称操作数
identity operation1_555x,y,z1
単位格子
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
非結晶学的対称性 (NCS)NCSドメイン:
IDEns-ID詳細
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ドメイン領域:
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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要素

#1: タンパク質
a novel designed pore protein


分子量: 7926.179 Da / 分子数: 10 / 由来タイプ: 組換発現 / 由来: (組換発現) synthetic construct (人工物) / 発現宿主: Escherichia coli BL21(DE3) (大腸菌)
#2: タンパク質 a novel designed pore protein


分子量: 7898.166 Da / 分子数: 2 / 由来タイプ: 組換発現 / 由来: (組換発現) synthetic construct (人工物) / 発現宿主: Escherichia coli BL21(DE3) (大腸菌)
#3: タンパク質・ペプチド affinity purification tag


分子量: 1871.095 Da / 分子数: 2 / 由来タイプ: 天然 / 由来: (天然) synthetic construct (人工物)
#4: 化合物
ChemComp-SO4 / SULFATE ION / 硫酸ジアニオン


分子量: 96.063 Da / 分子数: 5 / 由来タイプ: 合成 / 式: SO4
#5: 水 ChemComp-HOH / water


分子量: 18.015 Da / 分子数: 1077 / 由来タイプ: 天然 / 式: H2O
研究の焦点であるリガンドがあるかN

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実験情報

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実験

実験手法: X線回折 / 使用した結晶の数: 1

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試料調製

結晶マシュー密度: 1.96 Å3/Da / 溶媒含有率: 36.6 %
結晶化温度: 291 K / 手法: 蒸気拡散法, シッティングドロップ法 / pH: 6.6 / 詳細: 0.1 M MES pH 6.6, 17.5% PEG550MME

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データ収集

回折平均測定温度: 100 K / Serial crystal experiment: N
放射光源由来: シンクロトロン / タイプ: OXFORD DIFFRACTION NOVA / 波長: 0.98 Å
検出器タイプ: MARMOSAIC 325 mm CCD / 検出器: CCD / 日付: 2015年9月24日
放射プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: x-ray
放射波長波長: 0.98 Å / 相対比: 1
Reflection twinOperator: h,-k,-l / Fraction: 0.5
反射解像度: 2.5→54 Å / Num. obs: 77817 / % possible obs: 98.7 % / Observed criterion σ(F): 3 / Observed criterion σ(I): 3 / 冗長度: 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
反射 シェル解像度: 2.5→2.6 Å / 冗長度: 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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解析

ソフトウェア
名称バージョン分類
PHENIX1.11.1_2575精密化
Aimlessデータスケーリング
PDB_EXTRACT3.25データ抽出
iMOSFLMデータ削減
PHASER位相決定
精密化構造決定の手法: 分子置換
開始モデル: Rossetta model

解像度: 2.7→15.991 Å / 交差検証法: THROUGHOUT / σ(F): 0.01 / 位相誤差: 29.5 / 立体化学のターゲット値: TWIN_LSQ_F
Rfactor反射数%反射
Rfree0.2994 2084 5.2 %
Rwork0.2879 38025 -
obs0.2958 40063 95.74 %
溶媒の処理減衰半径: 0.9 Å / VDWプローブ半径: 1.11 Å / 溶媒モデル: FLAT BULK SOLVENT MODEL
原子変位パラメータBiso max: 166.7 Å2 / Biso mean: 47.9781 Å2 / Biso min: 4.2 Å2
精密化ステップサイクル: final / 解像度: 2.7→15.991 Å
タンパク質核酸リガンド溶媒全体
原子数6663 0 25 1077 7765
Biso mean--41.56 56.99 -
残基数----835
拘束条件
Refine-IDタイプDev ideal数
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-ID数Refine-IDRmsタイプ
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精密化 シェル

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

解像度 (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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