visualization-atp-synthase-dimers-mitochondria-electron-cryo

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Max Planck Institute of Biophysics

The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an alpha-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms.

Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase.

&#x767D;&#x4E09;&#x70EF; &#xFF08;LT&#xFF09; C(4) &#x5408;&#x6210;&#x9176;&#x3001; 18 kDa &#x79EF;&#x5206;&#x819C;&#x9176;&#x5171;&#x8F6D; LTA(4) &#x4E0E;&#x51CF;&#x5C11;&#x8C37;&#x80F1;&#x7518;&#x80BD;&#x5BF9;&#x8868;&#x5355; LTC(4)&#xFF0C;&#x7236;&#x590D;&#x5408;&#x7684;&#x6240;&#x6709;&#x534A;&#x80F1;&#x6C28;&#x9170;&#x767D;&#x4E09;&#x70EF;&#x5728;&#x652F;&#x6C14;&#x7BA1;&#x54EE;&#x5598;&#x7684;&#x75C5;&#x7406;&#x65B9;&#x9762;&#x53D1;&#x6325;&#x7740;&#x81F3;&#x5173;&#x91CD;&#x8981;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x6839;&#x636E;&#x6211;&#x4EEC;&#x7684;&#x8BA1;&#x7B97;&#xFF0C;&#x5206;&#x8FA8;&#x7387;&#x4E3A; 4.5 A &#x91CD;&#x7EC4;&#x4EBA;&#x7C7B; LTC(4) &#x5408;&#x9176;&#x6295;&#x5F71;&#x5730;&#x56FE;&#x7531;&#x7535;&#x5B50;&#x6676;&#x4F53;&#x5B66;&#xFF0C;&#x8FD9;&#x8868;&#x660E;&#x8FD9;&#x79CD;&#x9176;&#x662F;&#x4E09;&#x805A;&#x4F53;&#x3002;&#x5728; 7.5 A &#x5904;&#x622A;&#x65AD;&#x5730;&#x56FE;&#x76F4;&#x89C2;&#x663E;&#x793A;&#x56DB;&#x4E2A;&#x8DE8;&#x819C; &alpha; &#x87BA;&#x65CB;&#x86CB;&#x767D;&#x5355;&#x4F53;&#x6BCF;&#x3002;&#x5728;&#x6295;&#x5F71;&#x4E2D;&#x7684;&#x5BC6;&#x5EA6;&#x6307;&#x793A;&#x7684;&#x963F;&#x5C14;&#x6CD5;&#x87BA;&#x65CB;&#x5927;&#x591A;&#x6570;&#x8FD0;&#x884C;&#x51E0;&#x4E4E;&#x5782;&#x76F4;&#x4E8E;&#x5E73;&#x9762;&#x7684;&#x819C;&#x3002;&#x5728;&#x8FD9;&#x9879;&#x51B3;&#x8BAE;&#xFF0C;LTC(4) &#x5408;&#x9176;&#x662F;&#x60CA;&#x4EBA;&#x5730;&#x76F8;&#x4F3C;&#x5FAE;&#x7C92;&#x4F53;&#x8C37;&#x80F1;&#x7518;&#x80BD; S-&#x8F6C;&#x79FB;&#x9176; 1&#xFF0C;&#x5176;&#x4E2D;&#x5C5E;&#x4E8E;&#x540C;&#x4E00;&#x57FA;&#x56E0;&#x5BB6;&#x65CF;&#xFF0C;&#x4F46;&#x5728; LTC(4) &#x5408;&#x9176;&#x5BF9;&#x5E95;&#x7269;&#x4E13;&#x4E00;&#x6027;&#x627F;&#x62C5;&#x5C0F;&#x5E8F;&#x5217;&#x6807;&#x8BC6;&#x548C;&#x6CA1;&#x6709;&#x76F8;&#x4F3C;&#x4E4B;&#x5904;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x63D0;&#x4F9B;&#x7684;&#x7ED3;&#x6784;&#x548C;&#x529F;&#x80FD;&#x65F6;&#x82B1;&#x751F;&#x56DB;&#x70EF;&#x9178;&#x548C;&#x8C37;&#x80F1;&#x7518;&#x80BD;&#x4EE3;&#x8C22;&#x76F8;&#x5173;&#x7684;&#x819C;&#x86CB;&#x767D;&#x7684;&#x65B0;&#x89C1;&#x89E3;&#x3002;

&#x4EBA;&#x7C7B;&#x767D;&#x4E09;&#x70EF; C(4) &#x5408;&#x9176;&#x5728; 4.5 A &#x5206;&#x8FA8;&#x7387;&#x6295;&#x5F71;&#x4E2D;&#x3002;

Leukotriene (LT) C(4) synthase, an 18 kDa integral membrane enzyme, conjugates LTA(4) with reduced glutathione to form LTC(4), the parent compound of all cysteinyl leukotrienes that play a crucial role in the pathobiology of bronchial asthma. We have calculated a projection map of recombinant human LTC(4) synthase at a resolution of 4.5 A by electron crystallography, which shows that the enzyme is a trimer. A map truncated at 7.5 A visualizes four transmembrane alpha helices per protein monomer. The densities in projection indicate that most of the alpha helices run nearly perpendicular to the plane of the membrane. At this resolution, LTC(4) synthase is strikingly similar to microsomal glutathione S-transferase 1, which belongs to the same gene family but bears little sequence identity and no resemblance in substrate specificity to the LTC(4) synthase. These results provide new insight into the structure and function of membrane proteins involved in eicosanoid and glutathione metabolism.

Human leukotriene C(4) synthase at 4.5 A resolution in projection.

Leucotri&#xE8;nes (LT) C (4) synthase, une enzyme de 18 kDa membrane int&#xE9;grale, conjugu&#xE9;s LTA (4) avec le glutathion r&#xE9;duit pour former LTC (4), le compos&#xE9; parent de tous les leucotri&#xE8;nes cyst&#xE9;inyl qui jouent un r&#xF4;le crucial dans la physiopathologie de l&#x26;#39;asthme bronchique . Nous avons calcul&#xE9; une projection cartographique de la LTC humaine recombinante (4) synthase &#xE0; une r&#xE9;solution de 4,5 A par cristallographie &#xE9;lectronique, ce qui montre que l&#x26;#39;enzyme est un trim&#xE8;re. Une carte tronqu&#xE9;e &#xE0; 7,5 A visualise quatre h&#xE9;lices alpha transmembranaires par monom&#xE8;re de prot&#xE9;ine. Les densit&#xE9;s de projection indiquent que la plupart des h&#xE9;lices alpha ex&#xE9;cuter pr&#xE8;s perpendiculaire au plan de la membrane. &#xC0; cette r&#xE9;solution, LTC (4) synthase est &#xE9;tonnamment similaire &#xE0; microsomale glutathion S-transf&#xE9;rase 1, qui appartient &#xE0; la m&#xEA;me famille de g&#xE8;nes, mais porte une identit&#xE9; de s&#xE9;quence peu et ne ressemble en rien &#xE0; la sp&#xE9;cificit&#xE9; de substrat de la LTC (4) synthase. Ces r&#xE9;sultats fournissent un nouvel &#xE9;clairage sur la structure et la fonction des prot&#xE9;ines membranaires impliqu&#xE9;es dans des eicosano&#xEF;des et le m&#xE9;tabolisme du glutathion.

Human leucotri&#xE8;nes C (4) synthase &#xE0; 4,5 Une r&#xE9;solution de la projection

Menschliche Leukotrien-C (4)-Synthase bei 4,5 A Aufl&#xF6;sung in der Projektion.

Leucotrieni (LT) c sintasi, un enzima di membrana integrale 18 kDa, coniugati LTA(4) con Glutatione ridotto alla forma LTC(4), il composto capostipite di tutti i leucotrieni cisteinici che giocano un ruolo cruciale nel pathobiology dell&#39;asma bronchiale. Noi abbiamo calcolato una mappa di proiezione di ricombinante umano LTC(4) sintasi ad una risoluzione di 4.5 A cristallografia di elettroni, che dimostra che l&#39;enzima &egrave; un trimero. Mappa troncata a 7,5 A Visualizza quattro transmembrane &alpha; eliche al monomero della proteina. La densit&agrave; in proiezione indicano che la maggior parte di &alpha; eliche eseguire quasi perpendicolare al piano della membrana. A questa risoluzione, LTC(4) sintasi sono sorprendentemente simile ai microsomi glutathione S-transferase 1, che appartiene alla stessa famiglia del gene, ma reca poco identit&agrave; di sequenza e alcuna somiglianza nella specificit&agrave; di substrato per la sintasi LTC(4). Questi risultati forniscono una nuova visione della struttura e funzione delle proteine di membrana coinvolte nel metabolismo del glutatione ed eicosanoid.

Umano leucotriene c sintasi a 4,5 risoluzione in proiezione.

&#x30ED;&#x30A4;&#x30B3;&#x30C8;&#x30EA;&#x30A8;&#x30F3;&#xFF08;LT&#xFF09;C&#xFF08;4&#xFF09;&#x9175;&#x7D20;&#x3001;18 kDa&#x306E;&#x819C;&#x5185;&#x5728;&#x6027;&#x9175;&#x7D20;&#x3001;LTC&#xFF08;4&#xFF09;&#x3001;&#x6C17;&#x7BA1;&#x652F;&#x5598;&#x606F;&#x306E;&#x75C5;&#x7406;&#x751F;&#x7269;&#x5B66;&#x306B;&#x304A;&#x3044;&#x3066;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x308B;&#x3059;&#x3079;&#x3066;&#x306E;&#x30B7;&#x30B9;&#x30C6;&#x30A4;&#x30CB;&#x30EB;&#x30ED;&#x30A4;&#x30B3;&#x30C8;&#x30EA;&#x30A8;&#x30F3;&#x306E;&#x89AA;&#x5316;&#x5408;&#x7269;&#x3092;&#x5F62;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x9084;&#x5143;&#x578B;&#x30B0;&#x30EB;&#x30BF;&#x30C1;&#x30AA;&#x30F3;&#x3068;&#x306E;&#x30B3;&#x30F3;&#x30B8;&#x30E5;&#x30B2;&#x30FC;&#x30C8;LTA&#xFF08;4&#xFF09; &#x3002;&#x6211;&#x3005;&#x306F;&#x3001;&#x9175;&#x7D20;&#x304C;&#x4E09;&#x91CF;&#x4F53;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x96FB;&#x5B50;&#x7DDA;&#x7D50;&#x6676;&#x5B66;&#x306B;&#x3088;&#x308B;4.5&#x306E;&#x89E3;&#x50CF;&#x5EA6;&#x3067;&#x7D44;&#x63DB;&#x3048;&#x30D2;&#x30C8;LTC&#xFF08;4&#xFF09;&#x5408;&#x6210;&#x9175;&#x7D20;&#x306E;&#x6295;&#x5F71;&#x30DE;&#x30C3;&#x30D7;&#x3092;&#x8A08;&#x7B97;&#x3057;&#x305F;&#x3002; 7.5&#x3067;&#x5207;&#x308A;&#x6368;&#x3066;&#x30DE;&#x30C3;&#x30D7;&#x306F;&#x3001;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x5358;&#x91CF;&#x4F53;&#x3054;&#x3068;&#x306B;4&#x3064;&#x306E;&#x819C;&#x8CAB;&#x901A;&#x3B1;&#x30D8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x3092;&#x53EF;&#x8996;&#x5316;&#x3059;&#x308B;&#x3002;&#x6295;&#x5F71;&#x306E;&#x5BC6;&#x5EA6;&#x306F;&#x3B1;&#x30D8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x306E;&#x5927;&#x90E8;&#x5206;&#x306F;&#x819C;&#x306E;&#x5E73;&#x9762;&#x306B;&#x307B;&#x307C;&#x5782;&#x76F4;&#x306B;&#x8D70;&#x3063;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x89E3;&#x50CF;&#x5EA6;&#x3067;&#x3001;LTC&#xFF08;4&#xFF09;&#x9175;&#x7D20;&#x306F;&#x540C;&#x3058;&#x907A;&#x4F1D;&#x5B50;&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306B;&#x5C5E;&#x3057;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x5C11;&#x3057;&#x914D;&#x5217;&#x540C;&#x4E00;&#x6027;&#x3068;LTC&#xFF08;4&#xFF09;&#x5408;&#x6210;&#x9175;&#x7D20;&#x306E;&#x57FA;&#x8CEA;&#x7279;&#x7570;&#x6027;&#x306E;&#x306A;&#x3044;&#x4F3C;&#x3066;&#x3044;&#x308B;&#x30DF;&#x30AF;&#x30ED;&#x30BD;&#x30FC;&#x30E0;&#x30B0;&#x30EB;&#x30BF;&#x30C1;&#x30AA;&#x30F3;S-&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;1&#x3001;&#x3068;&#x9A5A;&#x304F;&#x307B;&#x3069;&#x4F3C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x30A8;&#x30A4;&#x30B3;&#x30B5;&#x30CE;&#x30A4;&#x30C9;&#x3068;&#x30B0;&#x30EB;&#x30BF;&#x30C1;&#x30AA;&#x30F3;&#x4EE3;&#x8B1D;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x819C;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x69CB;&#x9020;&#x3068;&#x6A5F;&#x80FD;&#x306B;&#x65B0;&#x305F;&#x306A;&#x6D1E;&#x5BDF;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

4.5&#x30D2;&#x30C8;&#x306E;&#x30ED;&#x30A4;&#x30B3;&#x30C8;&#x30EA;&#x30A8;&#x30F3;C&#xFF08;4&#xFF09;&#x5408;&#x6210;&#x9175;&#x7D20;&#x306E;&#x6295;&#x5F71;&#x306E;&#x89E3;&#x50CF;&#x5EA6;

Leukotriene (LT) C(4) synthase, 18 kDa &#xC815;&#xC218; &#xB9C9; &#xD6A8;&#xC18C; &#xAC10;&#xC18C; glutathione LTC(4), &#xAE30;&#xAD00;&#xC9C0; &#xCC9C;&#xC2DD;&#xC758; pathobiology&#xC5D0;&#xC11C; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD558;&#xB294; &#xBAA8;&#xB4E0; cysteinyl leukotrienes &#xBD80;&#xBAA8; &#xD654;&#xD569;&#xBB3C; &#xD615;&#xD0DC;&#xB97C; &#xAC00;&#xC9C4; LTA(4)&#xC744; &#xBCC0;&#xD654;. &#xC6B0;&#xB9AC; &#xD6A8;&#xC18C;&#xB294; &#xD2B8;&#xB9AC;&#xBA38; &#xBCF4;&#xC5EC;&#xC8FC;&#xB294; &#xC804;&#xC790; &#xACB0;&#xC815;&#xD559;, 4.5 A&#xC758; &#xD574;&#xC0C1;&#xB3C4;&#xC5D0;&#xC11C; &#xC7AC;&#xC870;&#xD569; &#xC778;&#xAC04; LTC(4) synthase&#xC758; &#xD504;&#xB85C;&#xC81D;&#xC158; &#xB9F5; &#xACC4;&#xC0B0;. 7.5 A&#xC5D0;&#xC11C; &#xC798;&#xB9B4; &#xC9C0;&#xB3C4; &#xC2DC;&#xAC01;&#xD654; &#xB2E8;&#xBC31;&#xC9C8; &#xB2E8;&#xC704;&#xCCB4; &#xB2F9; 4 &#xB9C9; &#xD6A1;&#xB2E8; &#xC54C;&#xD30C; &#xD3EC;&#xD568;. &#xD22C;&#xC601;&#xC5D0; &#xBC00;&#xB3C4; &#xC54C;&#xD30C; &#xD3EC;&#xD568; &#xB300;&#xBD80;&#xBD84; &#xAC70;&#xC758; &#xB9C9;&#xC758; &#xD3C9;&#xBA74;&#xC5D0; &#xC218;&#xC9C1;&#xC778; &#xC2E4;&#xD589;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xC774; &#xD574;&#xC0C1;&#xB3C4;&#xC5D0;&#xC11C; LTC(4) synthase&#xB294; &#xAE30; &#xB9C9;&#xD788;&#xAC8C; microsomal glutathione S-&#xC804;&#xC774; 1, &#xB3D9;&#xC77C;&#xD55C; &#xC720;&#xC804;&#xC790; &#xAC00;&#xC871;&#xC5D0; &#xC18D;&#xD574; &#xC788;&#xC9C0;&#xB9CC; LTC(4) synthase&#xC5D0; &#xAE30;&#xC9C8; &#xD2B9;&#xC774;&#xC131;&#xC5D0; &#xC791;&#xC740; &#xC2DC;&#xD000;&#xC2A4; id&#xC640; &#xC804;&#xD600; &#xB2EE;&#xC740; &#xACF0; &#xD558;&#xB294; &#xBE44;&#xC2B7;&#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; eicosanoid &#xBC0F; glutathione &#xC2E0;&#xC9C4; &#xB300;&#xC0AC;&#xC5D0; &#xAD00;&#xB828; &#xB41C; &#xB9C9; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xAE30;&#xB2A5;&#xACFC; &#xAD6C;&#xC870;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xD1B5;&#xCC30;&#xB825;&#xC744; &#xC81C;&#xACF5;&#xD569;&#xB2C8;&#xB2E4;.

&#xC778;&#xAC04;&#xC758; leukotriene C(4) synthase &#xD22C;&#xC601;&#xC5D0;&#xC11C; 4.5 A &#xD574;&#xC0C1;&#xB3C4;.

Leucotrieno (LT) C (4) sintasa, una enzima de 18 kDa de membrana integral, conjugados LTA (4) con glutati&#xF3;n reducido para formar LTC (4), el compuesto de origen de todos los cisteinil leucotrienos que juegan un papel crucial en la patobiolog&#xED;a del asma bronquial . Hemos calculado un mapa de proyecci&#xF3;n humana recombinante LTC (4) sintasa en una resoluci&#xF3;n de 4,5 A por cristalograf&#xED;a de electrones, lo que demuestra que la enzima es un tr&#xED;mero. Un mapa truncado en 7,5 Una visualiza cuatro h&#xE9;lices alfa transmembrana por mon&#xF3;mero prote&#xED;na. Las densidades de proyecci&#xF3;n indican que la mayor&#xED;a de las h&#xE9;lices alfa ejecutar casi perpendicular al plano de la membrana. En esta resoluci&#xF3;n, LTC (4) sintasa es sorprendentemente similar a microsomales glutation S-transferasa 1, que pertenece a la misma familia de genes, pero tiene una identidad de secuencia poco y no se parece en especificidad de sustrato para el LTC (4) sintasa. Estos resultados proporcionan una nueva comprensi&#xF3;n de la estructura y funci&#xF3;n de prote&#xED;nas de membrana que participan en el metabolismo de los eicosanoides y el glutati&#xF3;n.

Humanos leucotrieno C (4) sintasa en el 4,5 una resoluci&#xF3;n en la proyecci&#xF3;n

Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a three-dimensional map of yeast FAS at 5.9-A resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of approximately 18 A from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.

Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy.

The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I(1)III(2)IV(1) from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10-11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.

Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1.

Archaella are the archaeal motility structure, which are structurally similar to gram-negative bacterial type IV pili but functionally resemble bacterial flagella. Structural and biochemical data of archaellum subunits are missing. FlaX, a conserved subunit in crenarchaeal archaella, formed high molecular weight complexes that adapted a ring-like structure with an approximate diameter of 30 nm. The C terminus of FlaX was not only involved in the oligomerization, but also essential for FlaX interaction with FlaI, the bifunctional ATPase that is involved in assembly and rotation of the archaellum. This study gives first insights in the assembly apparatus of archaella.

FlaX, a unique component of the crenarchaeal archaellum, forms oligomeric ring-shaped structures and interacts with the motor ATPase FlaI.

The choice of equipment for an electron crystallography laboratory will ultimately be determined by the available budget; nevertheless, the ideal lab will have two electron microscopes: a dedicated 300 kV cryo-EM with a field emission gun and a smaller LaB(6) machine for screening. The high-end machine should be equipped with photographic film or a very large CCD or CMOS camera for 2D crystal data collection; the screening microscope needs a mid-size CCD for rapid evaluation of crystal samples. The microscope room installations should provide adequate space and a special environment that puts no restrictions on the collection of high-resolution data. Equipment for specimen preparation includes a carbon coater, glow discharge unit, light microscope, plunge freezer, and liquid nitrogen containers and storage dewars. When photographic film is to be used, additional requirements are a film desiccator, dark room, optical diffractometer, and a film scanner. Having the electron microscopes and ancillary equipment well maintained and always in optimum condition facilitates the production of high-quality data.

Choice and maintenance of equipment for electron crystallography.

Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure. DOI:http://dx.doi.org/10.7554/eLife.00218.001.

De novo modeling of the F420-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy.

Antibiotic therapy in response to Mycobacterium tuberculosis infections targets de novo fatty acid biosynthesis, which is orchestrated by a 1.9 MDa type I fatty acid synthase (FAS). Here, we characterize M. tuberculosis FAS by single-particle cryo-electron microscopy and interpret the data by docking the molecular models of yeast and Mycobacterium smegmatis FAS. Our analysis reveals a porous barrel-like structure of considerable conformational variability that is illustrated by the identification of several conformational states with altered topology in the multienzymatic assembly. This demonstrates that the barrel-like structure of M. tuberculosis FAS is not just a static scaffold for the catalytic domains, but may play an active role in coordinating fatty acid synthesis. The conception of M. tuberculosis FAS as a highly dynamic assembly of domains revises the view on bacterial type I fatty acid synthesis and might inspire new strategies for inhibition of de novo fatty acid synthesis in M. tuberculosis.

Structure and conformational variability of the mycobacterium tuberculosis fatty acid synthase multienzyme complex.

Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.

Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids.

The introduction of direct electron detectors with higher detective quantum efficiency and fast read-out marks the beginning of a new era in electron cryo-microscopy. Using the FEI Falcon II direct electron detector in video mode, we have reconstructed a map at 3.36 Å resolution of the 1.2 MDa F420-reducing hydrogenase (Frh) from methanogenic archaea from only 320,000 asymmetric units. Videos frames were aligned by a combination of image and particle alignment procedures to overcome the effects of beam-induced motion. The reconstructed density map shows all secondary structure as well as clear side chain densities for most residues. The full coordination of all cofactors in the electron transfer chain (a [NiFe] center, four [4Fe4S] clusters and an FAD) is clearly visible along with a well-defined substrate access channel. From the rigidity of the complex we conclude that catalysis is diffusion-limited and does not depend on protein flexibility or conformational changes. DOI: http://dx.doi.org/10.7554/eLife.01963.001.

Article	Year
Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase. Journal of molecular biology\| PubMed ID: 12144787	2002
Human leukotriene C(4) synthase at 4.5 A resolution in projection. Structure (London, England : 1993)\| PubMed ID: 15530365	2004
Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20231485	2010
Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1. The EMBO journal\| PubMed ID: 21909073	2011
FlaX, a unique component of the crenarchaeal archaellum, forms oligomeric ring-shaped structures and interacts with the motor ATPase FlaI. The Journal of biological chemistry\| PubMed ID: 23129770	2012
Choice and maintenance of equipment for electron crystallography. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 23132070	2013
De novo modeling of the F420-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy. eLife\| PubMed ID: 23483797	2013
Structure and conformational variability of the mycobacterium tuberculosis fatty acid synthase multienzyme complex. Structure (London, England : 1993)\| PubMed ID: 23746808	2013
Self-assembly of the general membrane-remodeling protein PVAP into sevenfold virus-associated pyramids. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24567401	2014
Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector. eLife\| PubMed ID: 24569482	2014

Deryck J. Mills

Biography

JoVE Journal Publications

Other Publications