Present†Current address: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Ing reaction center light-harvesting complex 1 (RC-LH1) iya ing core photosynthetic component da reng purple phototrophic bacteria. Pepakilala mi la reng adwang cryo-electron microscopy structures ning RC-LH1 complex ibat keng Rhodopseudomonas palustris. Ing 2.65-Å resolution structure ning RC-LH114-W complex atin yang 14 subunit LH1 loops makapadurut keng RC, na me-interrupt ya keng protein W, kabang ing complex a alang protein-W ganap yang RC composition makapadurut keng RC. makasara ya ing 16 subunit LH1 loop. Ing pamagkumpara kareng istrukturang deni mamye yang pamanalastas keng dinamika ning quinone keng RC-LH1 complex, kayabe na ing e pa meyakit a pamagbayu keng conformational nung makatali ya ing quinone keng RC QB site, ampo ing lokasyon da reng auxiliary quinone binding sites, na sasaup para ipasa la keng RC. Ing makatagulaling a istruktura ning W protein pipigilan na ing pamagsara ning LH1 loop, inya sa’t gagawang channel para king pamagpabilis king pamipalitan ning quinone/quinolone.
Ing enerhiyang bibie ning photosynthesis agyu nang sustentuan ing halus eganaganang bye keti king yatu, at atin yang maragul a potensyal para king solar biotechnology. Kabang susulung ne ing global photosynthesis, deng purple phototrophic bacteria magpakit lang miyayaliwang energy modes ampo metabolic capabilities. Malyari lang makaiwas keng photosynthesis ampo tumubu la bilang heterotrophic a bacteria keng madalumdum, malyari lang mag-ayus nitrogen ampo carbon dioxide, makagawa lang hydrogen, ampo maka-degrade kareng aromatic compounds (1-3). Para mika enerhiya para kareng prosesung deni, kailangan yang mabilis at masalese ing sala para maging chemical energy. Ing prosesung ayni magumpisa ya nung ing light-trapping antenna complex sisipsip yang sala at ilipat ne ing me-trap a enerhiya keng reaction center (RC), inya magumpisa ing pamikawani da reng charge (4 – 7). Ing basic unit ning photosynthesis kareng purple phototrophic bacteria bibilugan ne ning type 2 RC, makapadurut ya keng light-harvesting complex 1 (LH1), a bubuu keng RC-LH1 core complex. Ing LH1 mebuu ya kapamilatan ning metung a array da reng curved αβ heterodimers, a balang metung kareti makatali ya kareng adwang bacterial chlorophyll (BChl) a molecules ampo metung o adwang carotenoids (8-12). Ing pekasimpleng LH1 antenna atin yang 16 o 17 αβ heterodimers a makapadurut keng RC (9-13) keng metung a saradung loop, pero kareng aliwang core complexes, deng transmembrane peptides maka-interrupt la keng continuity ning makapadurut a LH1, inya sa't susulung ne ing quinol/quinone diffusion pilatan ning RC ampo ing cytochrome bc complex (1111, 13). Ing purple phototrophic plant Rhodopseudomonas (Rps.) metung yang modelung organismu na makaintindi keng enerhiya ampo electron transfer na susuporta keng photosynthesis. Ing mumunang kristal a istruktura ning Rps. Ing modelu ning palustris RC-LH1 complex yapin ing RC, makapadurut ya kareng 15 heterodimeric LH1 loops, na me-interrupt ya keng e balu a protina a ausan dang “Protein W” (14). Ing protein-W mekilala ya bilang RPA4402, na metung yang e pa mekilalang 10.5kDa a protina a maki atlung me-predict a transmembrane helices (TMH) (16). Imungkahi mi ing palitan ing lagyu ning rpa4402 gene encoding protein W para maging pufW para maging consistent ya keng nomenclature a gagamitan para kareng genes encoding RC-L, M (pufL, pufM) ampo LH1α, β (pufA, pufB) subunits. Ing makawili, ing protein-W atyu yamu keng 10% ning RC-LH1, papakit na ing Rps. ing palustris magproduce yang adwang miyayaliwang RC-LH1 complexes. keni, ireport mi ing high-resolution cryo-EM (cryo-EM) a istruktura da reng adwang core complexes, ing metung atin yang protein W ampo 14 αβ heterodimers, ing metung alang protein W ampo ing metung a makasaradung 16 Heterodimer LH1 loop. Ing kekaming istruktura magrepresenta yang pamagbayu keng pamanintindi keng RC-LH1 complex ning Rps. palustris, uling sinuri mi ing homogeneous a populasyun da reng balang variant at atin lang sapat a resolution para malino mi lang i-assign deng balang peptide ampo reng bound pigments ampo reng related lipids ampo quinones. Ing pamagkumpara kareng istrukturang deni papakit na ing atlung TMH proteins-W na e pa meyakit keng aliwang RC-LH1 complex angga ngeni maggenerate yang quinone channel para mabilis ing quinone/quinolone exchange. Metung a bilang da reng conserved a lipid ampo quinone binding sites ing mekilala, ampo atin kaming bayung conformational change kaybat ning pamisanmetung ning quinone ampo RC, na malyaring gamitan para keng photosystem II (PSII) RC da reng oxygenated phototrophic organisms. Ing kekaming akit mamye yang bayung panaun keng kinetics ning quinone/quinolone binding ampo exchange keng RC-LH1 core complex ning purple phototrophic bacteria.
Para asikasu ing detalyadung pamagaral kareng adwang complex a mayayakit keng Rps. palustris, i-isolate mi la reng balang RC-LH1 kapamilatan ning biochemical a paralan. Ing protein W-deficient complex (kaybat mayayaus yang ΔpufW) melinis ya ibat keng strain na alang pufW gene (16), at metung mung RC-LH1 complex ing malyaring gawan. Ing protein W-containing complex gagawan ne ning metung a strain. Ing protina W na niting strain me-modify ya keng 10x His tag keng kayang C-terminus, para ing protina W-containing complex malyari yang epektibung misasanmetung kareng keraklan a kulang a protina W kapamilatan ning pamag-immobilize metal. Ing complex epektibo yang makabukud (16) Affinity Chromatography (IMAC).
Antimo ing akakit tamu keng Figure 1, parehu lang complex deng atlung sub-unit RC (RC-L, RC-M ampo RC-H) a makapadurut keng LH1 antenna. Ing 2.80-A a istruktura ning complex a alang protein-W papakit yang 16 αβ heterodimers, gagawang metung a makasaradung LH1 loop a makapadurut keng RC, a kaybat mayayaus yang RC-LH116 complex. Ing 2.65Å a istruktura ning protein-W-containing complex atin yang 14-heterodimer LH1 a me-interrupt keng protein-W, kaybat ausan deng RC-LH114-W.
(A ampo B) Surface representation ning compound. (C ampo D) Bonded pigments a mipalabas keng rods. (E ampo F) Deng complex a mayayakit ibat keng cytoplasmic surface atin lang peptides ampo LH1 subunits a makarepresenta kareng cartoons, ampo makabilang la clockwise manibat keng protein-W gap [consistent keng Rba numbering. sphaeroides complex (13)]. Para keng LH1-α, ing kule ning protein subunit dilaw ya; para keng LH1-β, ing kule ning protein subunit asul ya; para keng protina-W, ing protina maputi ya; para keng RC-H, cyan ya; para keng RC-L, Orange ya; para keng RC-M, magenta. Deng cofactors makarepresenta la kareng rods, ing berdi mangabaldugan yang BChl ampo BPh a molecules, ing purple mangabaldugan yang carotenoids, ampo ing yellow mangabaldugan yang UQ10 molecules. (G ampo H) Magnified view ning protein-W gap keng equivalent a rehiyon ning RC-LH114-W complex (G) ampo RC-LH116 complex (H). Deng cofactors mipalabas la keng porma ning space filling, ing chelated quinone mipalabas ya keng asul. Ing protein-W gap maka highlight ya keng blue dashed line keng (G), ampo deng malating butas nung nukarin ya mikalat ing quinone/quinolol keng LH116 ring maka highlight ya keng black dashed line keng (H).
Ing Figure 1 (A ampo B) papakit ne ing RC a makapadurut kareng makabuklat o makasaradung array ning LH1αβ heterodimers, balang metung kareti makatali ya kareng adwang BChl ampo metung a carotenoid (Figure 1, C ampo D). Deng minunang pamagaral pepakit da na ing Rps ya ing LH1 complex. keng biosynthetic pathway ning spirulina xanthin, deng species a reni atin lang miyayaliwang populasyun da reng carotenoids (17). Makanyan man, ing spiropyrroxanthin ing dominanting carotenoid at ing kayang densidad makayagpang ya. Uli na niti, pinili ming imodel ing spiroxanthin kareng eganaganang LH1 binding sites. Deng alpha ampo beta polypeptides metung lang TMHs a maki makuyad a membrane outer regions (Figure 1, A, B, E, ampo F). Agyang e ya meyakit ing density da reng 17 residues keng C-terminus, ing alpha polypeptide meputut ya manibat Met1 angga Ala46 kareng adwang complex. Ing β polypeptide mebawas ya ibat Gly4 papunta Tyr52 keng RC-LH116, ampo ibat Ser5 papunta Tyr52 keng RC-LH114-W. Ala yang density a 3 o 4 N-terminal o 13 C-terminal residues ing ikit (Figure S1). Ing mass spectrometry analysis ning mixed RC-LH1 complex a mesadya ibat keng wild-type strain pepakit na ing mewala a rehiyon ing resulta ning heterologous cleavage da reng peptides a reni (Figure S1 ampo S2). Ing N-terminal formylation ning α-Met1 meyakit ya naman (f). Ing pamagsuri pepakit na ing α-peptide atin yang residues fMet1 anggang Asp42/Ala46/Ala47/Ala50, ampo ing β-peptide atin yang residues Ser2 anggang Ala53, na masanting a pamikasundu keng low-temperature EM density map.
Ing pamisanmetung ning α-His29 ampo β-His36 gagawan na lang arapan deng BChls; balang αβ heterodimer mag assemble ya kareng siping na para gawang open loop (RC-LH114-W) o closed loop (RC-LH116) makapadurut keng RC Ing exciton coupled pigment array (Figure 1, C ampo D). Nung ikumpara me keng 877 nm band ning RC-LH114-W, ing 880 nm absorption red shift ning RC-LH116 3 nm ya (Figure 2A). Makanyan man, ing circular dichroism spectrum halus parehu ya (Figure 2B), a papakit na agyang atin yang malino pamiyaliwa ing makabuklat ampo makasaradung loop, ing lokal a kapaligiran da reng BChls parehu ya. Ing absorption redshift malyari yang maging resulta ning mabawas a thermal motion ampo ing mas maragul a katatagan keng closed loop (18, 19), ing pamagbayu keng pigment coupling a sangkan ning closed loop (20, 21), o ing pamisanmetung da reng adwang epektu (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, deng taluktuk na makamarka la kareng karelang katumbas a pigment at me-normalize la keng BPh peak keng 775 nm. (B) Circular dichroism spectrum a me-normalize keng BChl absorbance keng 805 nm. (C ampo D) Mepiling ΔA spectra ibat keng time-resolved absorption spectra ning RC-LH114-W complex (C) ampo RC-LH116 complex (D). Para keng mas masanting a pamikumpara, deng eganaganang spectra me-normalize la keng ∆A ning −A keng 0.2 ps. (E) Ing rate ning cytochrome c2 oxidation kaybat ning irradiation keng presensya da reng miyayaliwang konsentrasyun ning UQ2 (lawen ya ing Figure S8 para keng raw data). (F) Kareng cell a meragul lalam ning mababang, katamtaman o matas a intensity a sala (10, 30 o 300μMm-2 s-1, respectively), ing protina W ampo RC-L subunits keng purified complex ampo ing separated membrane ratio. abalu me ing lebel ning protina kapamilatan ning SDS-polyacrylamide gel electrophoresis ampo immunoassay (lawen ya ing Figure S9 para keng raw data). abalu me ing ratio a makayagpang keng melinis a RC-LH114-W complex. Ing stoichiometric ratio ning RC-L keng protein-W ning complex 1:1 ya.
Deng BChls keng posisyun 1 keng deformed αβ14 loop ning RC-LH114-W (Figure 1, A, C, ampo E) mas malapit la keng RC primary donor (P) keng 6.8Å kesa kareng katumbas a BChls keng RC-LH116 (Figure. 1, B, D, ampo F, ampo S3); makanyan man, ing transient absorption kinetics da reng adwang complex papakit na para keng RC-LH114-W ampo RC-LH116, ing excitation energy transfer time constants manibat LH1 papunta RC 40 ±4 ampo 44±3 ps (Figure 2). , C ampo D, Figure S4 ampo Table S2). Ala ya namang makabaldugan a pamiyaliwa ing electronic transfer kilub ning RC (Figure S5 ampo ing kaugnayan a supplementary text). Suspetsa mi na ing malapit a pamiugnayan ning oras ning pamaglipat enerhiya pilatan ning LH1 ampo RC-P uli ning parehung distansya, anggulo ampo potensyal a enerhiya da reng keraklan kareng BChl kareng adwang LH1 loops. Balamu ing pamag explore keng LH1 energy pattern para miras keng minimum a distansya ali ya mas mabilis kesa keng direct energy transfer ibat keng suboptimal a lugal papunta keng RC. Ing open-loop LH1 loop keng RC-LH114-W malyari ya namang dumalan keng e makabaldugan a thermal motion keng mababang temperatura para keng structural analysis, ampo atin yang mas makaba a αβ14 ring conformation keng room temperature ibat keng pigmentation distance ning βBChls keng posisyun ning RC 1.
Ing RC-LH116 complex atin yang 32 BChls ampo 16 carotenoids, ampo ing kayang kabilugan a panga-ayus parehu ya keng mekwa keng Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) DB (PDB) strain (PDB07) (297) ampo green algae (Blc.viridis) (PDB ID 6ET5) (10). Kaybat ning pamag-align, ditak mung pamagbayu keng posisyun da reng αβ heterodimers ing ikit, lalu na ing 1-5, 15, ampo 16 (Figure S6). Ing presensya ning protein-W atin yang maragul a epektu keng istruktura ning LH1. Deng atlung TMHs na makasuglung la kareng makuyad a loop, ing N-terminal keng lumen side ning complex ampo ing C-terminal keng cytoplasmic side (Figures 1A ampo 3, A anggang D). Ing protein-W maragul yang hydrophobic (Figure 3B), at ing TMH2 ampo ing TMH3 makipag-ugnayan la keng LH1αβ-14 para gawang transmembrane surface (Figure 3, B ampo E anggang G). Ing interface keraklan metung yang Phe, Leu ampo Val residues keng transmembrane region. Deting residues makatumpak la kareng hydrophobic amino acids ampo αβ-14 pigments. Deng mapilan a polar residues makaambag la keng pamiugnayan, kayabe na ing hydrogen bond pilatan ning W-Thr68 ampo β-Trp42 keng babo ning complex cavity (Figure 3, F ampo G). keng babo ning cytoplasm, ing Gln34 makalapit ya keng keto group ning αβ-14 carotenoids. Dagdag pa, ing n-dodecyl β-d-maltoside (β-DDM) molecule me-resolba ya, at ing kayang hydrophobic tail mipalapad ya keng interface pilatan ning protein-W ampo αβ-14, ampo ing lipid tail malyari yang atyu keng katawan. apansinan mi mu rin na ing C-terminal resolution regions ning protein W ampo RCH malapit la, pero ali la keng scope ning pamaglalang specific interactions (Figure 1, A ampo E). Makanyan man, malyari lang mika pamiugnayan deng e pa meyari a C-terminal amino acids da reng adwang protina a reni, a malyaring mamye mekanismo para king pamag-recruit ning protina-W kabang mag-assemble ya ing RC-LH114-W complex.
(A) Ing protein-W, na makayarap keng interface ning LH1αβ14 keng cartoon form, atin yang rod-shaped side chain (pula), a makadisplay keng metung a bage ning electrostatic potential diagram (transparent grey surface a maki contour level a 0.13). (B) Protein-W a mipaintagun keng hydrophobic a maki kule a babo. Deng polar ampo charged a lugal maka display la keng cyan, deng hydrophobic a lugal maka display la keng maputi, ampo deng masikan a hydrophobic a lugal maka display la keng orange. (C ampo D) Protein-W a makarepresenta keng cartoon, ing orientation na parehu ya keng (A) (C), ampo mibalikid yang 180° (D). Agpang keng posisyun keng sequence, deng distinguishable residues gamitan de ing rainbow color scheme, nung nukarin ing N-terminal asul ya ampo ing C-terminal mapula ya. (E) Protein-W keng parehung view antimo ing (A), ampo reng residues keng interface ning protein-W:LH1 makarepresenta la kareng rods a maki makadikit a marka. (F) Ing protein-W mibalikid yang 90° relatibu keng (E) ampo LH1αβ14 keng cartoon representation, ampo relative kareng interface residues keng bar representation. Deng overhanging residues ibat keng beta polypeptide makalabel la. Ing cofactor mipalabas ya bilang bar a makatugma keng kule ning Figure 1, ing decomposed β-DDM mipalabas ya keng gray, ampo ing oxygen mipalabas ya keng red. (G) Ing view keng (F) mibalikid ya 180°, kareng prominenting residues ning makalabel a alpha polypeptide.
Ing protein-W papalitan ne ing αβ heterodimer (ing 15th keng Figure 1F), inya pin makaiwas ya ing loop closure ampo makatikdo ya kareng mumunang atlung αβ heterodimers. Ikit da na ing maximum a inclination angle ning mumunang αβ-1 heterodimer a maki kaugnayan keng film normal yapin ing 25° anggang 29° (Figure 1, A ampo E), na mebuu keng 2° anggang 8° a inclination ning αβ-1 keng RC A sharp contrast-LH116 (Figure 1, B ampo F). Ing kadwa ampo katlung heterodimers makahilig la keng 12° anggang 22° ampo 5° anggang 10°, agpang kareti. Uling keng steric hindrance ning RC, ing tilt ning αβ-1 e ya kayabe ing kaduang pares ning αβ (na maki kaugnayan keng 16th αβ keng Figure 1F), inya sa't mika malino yang gap keng LH1 ring (Figure 1, A ampo E) . Uling ala lang adwang αβ heterodimers, kayabe ning pangawala da reng apat a BChl ampo adwang carotenoids, alang metung man kareng carotenoids ing makagapus keng twisted αβ-1 subunit, a magresulta keng LH114-W ring a maki 13 carotenoids Vegetarian ampo 28 BChls. Ing local resolution estimates da reng adwang complex keng αβ1 anggang 7 a rehiyon mas mababa ya kesa kareng aliwang LH1 loop, na malyaring magreflect keng inherent plasticity ning LH1 subunit a makalapit keng RC QB site (Figure 4) .
Deng larawan ning RC-LH114-W (A ampo B) ampo RC-LH116 (C ampo D) pepakit la ibat keng parehung babo/side view (A ampo B) (A ampo C) ampo ing cavity surface ning Fig. 1. (B ampo D). Deng maki kule a susi makakit la keng wanan.
Ing metung mung aliwang katangian ning core complex a maki stoichiometric ratio a 1:14 ya pin ing Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13). Makanyan man, ing protein W ampo ing PufX ala lang malino homology, ampo atin lang maragul a epektu kareng karelang LH1 structures. Ing PufX metung yang TMH a maki N-terminal cytoplasmic domain a makipag-ugnayan king cytoplasmic side ning RC-H subunit (13) king posisyun a maki kaugnayan king Rps. palustris LH116αβ-16. Ing PufX gagawang channel para keng quinone/quinolone exchange pilatan ning RC-LH1 ampo ing cytochrome bcl complex ampo atyu ya keng eganaganang Rba. sphaeroides core complex (13). Agyang ing monomer-monomer interface atyu ya keng Rba. Ing sphaeroides RC-LH1-PufX dimer atyu ya keng binding position ning protein W keng RC-LH114-W, ampo ing gap a pepalwal ning PufX ampo protein-W atyu ya keng katumbas a posisyun (Figure S7A). Ing gap keng RC-LH114-W makalinya ya keng hypothetical quinone channel (8) ning Pseudomonas rosea LH1, na mebuu kareng peptides na ali makaugne keng protein W o PufX (Figure S7B). Dagdag pa, ing quinone channel keng Blc. Ing emerald green LH1 a mebuu kapamilatan ning pamaglakwan king metung a γ subunit (7) atyu ya king metung a posisyun (Figure S7C). Agyang mipapagsalita ya kareng miyayaliwang protina, ing pangakit da reng quinone/quinolol channels keng metung a karaniwan a posisyun keng RC-LH1 complex balamu metung yang alimbawa ning convergent evolution, a magpakit na ing gap a gewa ning protina W malyari yang maging quinone channel.
Ing gap keng LH114-W loop paintulutan na ing pamaglalang ning metung a patuloy a membrane region pilatan ning kilub a espasyu ning RC-LH114-W complex ampo ing bulk membrane (Figure 1G), kesa keng ikonekta la reng adwang domain kapamilatan ning metung a protein pore antimo reng protina. Ing RC-LH116 complex kalupa ne ning metung a makasaradung Tch. Needle-like complex (22) (Figure 1H). Uling ing diffusion ning quinone keng membrane mas mabilis ya kesa keng diffusion keng makitid a protein channel, ing makabuklat a LH114-W loop malyari yang paintulutan ing mas mabilis a RC turnover kesa keng makasaradung LH116 loop, at ing diffusion ning quinone keng RC malyari yang mas mabawal. Para asuri nung ing protein W makaapektu ya keng pamagbayu da reng quinones kapamilatan ning RC, ginawa kaming cytochrome oxidation assay keng metung a konsentrasyun ning ubiquinone 2 (UQ2) (metung a analogue ning natural UQ10 a maki mas makuyad a isoprene tail) (Figure 2E). Agyang ing presensya ning chelated quinone makasabat ya keng ustu a pamagdetermina ning apparent Michaelis constant (RC-LH114-W ampo RC-LH116 makayagpang la para keng 0.2±0.1μM ampo 0.5±0.2μM, respectively), ing maximum rate ning RC-LH114-W ( 4.6±0.2012-1-1) yapin ing s-LH114-W. 28±5% mas maragul kesa keng RC-LH116 (3.6±0.1 e-RC-1 s-1).
Ing mumuna ming estimate na ing protein-W atyu ya keng 10% ning core complex (16); keni, ing occupancy rates da reng low-light, medium-light, ampo high-light growth cells 15±0.6%, 11±1% ampo 0.9±0.5, respectively % (Figure 2F). Ing quantitative a pamagkumpara ning mass spectrometry pepakit na ing pamagdagdag king histidine tag e na pepababa ing relatibung karakal ning protein-W kumpara kareng wild-type strains (P = 0.59), inya deng antas a reni e la artifact ning modified protein-W (Figure S10). Makanyan man, ining mababang occupancy ning protein-W keng RC-LH1 complex malyari yang paintulutan ing mapilan a RCs na mag flip keng mabilis a rate, inya sa’t mabawas ing mas mabagal a pamipalitan ning quinone/quinolone keng RC-LH116 complex. apansinan mi na ing matas a light occupancy rate e ya makayagpang keng bayung transcriptomics data, na magpakit na ing pufW gene expression daragul ya lalam ning masikan a sulu (Figure S11) (23). Ing pamiyaliwa ning pufW transcription ampo ing protein-W incorporation keng RC-LH1 complex makalitu ya at malyari yang magreflect keng complex regulation ning protein.
Ing RC-LH114-W, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) ampo 29 β-DDM molecules ing mipagkalub ampo mimodelu la 6 CDLs, 24 POPCs, 2 POPGs ampo 12 DMDs. RC-LH116 (Figure 5, A ampo B). Kareng adwang istruktura a reni, ing CDL halus atyu ya keng cytoplasmic a gilid ning complex, kabang ing POPC, POPG ampo ing β-DDM keraklan atyu la keng luminal a gilid. Adwa lang lipid ampo detergent molecules ing me-isolate keng αβ-1 anggang αβ-6 a rehiyon ning RC-LH114-W complex (Figure 5A), ampo lima ing me-isolate keng equivalent a region ning RC-LH116 (Figure 5B ). Mas dakal a lipids ing ikit da keng aliwang gilid ning complex, keraklan CDL, a mitipun pilatan ning RC ampo αβ-7 anggang αβ-13 (Figure 5, A ampo B). Deng aliwang structurally resolved lipids ampo detergents atyu la keng kilwal ning LH1 ring, ampo deng well-resolved acyl chains makalawe la pilatan da reng LH1 subunits, tentatively designed as β-DDM keng RC-LH114-W, ampo medefine bilang β-DDM keng RC A mixture ning β-DDM ampo PO-LH166-LH16. Ing parehung posisyun da reng chelating lipids ampo detergents keng kekatamung istruktura papakit na ila reng physiologically relevant binding sites (Figure S12A). Deng posisyun da reng katumbas a molecule keng Tch atin lang masanting a consistency. Malumana at Trv. Ing strain 970 RC-LH1s (Figure S12, B to E) (9, 12) ampo reng hydrogen-bonding residues ning lipid head group pepakit da ing masanting a pamag-ingat keng sequence alignment (Figure S13), a magpakit na ing Conserved CDL a maka-bind keng RC (24), deng sites a reni malyari lang mi-conserve keng RCH-LH1 complex.
(A ampo B) RC-LH114-W (A) ampo RC-LH116 (B) peptides makarepresenta la kareng cartoons, ampo deng pigments makarepresenta la kareng rods, gamit ing kule scheme keng Figure 1. Deng lipids makalarawan la keng kule pula, ampo deng detergents makalarawan la keng kule abu. Ing UQ a ​​makatali keng RC QA ampo QB sites dilaw ya, kabang ing isolated UQ asul ya. (C ampo D) Parehu lang akakit deng (A) ampo (B), a alang lipid. (E to G) Mas maragul a view ning Q1(E), Q2(F) ampo Q3(G) ibat keng RC-LH116, a maki side chains na makaimpluwensya keng metung at metung. Deng hydrogen bonds makakit la bilang black dashed lines.
Ing RC-LH116, pareung RC QA ampo QB UQ, na makiabe keng pamaglipat electron keng prosesu ning pamikawani da reng charge, mesira la keng karelang binding sites. Makanyan man, keng RC-LH114-W, e ya pa me-resolba ing QB quinone at pag-usapan mi yang detalyadu king lalam. Dagdag pa kareng QA ampo QB quinones, adwa lang chelated UQ molecules (atyu la pilatan da reng RC ampo LH1 rings) ing makalage keng RC-LH114-W structure agpang kareng karelang well-resolved head groups (atyu la keng Q1 ampo Q2, respectively). espasyu). Figure 5C). Adwa lang isoprene units deng mipagkalub keng Q1, at ing density map lulutas ne ing kumpletung 10 isoprene tails ning Q2. Ing istruktura ning RC-LH116, atlung chelated UQ10 molecules (Q1 anggang Q3, Figure 5D) ing me-resolba, at ding sablang molecule atin lang malino density king mabilug a buntuk (Figure 5, D anggang G). kareng adwang istruktura, ing posisyun da reng quinone head groups ning Q1 ampo Q2 atin lang masanting a consistency (Figure S12F), ampo makipag-ugnayan la mu keng RC. Ing Q1 atyu ya keng pasbul ning W gap ning RC-LH114-W (Figure 1G ampo 5, C, D ampo E), ampo ing Q2 atyu ya malapit keng QB binding site (Figure 5, C, D) ampo F). Ing conserved L-Trp143 ampo L-Trp269 residues malapit la keng Q1 ampo Q2 ampo mamye lang potensyal a π-stacking interactions (Figure 5, E ampo F, ampo Figure S12). L-Gln88, 3.0 Å ibat keng distal oxygen ning Q1, mamye yang masikan a hydrogen bond (Figure 5E); ining residue maka-conserve ya kareng eganaganang RCs maliban keng pekamarayung relasyun (Figure S13). Ing L-Ser91 conservatively yang mipalitan para keng Thr kareng keraklan kareng aliwang RCs (Figure S13), 3.8 Angstroms ya ibat keng methyl oxygen ning Q1, at malyari yang mamye mahina a hydrogen bonds (Figure 5E). Ing Q3 ala yang partikular a pamiugnayan, pero atyu ya keng hydrophobic a rehiyon pilatan ning RC-M subunit ampo ing LH1-α subunit 5 anggang 6 (Figure 5, D ampo G). Q1, Q2 ampo Q3 o malapit a chelated quinones me-resolba la naman keng Tch. Malugud, Trv. Strain 970 at Blc. Ing iris structure (9, 10, 12) tuturu ya keng metung a conserved a auxiliary quinone binding site keng RC-LH1 complex (Figure S12G). Deng limang mesira UQs keng RC-LH116 masanting lang pamikasundu keng 5.8±0.7 ning balang complex a meyakit keng high performance liquid chromatography (HPLC), kabang deng atlung mesira UQs keng RC-LH114-W mas mababa la kesa keng ing mesukad a ulaga ning 6.2±0.3 (Fig. S14) papakit na ala lang solusyun deng molecule ning UQ. balangkas.
Deng pseudo-symmetric L ampo M polypeptides atin lang limang TMHs ampo bubuu lang heterodimer na magsamut keng metung a BChl dimer, adwang BChl monomers, adwang bacteriophage (BPh) monomers, ampo metung a non-Heme iron ampo metung o adwang UQ10 molecules. Kapamilatan ning presensya da reng hydrogen bonds keng terminal ketone group ampo ing balu nang pamitipun keng Rps, deng carotenoids miyabe la keng M-subunit, a mayayaus cis-3,4-dehydroorhodopin. Species (25). Ing outer membrane domain ning RC-H maka-angkla ya keng membrane kapamilatan ning metung a TMH. Ing kabilugan a RC structure kalupa ne ning atlung subunit RC da reng mikaugne a species (antimo ing Rba). sphaeroides (PDB ID: 3I4D). Deng macrocycles ning BChl ampo BPh, ing carotenoid backbone ampo ing non-heme iron mipapalibut la keng resolution range da reng istrukturang deni, antimo ing UQ10 head group keng QA site ampo ing QB quinone keng RC-LH116 (Figure S15).
Ing panga-atyu da reng adwang RC structures a miyayaliwang QB site occupancy rates mamye yang bayung oportunidad para asuri ing consistent a conformational a pamagbayu a kayabe ning QB quinone binding. Ing RC-LH116 complex, ing QB quinone atyu ya keng ganap a makagapus a “proximal” a posisyun (26), pero ing pamikawani ning RC-LH114-W ala yang QB quinone. Ala yang QB quinone ing RC-LH114-W, na makabigla uling ing complex aktibu ya, mas marakal kesa keng RC-LH116 complex a maki structurally resolved QB quinone. Agyang deng adwang LH1 rings chelate la reng anam a quinones, lima la reng structurally resolved keng closed RC-LH116 ring, kabang atlu la mu reng structurally limited keng open RC-LH114-W ring . Ing daragul a structural disorder malyari yang magreflect keng mas mabilis a pamagpalit da reng RC-LH114-W QB sites, mas mabilis a quinone kinetics keng complex, ampo mas daragul a posibilidad ning pamaglakbe keng LH1 loop. Imungkahi mi na ing kakulangan ning UQ keng RC QB site ning RC-LH114-W malyari yang maging resulta ning mas komplikadu at mas aktibung complex, at ing QB site ning RC-LH114-W agad yang me-freeze keng UQ turnover Ing specific a yugtu (ing pasbul keng QB site mesara ya) papakit na ing conformation na niting aktibidad.
nung alang QB, ing kayabe nang pamag-ikot ning L-Phe217 keng posisyun na e makayagpang keng UQ10 binding, uling magdalang spatial collision keng mumunang isoprene unit ning buntuk (Figure 6A). Dagdag pa, malino la reng malino a pamagbayu keng conformational, lalu na ing helix de (makuyad a helix keng loop pilatan ning TMH D ampo E) nung nukarin ing L-Phe217 mipalitan ya keng QB binding pocket ampo ing pamag-ikot ning L-Tyr223 (Figure 6A ) Para masira ing hydrogen bond kareng M-A4 framework at isara ne ing binding ning QB site (Figure 6B). Ing Helix de pivot ya keng base na, ing Cα ning L-Ser209 mipalitan ya 0.33Å, kabang ing L-Val221Cα mipalitan ya 3.52Å. Ala yang akit a pamagbayu ing TMH D ampo E, na maka-superimposable kareng adwang istruktura (Figure 6A). keng balu mi, ini ing mumunang istruktura keng natural a RC na magsara keng QB site. Ing pamagkumpara keng kumpletung (QB-bound) a istruktura papakit na bayu ya mabawas ing quinone, kailangan ing pamagbayu keng conformational para lungub ya keng quinone. Ing L-Phe217 mag-rotate ya para gawang π-stacking interaction keng quinone head group, at ing helix lilipat ya palabas, a magpaintulut keng skeleton ning L-Gly222 ampo ing side chain ning L-Tyr223 para gawang hydrogen bond network a maki stable a hydrogen bond structure (Figure 6, A ampo C).
(A) Overlapping cartoon ning hologram (L chain, orange/M chain, magenta) ampo apo (gray) a istruktura, nung nukarin ding key residues mipalabas la keng porma ning rod-like representation. Ing UQ10 makarepresenta ya keng dilaw a bar. Ing dotted line papakit na ing hydrogen bonds a mebuu keng mabilug a istruktura. (B ampo C) Ing surface representation ning apolipoprotein ampo ing mabilug a ring structure, papakit ne ing side chain oxygen ning L-Phe217 keng asul ampo ing L-Tyr223 keng red, respectively. Ing L subunit orange ya; ing M ampo H subunits ala lang kule. (D ampo E) Apolipoprotein (D) ampo ing mabilug (E) RC QB sites [kulay ning (A) balang metung] ampo Thermophilus thermophilus PSII (berdi, asul a maki plastic quinone; PDB ID: 3WU2) Align (58).
E asahan, agyang mapilang istruktura da reng QB-deficient RCs a alang LH1 ing magagamit, deng pamagbayu king conformational a ikit da king pamagaral a ini e la pa mibalita kanitang minuna. Kayabe la kareti deng QB depletion structure ibat keng Blc. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) ampo Rba. sphaeroides (PDB ID: 1OGV) (29), na halus parehu la ngan keng karelang kabilugan a QB structure. Ing malapit a pamagsuri king 3PRC pepakit na ing LDAO (Lauryl Dimethyl Amine Oxide) detergent molecules makagapus la king pasbul ning QB a posisyun, a malyaring makapigil king pamagbayu king metung a makasaradung conformation. Agyang e ya mabubuluk ing LDAO keng pareung posisyun keng 1EYS o 1OGV, deng RCs a reni makasadya la gamit ing pareung detergent at uli na niti malyari lang mika pareung epektu. Ing kristal a istruktura ning Rba. Ing Sphaeroides RC co-crystallized keng cytochrome c2 (PDB ID: 1L9B) atin ya namang makasaradung QB site. Makanyan man, keng kasu ayni, ing N-terminal a rehiyon ning RC-M polypeptide (makipag-ugnayan ya keng QB binding site kapamilatan ning H bond ning Tyr residue keng Q helix) mag-adopt yang e natural a conformation, at ing QB conformational change e ya pa mipabalu (30 ). Ing makabaldugan yapin ing ala tamung ikit a makanining deformation ning M polypeptide keng RC-LH114-W structure, na halus parehu ya keng N-terminal region ning RC-LH116 RC. Dapat mu namang papansinan na kaybat ning pamag-eradicate ning detergent-based LH1 antenna, ing apolipoprotein RCs keng PDB me-resolba ya, na mewala ing internal quinone pools ampo lipids keng gap pilatan ning RC ampo ing kilub ning makapadurut a LH1 ring (31, 32). Ing RC manatili yang functional uling panatilyan na la ngan deng cofactors, maliban keng decomposable QB quinone, na e masyadung matatag at maralas mawawala ya keng prosesu ning pamagsadya (33). Dagdag pa, balu tamu na ing pamaglako ning LH1 ampo reng natural a cyclic lipids ibat keng RC atin yang epektu kareng functions, antimo ing makuyad a bie ning charge-separated P+QB-state (31, 34, 35). Uli na niti, aisip mi na ing panga-atyu ning lokal a LH1 ring makapadurut king RC malyari yang magpanatili king “saradu” a QB site, inya sa’t mipanatili ing lokal a kapaligiran malapit king QB.
Agyang ing apolipoprotein (alang QB quinone) ampo ing kumpletung istruktura magrepresenta lang adwang snapshot ning pamagbayu ning QB site, kesa keng serye da reng events, atin yang indikasyon na ing binding malyari yang i-gate para aiwasan ing pamag-rebinding ning hydroquinone Para pigilan ing substrate inhibition. Ing pamiugnayan ning quinolol ampo quinone malapit keng QB site ning apolipoprotein malyari yang miyayaliwa, na magdalang pamagtakwil ning RC. Mababang panaun na ing mipanukala na ing pamagbayu keng conformational atin yang papel keng pamagtali ampo pamagbawas kareng quinones. Ing abilidad da reng frozen RCs para mabawas ing quinones kaybat ning dark adaptation mesira ya (36); Ing X-ray crystallography papakit na ing pangasira ayni uli ning QB quinones a makakulong keng “distal” a conformation mga 4.5 Å ibat keng aktibung proximal a posisyun (26) , 37). Isuggest mi na ining distal binding conformation metung yang snapshot ning intermediate state pilatan ning apolipoprotein ampo ing full ring structure, na tutuki keng mumunang pamiugnayan ning quinone ampo ing pamagbukas ning QB site.
Ing type II RC a mayayakit keng PSII complex da reng mapilan a phototrophic bacteria ampo cyanobacteria, algae ampo tanaman atin yang structural ampo functional a pamag-ingat (38). Ing structural alignment a akakit keng Figure 6 (D ampo E) papakit na ing pamikakametung da reng PSII RCs ampo ing QB site ning bacterial RC complex. Ing pamiyaliwa ayni metung yang modelu para keng pamagaral kareng malapit a sistema ning quinone binding ampo reduction. Deng minunang publikasyon sinabi da na ing pamagbayu keng conformational kayabe ne ing PSII a pamagbawas da reng quinones (39, 40). Uli na niti, nung isipan ing evolutionary conservation ning RC, ining e pa meyakit a binding mechanism malyari ya namang gamitan keng QB site ning PSII RC kareng oxygenated phototrophic a tanaman.
Rps ΔpufW (unlabeled pufW deletion) ampo PufW-His (C-terminal 10x His-tagged protein-W a mipalabas ibat keng natural a pufW locus) strains. ing palustris CGA009 me describe ya keng kekaming minunang obra (16). Deting strain ampo ing isogenic wild-type parent mebawi la keng freezer kapamilatan ning pamag-streak kareng ditak a cells keng PYE (balang 5 g liter -1) (maka-store keng LB keng -80 °C, atin yang 50% ( w/v) glycerol) protein, yeast extract ampo succinate) agar [1.5%) (w/v) plate. Ing plate mi-incubate ya keng mabilug a bengi keng madalumdum keng room temperature lalam ning anaerobic a kundisyun, at kaybat mi-illuminate ya keng maputing sulu (~50 μmolm-2 s-1) a binie ning OSRAM 116-W halogen bulbs (RS Components, UK) kilub ning 3 anggang 5 aldo anggang metung a colony ing linwal. Metung a colony ing ginamit para i-inoculate ing 10 ml ning M22+ medium (41) a midagdag 0.1% (w/v) casamino acids (kaybat ausan deng M22). Ing kultura meragul ya lalam ning mababang oxygen a kundisyun keng madalumdum keng 34°C a maki yugse keng 180 rpm kilub ning 48 oras, at kaybat 70 ml ning kultura ing mi-inoculate lalam ning pareung kundisyun kilub ning 24 oras. Ing semi-aerobic culture a maki volume a 1 ml magagamit ya para i-inoculate ing 30 ml ning M22 medium keng 30 ml a universal screw-top transparent glass bottle ampo mi-irradiate ya keng agitation (~50μmolm-2 s-1) kilub ning 48 oras kapamilatan ning sterile magnetic force Stirring rod. Kaybat 30 ml ning kultura ing mi-inoculate kareng 1 litru ning kultura lalam ning pareung kundisyun, na kaybat ginamit para i-inoculate ing mga 9 litru ning kultura a mi-illuminate keng ~200 μmolm-2 s-1 kilub ning 72 oras. Deng cells me-ani la kapamilatan ning centrifugation keng 7132 RCF kilub ning 30 minutu, mi-resuspended la keng ~10 ml ning 20 mM tris-HCl (pH 8.0), ampo mi-store la keng -20°C anggang kailangan.
Kaybat nang metunaw, dagdagan la reng mapilan a kristal ning deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) ampo reng adwang Roche holoenzyme protease inhibitor tablets (Merck, UK) kareng resuspended a cell. keng 20,000 psi French pressure cell (Aminco, USA), mesira la reng cell 8 anggang 12 a besis. Kaybat dang leko deng e mesira a cell ampo reng e matunaw a debris kapamilatan ning centrifugation king 18,500 RCF kilub ning 15 minutu king 4°C, ing membrane me-precipitate ya ibat king pigmented lysate kapamilatan ning centrifugation king 113,000 RCF kilub ning 2 oras king 43,000°C. Iyugse me ing soluble fraction at i-resuspend me ing colored membrane keng 100 anggang 200 ml ning 20 mM tris-HCl (pH 8.0) at i-homogenize me anggang ala nang akakit a aggregates. Ing makabitin a membrane mi-incubate ya keng 20 mM tris-HCl (pH 8.0) (Anatrace, USA) a maki 2% (w/v) β-DDM kilub ning 1 oras keng madalumdum keng 4°C a maki banayad a pamag-stir. Kaybat i-centrifuge ya keng 70°C para matunaw ing 150,000 RCF keng 4°C kilub ning 1 oras para milako la reng mitagan a e matunaw.
Ing solubilizing membrane ibat keng ΔpufW strain mipatupad ya keng 50 ml DEAE Sepharose ion exchange column a maki atlung column volumes (CV) ning binding buffer [20 mM tris-HCl (pH 8.0) a maki 0.03% (w / v) β-DDM]. Hugasan me ing column kareng adwang CV binding buffers, at kaybat hugasan me ing column kareng adwang binding buffers a maki 50 mM NaCl. Ing RC-LH116 complex me-elute ya keng linear gradient a 150 anggang 300 mM NaCl (keng binding buffer) keng 1.75 CV, at ing mitagan a binding complex me-elute ya keng binding buffer a maki 300 mM NaCl keng 0.5 CV. Tipunan me ing absorption spectrum pilatan ning 250 ampong 1000 nm, panatilyan me ing fraction a maki absorbance ratio (A880/A280) a mas maragul kesa 1 king 880 anggang 280 nm, dilute meng adwang besis king binding buffer, at gamitan me ing pareung paralan pasibayu king DEAE column On purification. I-dilute la reng fractions a maki A880/A280 ratios a mas matas kesa 1.7 ampo A880/A805 ratios a mas matas kesa 3.0, gawan ing katlung round ning ion exchange, at panatilyan la reng fractions a maki A880/A280 ratios a mas matas kesa 2.2 ampo A880/A805 ratios mas matas kesa 5.0.0. Ing partially purified complex me concentrate ya keng ~2 ml keng Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK), ampo mikarga ya keng Superdex 200 16/600 size exclusion column (GE Healthcare, US) a maki 200 mM NaCl, kaybat mibuffer ya keng parehung buffer. CV. Tipunan la reng absorption spectra ning size exclusion fraction, at i-concentrate la reng absorption spectra kareng A880/A280 ratios babo ning 2.4 ampo A880/A805 ratios babo ning 5.8 anggang 100 A880, at agad lang gamitan para keng cryo-TEM grid preparation o pamag-imbak Itabi keng -80°C anggang kailangan.
Ing solubilizing membrane ibat keng PufW-His strain mipatupad ya keng 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) a maki 200 mM NaCl ampo 0.03% (w/w)) keng IMAC buffer ( GE Healthcare). v) β-DDM]. Ing column mehugas ya kareng limang CVs ning IMAC buffer, at kaybat kareng limang CVs ning IMAC buffer a maki 10 mM histidine. Ing core complex me-elute ya ibat keng column gamit ing limang IMAC buffers a maki 100 mM histidine. Ing fraction a maki RC-LH114-W complex makakonsentra ya keng ~10 ml keng metung a stirred tank a maki Amicon 100,000 MWCO filter (Merck, UK), midilute yang 20 besis keng binding buffer, at kaybat midagdag ya keng 25 ml keng DEAE Sepharose column, apat a CVs ing gagamitan da keng b buffer. Hugasan me ing column kareng apat a CV binding buffers, kaybat i-elute me ing complex kareng walung CVs keng linear gradient ning 0 anggang 100 mM NaCl (keng binding buffer), ampo reng mitagan a apat a CVs a maglaman 100 mM binding buffer Deng residual complexes a me-elute keng sodium chloride a miyabe keng A888/2000 ratio mas matas kesa keng ratio. Ing A880/A805 ratio a mas matas kesa 4.6 fractions me concentrate ya keng ~2 ml keng Amicon 100,000 MWCO centrifugal filter, ampo mepuno ya keng 1.5 CV IMAC keng mumuna Buffer equilibrated Superdex 200 16/600 size exclusion column, at kaybat me elute ya keng parehung buffer keng babo ning 15 CV. Tipunan la reng absorption spectra da reng size-exclusion fractions at i-concentrate la reng absorption spectra kareng A880/A280 ratios babo ning 2.1 ampo A880/A805 ratios babo ning 4.6 anggang 100 A880, na agad dang gamitan para keng frozen TEM grid preparation o ibili keng -80°C anggang kailangan.
Ing Leica EM GP immersion freezer ginamit ya para isadya ing low temperature TEM grids. Ing complex me dilute ya keng IMAC buffer keng A880 ning 50, at kaybat 5μl me load ya keng bayung glow-discharged QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK). I-incubate me ing grid keng 20°C ampo 60% relative humidity kilub ning 30 s, kaybat i-blot me keng 3 s, at kaybat i-quench me keng liquid ethane keng -176°C.
Ing data ning RC-LH114-W complex me-record ya keng eBIC (Electronic Bioimaging Center) (British Diamond Light Source) gamit ing Titan Krios microscope, na magobra keng acceleration voltage a 300kV, a maki nominal magnification a 130,000× ampo ing energy a Choo-se a gap a 20V. Ing Gatan 968 GIF Quantum a maki K2 peak detector ginamit ya para magrecord kareng larawan keng counting mode para makakolekta data. Ing calibrated a sukad ning pixel 1.048Å ya, at ing dose rate 3.83 e-Å-2s-1 ya. Tinipun ke ing pelikula kilub ning 11 a sigundu at piparti-parti ke keng 40 a bage. Gamitan me ing carbon-coated area para i-refocus me ing microscope, at kaybat magkolekta kang atlung pelikula balang butul. Ing kabilugan, 3130 a pelikula ing mekolekta, a maki defocus a ulaga pilatan ning -1 ampong -3μm.
Ing data para keng RC-LH116 complex mekolekta ya gamit ing parehung microscope keng Asterbury Biostructure Laboratory (University of Leeds, UK). Ing data mekolekta ya keng counting mode a maki magnification a 130 k, at ing sukad ning pixel me-calibrate ya keng 1.065 Å a maki dose a 4.6 e-Å-2s-1. Ing pelikula me-record ya kilub ning 12 segundo at me-divide ya keng 48 a bage. Ing kabilugan, 3359 a pelikula ing mekolekta, a maki defocus values ​​pilatan ning -1 ampong -3μm.
Ing eganaganang pamagprosesu data gagawan de keng Relion 3.0 pipeline (42). Gamitan me ing Motioncorr 2 (43) para i-correct ing beam motion kapamilatan ning dose weighting, at kaybat gamitan me ing CTFFIND 4.1 (44) para abalu me ing CTF (contrast transfer function) parameter. Deng tipikal a photomicrographs kaybat da reng mumunang yugtu ning pamagprosesu mayayakit la keng Figure 2. S16. Ing automatic selection template megawa ya kapamilatan ning manual a pamamili kareng 250 pixels ning 1000 particles keng 250-pixel frame ampo alang reference a two-dimensional (2D) a klasipikasyun, inya sa't e na la a-reject deng klasipikasyun a makatupad keng sample contamination o alang akit a katangian. Kaybat, ing automatic selection megawa ya kareng eganaganang microphotographs, at ing RC-LH114-W 849,359 particles ya, ampo ing RC-LH116 complex 476,547 particles ya. Deng eganaganang mepiling particles dinalan lang adwang round ning non-reference 2D classification, at kaybat ning balang pamagdalagan, deng particles a makatupad keng carbon area, sample contamination, alang malino features o masikan a mipapatung particles ila reng me-reject, a magresulta keng 772,033 (90.9%) ampo 359,678 (75.5) particles ing ginamit para keng 3D classification ning 3D classification. RC-LH114-W ampo RC-LH116 balang metung. Ing mumunang 3D reference model megawa ya gamit ing stochastic gradient descent method. Gamit ing mumunang modelu bilang reperensya, ding mepiling particles me-classify la kareng apat a kategorya keng 3D. Gamit ing model keng kategoryang ayni bilang reperensya, gawa kang 3D refining kareng particles keng pekamaragul a kategorya, kaybat gamitan me ing mumunang 15Å low-pass filter para takpan ing solvent area, dagdagan me ing 6 pixels da reng malambut a gilid, ampo i-post-process la reng pixels para i-correct ing Gatan K2 peak Modulation transfer function ning top detector. Para keng RC-LH114-W dataset, ining mumunang model me-modify ya kapamilatan ning pamaglako king masikan a density kareng gilid ning mask (me-disconnect ya keng core complex density keng UCSF Chimera). Deng modelung meging resulta (deng resolusyun ning RC-LH114-W ampo RC-LH116 3.91 ampo 4.16 Å, agpang) ginamit la bilang reperensya para keng kaduang round ning 3D classification. Deng particles a ginamit makagrupu la keng mumunang 3D class at ala lang masikan a correlation keng siping bale. Overlap o kulang keng malino a structural features. Kaybat ning kaduang gulis ning 3D classification, ing kategorya a maki pekamatas a resolution ing mepili [Para king RC-LH114-W, metung a kategorya ing 377,703 a particles (44.5%), para king RC-LH116, atin yang adwang kategorya, a maki kabilugan a 260,752 a particles (54.7% W%), nung parehu la mu kaybat deng makaligne kaybat na nita. mumunang pamag-ikot a maki ditak a pamiyaliwa]. Deng mepili a particles mibalik la keng 400-pixel a kahun ampo mi-refine la kapamilatan ning 3D refining. Ing solvent mask megawa ya gamit ing mumunang 15Å low-pass filter, 3 pixel map expansion ampo 3 pixel soft mask. Gamit ing per-particle CTF refinement, per-particle motion correction ampo ing kaduang round ning per-particle CTF refinement, ing 3D refinement, solvent masking ampo ing post-processing gagawan de kaybat ning balang step para mas ma-refine ing resulta ning texture. Gamit ing FSC (Fourier Shell Correlation Coefficient) cut-off value a 0.143, ing resolusyun da reng tauling modelu ning RC-LH114-W ampo RC-LH116 2.65 ampo 2.80Å, agpang kareti. Ing FSC curve ning tauling model mayayakit ya keng Figure 2. S17.
Deng eganaganang protein sequences me-download la keng UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5); LH1-α (PufA; UniProtID: Q6N9L4); RC-L (PufL; UniProt ID: O83005); RC-M (PufM; UniProt ID: A0A4Z7); RC-H (PuhA; UniProt ID: A0A4Z9); Protein-W (PufW; UniProt ID: Q6N1K3). Ing SWISS-MODEL (45) ginamit ya para gawan ing homology model ning RC, na atin yang protein sequences ning RC-L, RC-M ampo RC-H ampo ing crystal structure ning Rba. sphaeroides RC ing ginamit bilang template (PDB ID: 5LSE) (46). Gamitan me ing "fit map" tool keng UCSF Chimera para i-fit ing model a me-generate keng mapa (47), a-improve ing protein structure, ampo ing cofactor [4×BChl a (monomer library residue name = BCL), 2×BPh a (BPH), metung o adwang uri ning UQ10 (U10), metung a non-heme iron (Fe) ampo metung a-hexa-hexa, 44 (QAK)] gamitan me ing Coot (48) para dagdagan. Uling e ya available ing QAK keng monomer library, me-parameterize ya gamit ing eLBOW tool keng PHENIX (49).
Kaybat, ing LH1 subunit megawa ya. Nung mumuna, ing automatic construction tool keng PHENIX (49) ginamit ya para automatic yang gawan ing bage ning LH1 sequence gamit ing mapa ampo ing LH1-α ampo LH1-β protein sequences bilang input. Pilinan me ing peka kumpletung LH1 subunit, i-extract me at i-load me keng Coot, idagdag me ing kulang a sequence, at i-refine me ing mabilug a istruktura bayu me idagdag ing adwang BCls a (BCL) ampo ing spirilloxanthin (CRT) [agpang keng relevant Rps Ing densidad ning LH1 complex ampo ing balu nang carotenoid content. Species (17)]. Kopya me ing kumpletung LH1 subunit, at gamitan me ing UCSF Chimera “Docking Map Tool” para i-dock ing siping nang non-model area ning LH1 density, at kaybat i-refine me keng Coot; ulit-ulitan ing prosesu anggang e la ngan me-model deng LH1 subunits. Para keng RC-LH114-W structure, kapamilatan ning pamag extract keng unallocated density keng Coot, ing protina me segment ya ibat kareng mitagan a non-protein components keng USCF Chimera map ampo ing Autobuild tool ginamit ya para mitatag ing mumunang model, ampo reng mitagan a subunits (protein-W) Modeling. keng PHENIX (49). Dagdagan me ing nanu mang kulang a sequence keng resulta ning model keng Coot (48), at kaybat manual meng i-refine ing mabilug a subunit. Ing mitagan a e mipagkalub a densidad makayagpang ya keng kombinasyun da reng lipids (PDB monomer library ID ning CDL = CDL, POPC = 6PL ampo POPG = PGT), β-DDM detergent (LMT) ampo UQ10 molecules (U10). Gamitan me ing PHENIX optimization (49) ampo ing manual optimization keng Coot (48) para maging perpektu ing kumpletung mumunang model anggang ing model statistics ampo ing visual quality ning fit e na la malyaring isundu pa. Katataulian, gamitan me ing LocScale (50) para patasan ing lokal a mapa, at kaybat gawan me ing mapilan pang siklo ning pamagmodelu king e mipagkalub a densidad at automatic at manual a pamag-optimize.
Deng peptides, cofactors ampo reng aliwang lipids ampo quinones a maka-dock keng karelang density makalage la keng Figures 1 ampo 2. S18 anggang S23. Ing statistical a impormasyun ning tauling model mayayakit ya keng Table S1.
nung ali ya makasulat, ing UV/Vis/NIR absorption spectra mekolekta ya keng Cary60 spectrophotometer (Agilent, USA) keng 1 nm intervals manibat 250 nm anggang 1000 nm ampo ing integration time a 0.1s.
I-dilute me ing sample keng quartz cuvette a maki 2 mm a dalan papunta A880 ning 1, at tipunan me ing absorption spectrum pilatan ning 400 ampong 1000 nm. Ing circular dichroic spectra mekolekta ya keng Jasco 810 spectropolarimeter (Jasco, Japan) keng 1 nm intervals pilatan ning 400 nm ampo 950 nm keng scan rate a 20 nm min-1.
Ing molar extinction coefficient abalu ya kapamilatan ning pamaglaso keng core complex keng A880 a manga 50. Lasawan me ing 10μl a volume keng 990μl a binding buffer o methanol, at tipunan me agad ing absorption spectrum para mabawas ing BChl degradation. Ing BChl content ning balang methanol sample mekwa ya kapamilatan ning extinction coefficient king 771 nm ning 54.8 mM-1 cm-1, at ing extinction coefficient meyakit ya (51). Ibye me ing mesukad a BChl concentration keng 32 (RC-LH114-W) o 36 (RC-LH116) para abalu ing core complex concentration, na gamitan para abalu ing absorption spectrum ning parehung sample a tinipun keng buffer Extinction coefficient. mala-kalapal. Atlung paulit-ulit a sukad ing mekwa para keng balang sample, at ing average a absorbance ning BChl Qy maximum ing ginamit para keng pamagkwenta. Ing extinction coefficient ning RC-LH114-W a sukad keng 878 nm 3280±140 mM-1 cm-1 ya, kabang ing extinction coefficient ning RC-LH116 a sukad keng 880 nm 3800±30 mM-1 cm-1 ya.
Ing UQ10 me-quantify ya agpang keng paralan keng (52). king makuyad a salita, ing reverse phase HPLC (RP-HPLC) megawa ya gamit ing Agilent 1200 HPLC system. I-dissolve ing 0.02 nmol ning RC-LH116 o RC-LH114-W keng 50μl ning 50:50 methanol:chloroform a maki 0.02% (w/v) ferric chloride, at i-inject ing pre-equilibrated Beckman Coulter Ultrasphere ODS 4 mml keng 1-1-1-ml. 40°C keng HPLC solvent (80:20 methanol:2-propanol) keng ×25 cm a column. Gawan ing isocratic elution keng HPLC solvent para abalu ing absorbance keng 275 nm (UQ10), 450 nm (carotenoids) ampo 780 nm (BChl) kilub ning 1 oras. Ing peak keng 275 nm chromatogram keng 25.5 minutus metung ya, na ala yang aliwang akit a compound. Ing integrated area magagamit ya para makalkula ing molar a dagul ning UQ10 a me-extract agpang keng calibration curve a me-calculate ibat keng injection da reng pure standards manibat 0 anggang 5.8 nmol (Figure S14). Balang sample me-analisa ya keng atlung replicates, at ing reported error makayagpang ya keng SD ning average.
Ing solusyun a maki RC-LH1 complex a maki maximum Qy absorption a 0.1 mesadya ya king 30 μM a mebawas horse heart cytochrome c2 (Merck, UK) ampo 0 anggang 50 μMUQ2 (Merck, UK). Atlung 1-ml a sample ing misadia keng balang UQ2 concentration ampo mi-incubate magdamag keng madalumdum keng 4°C para siguraduan ing kumpletung pamagbayu keng madalumdum bayu ing sukad. Ing solusyun mikarga ya keng OLIS RSM1000 modular spectrophotometer a ating 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle ampo 0.6 mm outlet slits. Ing 600 nm a long pass filter makabili ya keng pasbul ning sample phototube ampo ing reference photomultiplier tube para e milub ing excitation light. Ing absorbance me monitor ya keng 550 nm a maki integration time a 0.15 s. Ing excitation light lulual ya keng 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) kapamilatan ning fiber optic cable keng 90% intensity kapamilatan ning DC2200 controller (Thorlabs Ltd., UK) ampo lulual ya keng light source keng angle a 90°. Ing sukad a sinag makayaliwa ya keng salamin para ibalik ing nanu mang sala na e mesipsip keng sample. Bantayan ing absorbance 10 s bayu ing illuminance ning 50 s. Kaybat ing absorbance me-monitor ya pa kilub ning 60 s keng madalumdum para abalu nung angga nukarin ing quinolol spontaneously babawasan ne ing cytochrome c23 + (lawen ya ing Figure S8 para keng raw data).
Ing data meprosesu ya kapamilatan ning pamag-fit keng linear initial rate kilub ning 0.5 anggang 10 s (dependi keng UQ2 concentration) ampo ing pamag-average kareng rate da reng atlung sample keng balang UQ2 concentration. Ing RC-LH1 concentration a mekwa keng balang extinction coefficient ginamit ya para i-convert ing rate keng catalytic efficiency, me-plot ya keng Origin Pro 2019 (OriginLab, USA), ampo me-fit ya keng Michaelis-Menten model para abalu ing apparent Km ampo Kcat values.
Para kareng transient absorption measurements, ing RC-LH1 sample me dilute ya keng ~2μM keng IMAC buffer a maki 50 mM sodium ascorbate (Merck, USA) ampo 0.4 mM Terbutin (Merck, USA). Ing ascorbic acid magagamit ya bilang sacrificial electron donor, at ing tert-butaclofen magagamit ya bilang QB inhibitor para siguraduan na ing main RC donor manatili yang mabawas (yapin, ali ya photooxidized) keng mabilug a prosesu ning pamanuknang. Manga 3 ml ning sample ing miyabe keng custom rotating cell (mga 0.1 m keng diametru, 350 RPM) a maki 2 mm optical path length para siguraduan na ing sample keng laser path atin yang sapat a oras para keng dark adaptation pilatan da reng excitation pulses. Gamitan me ing ~100-fs laser pulses para amplify ing Ti: Sapphire laser system (Spectra Physics, USA) para i-excite ing sample keng 880 nm keng repetition rate a 1 kHz (20 nJ para keng NIR o 100 nJ para keng Vis). Bayu ka magkolekta data, i-expose me ing sample keng excitation light kilub ning 30 minutus. Ing exposure maging sangkan ne ning QA inactivation (malyari yang mabawas ing QA misan o adwang besis). Pero pakibaluan mu na ing prosesung ayni malyari yang ibalik uling kaybat ning makabang panaun ning dark adaptation, ing RC mabagal yang mibalik keng QA activity. Ing Helios spectrometer (Ultrafast Systems, USA) ginamit ya para sukatan ing transient spectra a maki delay a -10 anggang 7000 ps. Gamitan me ing Surface Xplorer software (Ultrafast Systems, USA) para i-ungroup la reng data sets, kaybat isamut me at i-standardize. Gamitan me ing CarpetView software package (Light Conversion Ltd., Lithuania) para gamitan me ing metung a data set para makakwa kang differential spectra a maki kaugnayan keng decay, o gamitan me ing function na mag convolve kareng dakal a exponents a maki instrument response para makayabe ya keng single-wavelength spectral evolution keng Origin (OriginLab, USA).
Antimo ing sinabi ku babo (53), metung a photosynthetic film a maki LH1 complex a alang RC ampo peripheral LH2 antenna ing misadia. Ing membrane me-dilute ya keng 20 mM tris (pH 8.0) at kaybat mi-load ya keng quartz cuvette a maki 2 mm optical path. Ing 30nJ laser pulse ginamit ya para i-excite ing sample keng 540 nm a maki delay time a -10 anggang 7000 ps. Iprosesu ing data set antimo ing makasulat para keng Rps. pal sample.
Ing membrane me-pellet ya kapamilatan ning centrifugation keng 150,000 RCF kilub ning 2 oras keng 4°C, at kaybat ing kayang absorbance keng 880 nm me-resuspended ya keng 20 mM tris-HCl (pH 8.0) ampo 200 mM NaCl. Lasawan me ing membrane kapamilatan ning mabagal a pamag-stir keng 2% (w/v) β-DDM kilub ning 1 oras keng madalumdum keng 4°C. Ing sample me dilute ya keng 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) keng protein concentration a 2.5 mg ml-1 (Bio-Rad analysis). Ing karagdagang pamagprosesu megawa ya ibat keng minunang mibulalag a paralan (54), magumpisa keng pamaglaso ning 50 μg a protina keng kabilugan a 50 μl TEAB a maki 1% (w/v) sodium laurate (Merck, UK). Kaybat ning sonication kilub ning 60 s, mebawas ya keng 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) keng 37°C kilub ning 30 minutu. Para keng S-alkylation, i-incubate me ing sample keng 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) ampo idagdag me ibat keng 200 mM isopropanol stock solution kilub ning 10 minutu keng room temperature. Ing proteolytic digestion megawa ya kapamilatan ning pamagdagdag 2 μg trypsin/endoproteinase Lys-C mixture (Promega UK) at mi-incubate ya keng 37°C kilub ning 3 oras. Ing laurate surfactant me-extract ya kapamilatan ning pamagdagdag 50 μl ethyl acetate ampo 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) ampo me-vortexing kilub ning 60 s. Ing phase separation mipalabung ya kapamilatan ning centrifugation keng 15,700 RCF kilub ning 5 minutu. Agpang keng protocol ning manufacturer, metung a C18 spin column (Thermo Fisher Scientific, UK) ing ginamit para maingat a aspirate ampo desalt ing lower phase a maki peptide. Kaybat nang me-dry kapamilatan ning vacuum centrifugation, ing sample me-dissolve ya king 0.5% TFA ampo 3% acetonitrile, at 500 ng ing me-analisa kapamilatan ning nanoflow RP chromatography a kayabe ning mass spectrometry gamit ing system parameters a midetalye kanitang minuna.
Gamitan me ing MaxQuant v.1.5.3.30 (56) para keng pamangilala kareng protina ampo pamanintun para keng Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Ing mass spectrometry proteomics data mideposit ya keng ProteomeXchange Alliance kapamilatan ning PRIDE partner repository (http://proteomecentral.proteomexchange.org) lalam ning dataset identifier PXD020402.
Para keng pamagsuri kapamilatan ning RPLC a kayabe ning electrospray ionization mass spectrometry, ing RC-LH1 complex mesadya ya ibat keng wild-type Rps. Gamit ing minunang mibulalag a paralan (16), ing konsentrasyun ning protina a megawa kareng palustris cells 2 mg ml-1 ya keng 20 mM Hepes (pH 7.8), 100 mM NaCl ampo 0.03% (w/v) β- (Bio-Rad analysis) ) DDM. Agpang keng protocol ning manufacturer, gamitan me ing 2D purification kit (GE Healthcare, USA) para makalual kang 10 μg a protina kapamilatan ning precipitation method, at lasawan me ing precipitate keng 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile ampo 20% (v/v) danum. Limang microliters ing me-analisa kapamilatan ning RPLC (Dionex RSLC) a kayabe ning mass spectrometry (Maxis UHR-TOF, Bruker). Gamitan me ing MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) para keng pamikawani keng 60°C ampo 100μlmin -1, a maki gradient a 85% (v / v) solvent A [0.1% (v / v) FA ampo 0.02% (V/v) TFA aqueous solution] keng 5% Bv/v) solvent [0.1%(v/v) FA ampo 0.02%(v/v) keng 90%(v/v) acetonitrile TFA] Gamit ing standard electrospray ionization source ampo default parameters kilub ning maigit 60 minutu, ing mass spectrometer makakwa yang 100 anggang 2750 m/z (mass ratio-charge). King saup ning ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/), i-map me ing mass spectrum kareng subunits ning complex.
Deng cell meragul la kilub ning 72 oras lalam ning 100 ml NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) o high (300μMm-2 s-1) a sulu. M22 medium (M22 medium nung nukarin ing ammonium sulfate mewala ya at ing sodium succinate mipalitan ya keng sodium acetate) keng 100 ml a screw-top a bote (23). King limang 30-s a siklo, 0.1 micron a glass beads ing me-bead king volume ratio a 1:1 para lyse la reng cells at mipalamig la king yelo kilub ning 5 minutu. Ing e matunaw a bage, e mesira a cell ampo reng glass beads mewala la kapamilatan ning centrifugation keng 16,000 RCF kilub ning 10 minutu keng benchtop microcentrifuge. Ing membrane mekawani ya keng Ti 70.1 rotor a maki 100,000 RCF keng 20 mM tris-HCl (pH 8.0) a maki 40/15% (w/w) sucrose gradient kilub ning 10 oras.
Antimo ing misulat keng minunang obra, ing immunodetection ning His tag keng PufW (16). Ing makuyad a salita, ing melinis a core complex (11.8 nM) o ing membrane a maki pareung konsentrasyun ning RC (a meyakit kapamilatan ning oxidation a babawasan ing mebawas a pamiyaliwa ning spectrum at ing pamakisanmetung king load king me-stained a gel) king 2x SDS loading buffer (Merck, UK) a me-dilute adwang besis. Deng protina mikawani la keng replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK). Ing gel me-stain ya keng Coomassie Brilliant Blue (Bio-Rad, UK) para i-load ampo akit ing RC-L subunit. Ing protina keng kaduang gel mipalitan ya keng methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) para keng immunoassay. Ing PVDF membrane me-block ya keng 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 ampo 5% (w / v) skimmed milk powder, at kaybat me-incubate ya keng anti-His primary antibody (keng Dilute ing antibody buffer [500 mM tris-HCl) 7.6), 150 mM NaCl ampo 0.05% (v/v) Tween-20] keng 1:1000 A190-114A, Bethyl Laboratories, USA) kilub ning 4 oras. Kaybat nang me hugas atlung besis kilub ning 5 minutu keng antibody buffer, ing membrane metung ya kareng horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (diluted 1:10,000 keng antibody buffer) I-incubate para akit (5 minutu kaybat ning atlung pamaghugas keng antibody buffer) gamit ing WAR 2000. chemiluminescence substrate (Cyanagen, Italy) ampo Amersham Imager 600 (GE Healthcare, UK).
King pamagdrowing king intensity distribution ning balang stained gel o immunoassay lane, pamisanmetung king lugal lalam ning peak at pamagkwenta king intensity ratio ning RC-L (stained gel) ampo Protein-W (immunoassay), king ImageJ (57) Iprosesu ing larawan. Deting ratios mibayu la keng molar ratios kapamilatan ning pamag-assume na ing ratio ning RC-L keng protein-W keng purong RC-LH114-W sample 1:1 ya ampo ing pamag-normalize keng mabilug a data set agpang kaniti.
Para kareng karagdagang materyales para kening artikulu, lawan me ing http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1
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David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirma, Hicoil Necoil, Andrew C.
Ing matas a resolusyun a istruktura ning light trap 1 complex keng reaction center mamye yang bayung panaun keng quinone dynamics.
David JK Swainsbury, Park Qian, Philip J. Jackson, Kaitlyn M. Faries, Dariusz M. Niedzwiedzki, Elizabeth C. Martin, David A. Farmer, Lorna A. Malone, Rebecca F. Thompson, Neil A. Ranson, Daniel P Canniffe , Mark J. Dickman, Dewey Holten, Christine Kirma, Hicoil Necoil, Andrew C.
Ing matas a resolusyun a istruktura ning light trap 1 complex keng reaction center mamye yang bayung panaun keng quinone dynamics.
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