Atu†A tu umnak: OX11 0DE, UK, Diamond Building, Harwell Science and Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center.
Reaction center ceunak-laknak complex 1 (RC-LH1) cu a senmi ceunak a ngeimi bacteria hna i a hrampi ceunak sernak thilri a si. Rhodopseudomonas palustris in RC-LH1 complex i cryo-electron microscopy sining pahnih kan chuahpi. RC-LH114-W complex i 2.65-Å resolution structure cu RC pawngkam ah a ummi subunit LH1 loop 14 in a um, cucu protein W nih a kham, cu lio ah protein-W a ngei lomi complex cu RC nih a kulhmi RC composition tling a si. LH1 loop 16 khar. Hi sining hna tahchunhnak nih RC-LH1 complex chung i quinone a cawlcanghning kong ah theihhngalhnak a pek, RC QB hmun ah quinone aa pehtlaih tikah a hlan ah hngalh khawh lomi sining thlennak hna aa tel, cun bawmtu quinone aa pehtlaimi hmun hna umnak zong aa tel, cu nih cun RC ah a kalpi hna. W protein a dang tein a ummi nih LH1 loop kharnak kha a kham, cucaah quinone/quinolone i thlennak a rangtertu lam a ser.
Photosynthesis nih a chuahpimi thazaang nih vawlei cung nunnak vialte a dirpi khawh, cun ni ceunak biotechnology caah thil ti khawhnak tampi a ngei. Vawleicung pumpi ceunak sernak a thanchoter lio ah, a senmi ceunak a ngeimi bacteria zong nih thazaang phunphun le thilri thlennak thilti khawhnak a langhter. Photosynthesis an hrial khawh i mui lakah heterotrophic bacteria bantukin an ṭhang kho, nitrogen le carbon dioxide kha an remh khawh, hydrogen an chuah khawh, cun rimhmui thilri pawl kha an hrawh khawh (1-3). Hi thil kalning hna caah thazaang pek khawhnak dingah, ceunak cu rang tein le ttha tein chemical thazaang ah thlen a hau. Hi thil kalning cu ceunak a tlaihmi antenna complex nih ceunak a lak i a tlaihmi thazaang kha lehrulhnak hmun (RC) ah a thlen tikah aa thawk, cucaah charge tthennak (4 – 7) a thawk. Purple phototrophic bacteria ah ceunak sernak hrampi cu type 2 RC in sermi a si, ceunak-laknak complex 1 (LH1) nih a kulh, RC-LH1 core complex a ser. LH1 cu a tlikmi αβ heterodimers phunphun in sermi a si, cu hna nih cun bacterial chlorophyll (BChl) a molecule pahnih le carotenoids pakhat asiloah pahnih (8-12) an pehtlaih hna. A sawhsawh bikmi LH1 antenna cu 16 asiloah 17 αβ heterodimers nih RC (9-13) kha a kharmi loop in a kulh, asinain a dang core complexes ahcun, transmembrane peptides nih a pawngkam LH1 pehzulhnak kha a kham, cucaah RC le cytochrome bc complex karlak ah quinol/quinone karhternak a thanchoter (1111, 13). A senmi ceunak a ngeimi thingram Rhodopseudomonas (Rps.) cu ceunak sernak a bawmtu thazaang le electron thlennak a hngalhthiam khomi zohchunh awk thilnung pakhat a si. Rps i a hmasa bik lungvar sining. Palustris RC-LH1 complex model cu RC a si, heterodimeric LH1 loop 15 nih a kulh, cu hna cu “Protein W” (14) timi theih lomi protein nih a kham hna. Protein-W cu a hnu ah RPA4402 tiah hngalh a si, cucu 10.5kDa protein a si i a hlankan in chimchungmi transmembrane helices (TMH) pathum a ngei (16). RC-L, M (pufL, pufM) le LH1α, β (pufA, pufB) subunits a codemi genes caah hmanmi min saknak he aa tlak nakhnga rpa4402 gene code protein W kha pufW ah thlen ding in ruahnak kan cheuh. Hngalh a nuammi cu, protein-W cu RC-LH1 10% hrawng lawng ah a um, Rps a langhter. palustris nih RC-LH1 phun hnih a chuahter. Hika ah, core complex pahnih, pakhat cu protein W le 14 αβ heterodimers he, a dang pakhat cu protein W tel loin le kharmi 16 Heterodimer LH1 loop he, a sangmi cryo-EM (cryo-EM) sining hna kha kan langhter. Kan dirhmun nih Rps i RC-LH1 complex hngalhthiamnak ah step thlennak a aiawh. palustris, zeicahtiah phun dangdang pakhat cio i aa khatmi milu kha kan hlathlai cang i peptide pakhat cio le aa pehtlaimi rong le aa pehtlaimi lipids le quinones kha fiang tein thleidan khawhnak ding caah a za tawkmi resolution kan ngei. Hi sining hna tahchunhnak nih a langhter mi cu RC-LH1 complex dang ah hmuh rih lomi TMH protein-W pathum nih quinone/quinolone i thlennak a rangtertu quinone lam an ser. A kilvenmi lipid le quinone pehtlaihnak hmun tampi cu hmuh an si cang, cun quinone le RC fonh hnu ah thlennak thar kan hmuh, cucu oxygen a ngeimi phototrophic thilnung hna i photosystem II (PSII) RC caah aa tlakmi a si kho men. Kan hmuhmi nih hin purple phototrophic bacteria RC-LH1 core complex ah quinone/quinolone pehtlaihnak le i thlennak a cawlcangh ning kong ah hmuhnak thar a kan pek.
Rps ah hmuhmi complex pahnih kong ttha tein hlathlainak tuah khawhnak ding caah. palustris, RC-LH1 pakhat cio kha biochemical lam in kan thleidan hna. Protein W-tlawmmi complex (a hnu ah ΔpufW tiah auhmi) cu pufW gene (16) a ngei lomi phun in thianh a si, cun RC-LH1 complex pakhat lawng chuah khawh a si. Protein W aa telmi complex cu phun pakhat nih a chuah. Hi phun i protein W cu a C-terminus ah 10x His tag in remh a si, cucaah protein W aa telmi complex cu a tlawm deuhmi protein W he thir kha a cawl kho lonak thawngin ttha tein fonh khawh a si. A harmi cu ttha tein aa ṭhen (16) Affinity Chromatography (IMAC).
Figure 1 ah hmuh khawhmi bantuk in, complex pahnih nih hin LH1 antenna nih a kulhmi sub-unit RC (RC-L, RC-M le RC-H) pathum an ngei. Protein-W a ngei lomi complex i 2.80-A sining nih 16 αβ heterodimers a langhter, RC a kulhmi kharmi LH1 loop a ser, a hnu ah RC-LH116 complex tiah auhmi a si. Protein-W aa telmi thilri 2.65Å sining nih protein-W nih a khammi 14-heterodimer LH1 a ngei, hi hnu ah RC-LH114-W tiah auhmi a si.
(A le B) Compound a cunglei langhternak. (C le D) Rod ah langhtermi aa pehtlaimi rong hna. (E le F) Cytoplasmic cunglei in hmuhmi complex pawl cu cartoon ah langhtermi peptides le LH1 subunits an ngei, cun protein-W gap in suimilam lei hoih in nambar an pek hna [Rba nambar he aa tlak. sphaeroides complex (13)]. LH1-α caah cun, protein subunit rong cu a sen; LH1-β caah cun, protein subunit rong cu a sen; protein-W caah cun, protein cu a sen; RC-H caah cun, a senmi a si; RC-L caah cun, Orange a si; RC-M caah, magenta. Cofactors cu fung nih a langhter, a sen nih BChl le BPh a molecule a aiawh, sen nih carotenoids a aiawh, cun a sen nih UQ10 molecule a aiawh. (G le H) RC-LH114-W complex (G) le RC-LH116 complex (H) i aa tlukmi hmun ah protein-W gap a ngan deuhmi hmuhnak. Cofactors cu hmunhma tlingternak phun in langhter an si, chelated quinone cu a sen in langhter an si. Protein-W gap cu (G) ah a senmi hri in langhter a si, cun LH116 ring cung i quinone/quinolol a karhnak hmun hmete hna cu (H) ah a senmi hri in langhter a si.
Figure 1 (A le B) nih RC cu LH1αβ heterodimers aa ongmi asiloah aa kharmi nih a kulhmi a langhter, cu hna nih cun BChl pahnih le carotenoid pakhat cio an i pehtlaih (Figure 1, C le D). Hlan lio hlathlainak nih Rps cu LH1 complex a si tiah a langhter. Spirulina xanthin sernak lam ah, hi phun hna nih hin carotenoids phunphun an ngei (17). Sihmanhsehlaw, spiropyrroxanthin cu a ukmi carotenoid a si i a rit ning cu a ttha. Cucaah, LH1 pehtlaihnak hmun vialte ah spiroxanthin model tuah ding in kan i thim. Alpha le beta polypeptides cu a tawimi membrane lenglei hmun a ngeimi TMH pakhat lawng an si (Figure 1, A, B, E, le F). C-terminus ah a tangmi 17 an tamnak cu hmuh a si lo nain, alpha polypeptide cu Met1 in Ala46 tiang complex pahnih ah an i tthen. β polypeptide cu RC-LH116 ah Gly4 in Tyr52 ah a zor, cun RC-LH114-W ah Ser5 in Tyr52 ah a zor. N-terminal 3 asiloah 4 asiloah C-terminal 13 a tangmi an tlawm lo (Figure S1). Ramlak phun in timhtuahnak cawhmi RC-LH1 complex i mass spectrometry hlathlainak nih a tlaumi hmun cu hi peptide pawl i aa dangmi tthennak ruangah a si ti a langhter (Figure S1 le S2). α-Met1 i N-terminal formylation zong hmuh a si (f). Hlathlainak nih a langhter mi cu α-peptide cu fMet1 in Asp42/Ala46/Ala47/Ala50 tiang a tangmi a si, cun β-peptide cu Ser2 in Ala53 tiang a tangmi a si, cucu a niammi EM density map he aa tlak ngai.
α-His29 le β-His36 i bawmhnak nih BChls cu hmaitonh in a tuah hna; αβ heterodimer pakhat cio cu a pawngkam hna he an i fonh i RC pawngkam ah a ongmi hri (RC-LH114-W) asiloah aa kharmi hri (RC-LH116) an ser. Exciton coupled pigment array (Figure 1, C le D). RC-LH114-W i 877 nm band he tahchunh ahcun, RC-LH116 i 880 nm laknak sen thlennak cu 3 nm a si (Figure 2A). Sihmanhsehlaw, a tlongmi dichroism spectrum cu aa khat dih (Figure 2B), a langhtermi cu a ongmi le a kharmi tthennak karlak ah fiang tein thleidannak a um ko nain, BChls pawngkam cu aa lo ngai. Absorption redshift cu kharmi hri cung i lumnak cawlcanghnak a zormi le a fekmi a karhmi (18, 19), kharmi hri nih a chuahtermi rong pehtlaihnak thlennak (20, 21), asiloah hi thil pahnih fonhmi (11) ruangah a si kho.
(A) Ultraviolet/hmuh khawhmi/infrared he aa naihmi laknak spectrum, a sangbik hna cu aa pehtlaimi rong hna he hmelchunhnak an tuah i 775 nm ah BPh sangbik ah an thlen. (B) 805 nm ah BChl laknak ah a tlarimi dichroism spectrum. (C le D) RC-LH114-W complex (C) le RC-LH116 complex (D) i caan-a thlenmi laknak spectra chungin thimmi ΔA spectra. A ttha deuhmi tahchunhnak caah, spectra vialte cu 0.2 ps ah −A ∆A ah an i tluk. (E) UQ2 a phunphun a um lio ah ceunak pek hnu ah cytochrome c2 oxidation rate (raw data caah Figure S8 zoh). (F) A niam, a karlak asiloah a sangmi ceunak (10, 30 asiloah 300μMm-2 s-1, ciocio) tangah a ṭhangmi cells ah, thianhmi complex chung i protein W le RC-L subunits le aa ṭhenmi membrane ratio. SDS-polyacrylamide gel electrophoresis le immunoassay hmangin protein dat kha tuak (raw data caah Figure S9 zoh). Thianhmi RC-LH114-W complex he aa pehtlaimi zat kha tuak. RC-L le protein-W i an i tluknak cu 1:1 a si.
RC-LH114-W (Figure 1, A, C, le E) i a rawkmi αβ14 loop chung i position 1 ah a ummi BChls cu RC-LH116 chung i aa tlukmi BChls nakin 6.8Å in RC primary donor (P) he an naih deuh (Figure. 1, B, D, le F, le S3); sihmanhsehlaw, complex pahnih hna i a caan tawite chung laknak kinetics nih RC-LH114-W le RC-LH116 caah, LH1 in RC tiang thazaang thlennak caan thlennak cu 40 ±4 le 44±3 ps a si ti a langhter (Figure 2). , C le D, Figure S4 le Table S2). Cun RC chung ah electronic thlennak ah a biapi mi thleidannak a um lo (Figure S5 le aa pehtlaimi chapmi cauk). LH1 le RC-P karlak ah thazaang thlennak caan aa naih tukmi cu LH1 loop pahnih chung i BChl tam deuh i an hlatnak, an angle le an potential thazaang aa khatmi ruangah a si lai tiah kan lunghrinh. A niam bik hlatnak phanh khawhnak ding caah LH1 thazaang sining kawlnak cu a ttha lomi hmun in RC ah direct thazaang thlennak nakin a rang deuh lo tiah a lang. RC-LH114-W chung i a ongmi LH1 loop zong nih a sining hlathlainak caah a niammi lumnak dirhmun tangah a biapi lomi lumnak cawlcanghnak a tong kho, cun RC 1 dirhmun ah βBChls i a rong aa hlatnak in khaan lumnak ah αβ14 ring sining a sau deuhmi a um.
RC-LH116 complex ah 32 BChls le 16 carotenoids aa tel, cun a dihlak in a dirhmun cu Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) DB strain (IDC 297) (297) in hmuhmi he aa khat. le green algae (Blc.viridis) (PDB ID 6ET5) (10). A tlukruannak hnu ah, αβ heterodimers dirhmun ah aa dangnak hmete lawng hmuh a si, a hlei in 1-5, 15, le 16 (Figure S6). Protein-W a ummi nih LH1 sining cungah tampi hnahnawhnak a pek. A TMH pathum cu a tawimi hri in an i pehtlai, N-terminal cu complex lumen lei ah a um i C-terminal cu cytoplasmic lei ah a um (Figure 1A le 3, A in D tiang). Protein-W cu a tam deuh cu ti a doh (Figure 3B), cun TMH2 le TMH3 nih LH1αβ-14 he an i pehtlaih i transmembrane cunglei an ser (Figure 3, B le E in G tiang). A pehtlaihnak cu a bik in transmembrane peng ah Phe, Leu le Val a tangmi hna in sermi a si. Hi a tangmi hna cu ti a do khomi amino acid le αβ-14 rong hna he an i cawh. A cheu polar residues zong nih pehtlaihnak ah an bawmh, W-Thr68 le β-Trp42 karlak ah a harmi khur cunglei ah hydrogen pehtlaihnak zong aa tel (Figure 3, F le G). Cytoplasm cunglei ah, Gln34 cu αβ-14 carotenoids keto phu he aa naih. Cun, n-dodecyl β-d-maltoside (β-DDM) molecule cu a ttha, cun a ti a dohmi hngawng cu protein-W le αβ-14 karlak ah a karh, cun lipid hngawng cu pum chung ah a um kho men. Cun, protein W le RCH i C-terminal resolution regions pawl cu an i naih tuk ti zong kan hmuh, asinain a hleiin pehtlaihnak sernak chung ah an um lo (Figure 1, A le E). Sihmanhsehlaw, hi protein pahnih hna i a phichuak a um rih lomi C-terminal amino acid ah pehtlaihnak a um kho, cu nih cun RC-LH114-W complex fonh lio ah protein-W laknak caah lam a ser khawh.
(A) Protein-W, cu cartoon bantuk in LH1αβ14 he aa pehtlaimi a si i, fung bantuk a simi a kam hri (sen) a ngei, electrostatic potential diagram (0.13 a tluknak he a fiangmi a senmi cunglei) ah langhter a si. (B) Protein-W cu ti a huat lomi rong a ngeimi cunglei nih a aiawh. Polar le charged hmun pawl cu cyan in langhter an si, ti a huat lomi hmun pawl cu rang in langhter an si, cun ti a huat tukmi hmun pawl cu orange in langhter an si. (C le D) Cartoon ah langhtermi Protein-W, a dirhmun cu (A) (C) he aa khat, cun 180° in aa mer (D). A zulmi dirhmun ning in, thleidan khawhmi a tangmi hna nih arfi rong an hman, cuka ahcun N-terminal cu a sen i C-terminal cu a sen. (E) Protein-W cu (A) he aa lo tein hmuh khawh a si, cun protein-W:LH1 karlak i a tangmi hna cu hmelchunhnak aa pehmi fung hna nih an langhter. (F) Protein-W cu cartoon langhternak ah (E) le LH1αβ14 he tahchunh in 90° in aa mer, cun bar langhternak ah aa pehtlaimi a tangmi he tahchunh in. Beta polypeptide chungin a tangmi a tangmi hna cu min an bunh hna. Cofactor cu hmanthlak 1 rong he aa tlakmi bar in langhter a si, a rawkmi β-DDM cu a sen in langhter a si, cun oxygen cu sen in langhter a si. (G) (F) chung i hmuhnak cu 180° in aa mer, min bunhmi alpha polypeptide a langmi a tangmi hna he.
Protein-W nih αβ heterodimer (Figure 1F ah 15nak) kha a thlen, cucaah loop kharnak kha a kham i a hmasa bik αβ heterodimer pathum kha a tlikter hna. A hmasa bik αβ-1 heterodimer i a sangbik a tliknak angle cu film normal he tahchunh tikah 25° in 29° tiang a si ti kha hmuh a si (Figure 1, A le E), cucu RC A sharp contrast-LH116 (Figure 1, B le F) ah αβ-1 2° in 8° tiang a tliknak nih a sermi a si. Pahnihnak le pathumnak heterodimers cu 12° in 22° le 5° in 10° tiang an i mer. RC i a tthawngmi dawnkhantu ruangah, αβ-1 i a tliknak nih αβ pahnihnak pahnih (cucu Figure 1F chung i 16th αβ he aa tlak) aa tel lo, cucaah LH1 ring ah a fiangmi gap a ser (Figure 1, A le E). αβ heterodimers pahnih an um lo caah, BChl pali le carotenoids pahnih an tlau caah, carotenoids pakhat hmanh nih a tlikmi αβ-1 subunit ah an i peh lo, cu nih cun LH114-W ring ah carotenoids 13 Vegetarian le 28 BChls aa telmi a chuahter. αβ1 in 7 tiang ah a ummi complex pahnih hna i an hmunhma thlennak tuaktannak cu LH1 loop a dang vialte nakin a niam deuh, cucu RC QB hmun he aa pehtlaimi LH1 subunit i a ummi plasticity kha a langhter men lai (Figure 4).
RC-LH114-W (A le B) le RC-LH116 (C le D) hmanthlak hna cu a cunglei zohnak/a kam zohnak (A le B) (A le C) le Fig. 1. (B le D) i a chunglei zohnak in langhter an si. A rong aa dangmi tawhfung pawl cu kehlei ah langhter an si.
Stoichiometric ratio 1:14 a ngeimi a dang sining a ngeimi core complex pakhat lawng a ummi cu Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13) a si. Sihmanhsehlaw, protein W le PufX cu a fiangmi aa lawhnak an ngei lo, cun anmah le anmah LH1 sining cungah hnahnawhnak nganpi an ngei. PufX cu N-terminal cytoplasmic domain a ngeimi TMH pakhat lawng a si i Rps he aa tlakmi hmun ah RC-H subunit (13) i cytoplasmic lei he aa pehtlaimi a si. palustris LH116αβ-16. PufX nih RC-LH1 le cytochrome bcl complex karlak ah quinone/quinolone i thlennak caah lam a ser i Rba vialte ah a um. sphaeroides core complex (13). Monomer-monomer pehtlaihnak cu Rba ah a um ko nain. Sphaeroides RC-LH1-PufX dimer cu RC-LH114-W chung i protein W i pehtlaihnak hmun ah a um, cun PufX le protein-W nih a chuahtermi gap cu aa tlukmi hmun ah a um (Figure S7A). RC-LH114-W chung i a ummi gap cu Pseudomonas rosea LH1 i a ummi quinone channel (8) he zong aa tlak, cucu protein W asiloah PufX he aa pehtlaimi a si lomi peptide nih a sermi a si (Figure S7B). Cun, Blc chung i quinone channel. γ subunit pakhat (7) telh loin sermi emerald green LH1 cu aa lo ngaimi dirhmun ah a um (Figure S7C). Protein phunphun nih a hruai ko nain, RC-LH1 complex ah aa khatmi hmun ah hi quinone/quinolol channel pawl an langhnak cu aa fonhmi thlennak tahchunhnak a lo, cu nih cun protein W nih a sermi gap cu quinone channel bantukin rian a ttuan kho ti a langhter.
LH114-W loop chung i a ummi tthennak nih RC-LH114-W complex chunglei hmun le bulk membrane (Figure 1G) karlak ah a pehzulhmi membrane pengtlang sernak a tuah khawh, cu nakin protein hna bantuk in protein pore in domain pahnih kha a pehtlaih hna lo. RC-LH116 complex cu kharmi Tch he aa lo. Hriamnam bantuk a harmi (22) (Hmanthlak 1H). Membrane chungin quinone a karhnak cu a kau lomi protein channel chungin a karhnak nakin a rang deuh caah, a ongmi LH114-W loop nih a kharmi LH116 loop nakin RC thlennak a rang deuhmi a tuah khawh, cun RC chungah quinone a karhnak cu a kham deuh men lai. Protein W nih RC hmangin quinones thlennak a hnorsuan maw ti hneksaknak caah, ubiquinone 2 (UQ2) (a tawi deuhmi isoprene hngawng a ngeimi natural UQ10 he aa lo) tlawmpal ah cytochrome oxidation hneksaknak kan tuah (Figure 2E). Chelated quinone a ummi nih a langmi Michaelis aa thleng lomi (RC-LH114-W le RC-LH116 cu 0.2±0.1μM le 0.5±0.2μM caah aa tlakmi an si) a dikthliar in hngalh khawhnak a kham ko nain, RC-LH114-W ( 4.6±0.20.2μM) a tam bik rate cu s-LH114-W a si. RC-LH116 (3.6±0.1 e-RC-1 s-1) nakin 28±5% a ngan deuh.
A hramthawk ahcun protein-W cu core complex 10% hrawng ah a um tiah kan rak tuak (16); hika ah, ceunak tlawm, ceunak tlawmpal, le ceunak sang tthanchonak cells hna i an umnak rate cu 15±0.6%, 11±1% le 0.9±0.5, ciocio % an si (Figure 2F). Mass spectrometry i zatlang tahchunhnak nih a langhter mi cu histidine tag chapnak nih ramlak phun he tahchunh tikah protein-W a tlawmter lo (P = 0.59), cucaah hi tlukruannak hna hi remhmi protein-W thilri an si lo (Figure S10). Sihmanhsehlaw, RC-LH1 complex chung i protein-W tlawmte lawng a ummi nih hin RC cheukhat kha a rang deuhmi rate in a thlen khawh hna, cucaah RC-LH116 complex chung i a tlawm deuhmi quinone/quinolone i thlennak kha a zorter khawh. Ceunak a tammi umnak rate cu naite transcriptomics data he aa kalh ti kha kan hmuh, cucu pufW gene langhternak cu ceunak tthawng tangah a karh ti a langhter (Figure S11) (23). pufW ṭialmi le RC-LH1 complex chungah protein-W luhternak karlak i aa dannak cu a fiang lo i protein a harmi uknak kha a langhter men lai.
RC-LH114-W ah, 6 cardiolipin (CDL), 7 phosphatidylcholine (POPC), 1 phosphatidylglycerol (POPG) le 29 β-DDM molecule pawl kha an chiah hna i 6 CDLs, 24 POPCs, 2 POPGs le 12βDs hna kha an suai hna. RC-LH116 (Hmanthlak 5, A le B). Hi sining pahnih ah hin, CDL cu complex cytoplasmic lei ah a um deuh, cu lio ah POPC, POPG le β-DDM cu luminal lei ah an um deuh. RC-LH114-W complex (Figure 5A) i αβ-1 in αβ-6 tiang ah lipid le detergent molecule pahnih an thleidan hna, cun RC-LH116 he aa tlukmi region ah panga an thleidan hna (Figure 5B ). Complex khatlei ah lipids tam deuh an hmuh, a bik in CDL, RC le αβ-7 in αβ-13 karlak ah an i pum (Figure 5, A le B). A dang sining tein aa thlengmi thau le thil sawhnak sii pawl cu LH1 ring lenglei ah an um, cun a ttha tein aa thlengmi acyl hri pawl cu LH1 subunits karlak ah an karh, RC-LH114-W ah β-DDM tiah a hlankan in an langhter, cun RC A ah β-DDM le PO-LH1616 cawhmi ah β-DDM tiah an fianter. Kan dirhmun ah chelating lipids le detergents hna an dirhmun aa khatmi nih hin pumsa lei in aa pehtlaimi pehtlaihnak hmun an si ti a langhter (Figure S12A). Tch chung i aa tlukmi molecule hna dirhmun zong nih aa khatmi a ttha. Nemnak le Trv. Strain 970 RC-LH1s (Figure S12, B in E) (9, 12) le lipid lu phu i hydrogen-bonding residues nih sequence alignment ah ttha tein kilvennak an langhter (Figure S13), cu nih cun RC (24) he aa pehtlaimi Conserved CDL, hi hmun hna hi RCH-LH complex ah kilven khawh a si ti a langhter.
(A le B) RC-LH114-W (A) le RC-LH116 (B) peptides cu cartoon in langhter an si, cun rong pawl cu rods in langhter an si, Figure 1 chung i rong suaisamnak hmangin. Lipids cu sen in langhter an si, cun thil sawhnak sii pawl cu rang in langhter an si. RC QA le QB hmun ah aa pehtlaimi UQ cu a sen, a dang tein a ummi UQ cu a sen. (C le D) (A) le (B) he aa khatmi hmuhning, lipids telh loin. (E in G) RC-LH116 in Q1(E), Q2(F) le Q3(G) a ngan deuhmi hmuhnak, pakhat le pakhat aa thlengmi a kam hri hna he. Hydrogen pehtlaihnak cu a senmi hri in langhter a si.
RC-LH116 ah, RC QA le QB UQ pahnih, charge tthennak ah electron thlennak ah aa telmi hna cu an i pehtlaihnak hmun ah an rawk. Sihmanhsehlaw, RC-LH114-W ah, QB quinone cu a ttha rih lo i a tanglei ah fiang tein kan i ruah lai. QA le QB quinones lengah, chelated UQ molecule pahnih (RC le LH1 ring karlak ah a ummi) cu RC-LH114-W sining ah an lu phu (Q1 le Q2 ah a ummi, ciocio) ningin an i tthen. hmun). Hmanthlak 5C). Isoprene unit pahnih cu Q1 ah chiah an si, cun density map nih Q2 i isoprene tail 10 tling tein a tawlrel. RC-LH116 dirhmun ah, chelated UQ10 molecule pathum (Q1 in Q3, Figure 5D) cu an i tthen, cun molecule vialte cu a hngawng chung vialte ah a fiangmi density an ngei (Figure 5, D in G). Mah sining pahnih ah, Q1 le Q2 i quinone lu phu hna dirhmun cu aa khatmi an si (Figure S12F), cun RC he lawng an i pehtlaih. Q1 cu RC-LH114-W W gap luhnak ah a um (Figure 1G le 5, C, D le E), cun Q2 cu QB pehtlaihnak hmun pawng ah a um (Figure 5, C, D) le F). A kilvenmi L-Trp143 le L-Trp269 a tangmi hna cu Q1 le Q2 he an i naih tuk i π-stacking pehtlaihnak a chuahter khomi an si (Figure 5, E le F, le Figure S12). L-Gln88, Q1 i a hlatnak oxygen in 3.0 Å nih a fekmi hydrogen pehtlaihnak a pek (Figure 5E); hi a tangmi cu a hlat bikmi pehtlaihnak ti lo cu RC vialte ah a um (Figure S13). L-Ser91 cu a dang RCs tam deuh ah Thr caah aa thlengmi a si (Figure S13), Q1 i methyl oxygen in 3.8 Angstroms a si, cun a derthawmmi hydrogen pehtlaihnak a pek khawh (Figure 5E). Q3 cu a hleiin pehtlaihnak a ngei lo, asinain RC-M subunit le LH1-α subunit 5 in 6 karlak ti a dohmi hmun ah a um (Figure 5, D le G). Q1, Q2 le Q3 asiloah a pawng i a ummi chelated quinones zong Tch ah an i remh cang. Nem tein, Trv. Strain 970 le Blc. Iris sining (9, 10, 12) nih RC-LH1 complex chung i a kilvenmi bawmtu quinone pehtlaihnak hmun kha a langhter (Figure S12G). RC-LH116 chung i a rawkmi UQ panga cu riantuannak sang a ngeimi ti chromatography (HPLC) in a tuakmi complex pakhat cio i 5.8±0.7 he aa tlak ngai, cu lioah RC-LH114-W chung i a rawkmi UQ pathum cu 6.2±0.3 tahmi man (Fig. S14) nakin a niam deuh. inn sakning.
Pseudo-symmetric L le M polypeptides nih TMHs panga cio an ngei i BChl dimer pakhat, BChl monomers pahnih, bacteriophage (BPh) monomers pahnih, le Heme a si lomi thir pakhat le UQ10 molecule pakhat asiloah pahnih aa fonhmi heterodimer an ser. A donghnak ketone phu cung i hydrogen pehtlaihnak a ummi le Rps ah a hngalhmi a khonnak thawngin, carotenoids cu M-subunit ah an i tel, cucu cis-3,4-dehydroorhodopin tiah min an sak. Phun (25). RC-H i a lenglei membrane domain cu TMH pakhat lawng nih membrane ah a hren. A dihlak in RC dirhmun cu aa pehtlaimi phun (Rba tibantuk) hna i subunit RC pathum he aa lo. sphaeroides (PDB ID: 3I4D). BChl le BPh, carotenoid hngawng le heme a si lomi thir hna i an macrocycle hna cu hi thilri hna i an resolution chung ah an i tluk, cu bantuk cun QA hmun ah UQ10 lu phu le RC-LH116 ah QB quinone zong an i tluk (Figure S15).
QB hmunhma hmannak rate aa dangmi RC sining pahnih hmuh khawhnak nih QB quinone pehtlaihnak he aa pehtlaimi aa khatmi sining thlennak zohfelnak caantha thar a pek. RC-LH116 complex ah, QB quinone cu a tlingmi “a naih” dirhmun ah a um (26), asinain RC-LH114-W tthennak nih QB quinone a ngei lo. RC-LH114-W ah QB quinone a um lo, cucu khuaruahhar a si, zeicahtiah complex cu a cawlcang, RC-LH116 complex nakin a ttha deuhmi QB quinone he. LH1 ring pahnih nih quinones paruk hrawng an chelate ko nain, panga cu kharmi RC-LH116 ring ah an dirhmun in an i remh, cu lioah pathum lawng cu a ongmi RC-LH114-W ring ah an dirhmun in an tlawm. Hi a karhmi dirhmun rawhralnak nih hin RC-LH114-W QB hmunhma pawl a rang deuhmi thlennak, complex chung i quinone kinetics a rang deuhmi le LH1 loop tan khawhnak a karhmi a langhter men lai. RC-LH114-W i RC QB hmun ah UQ a ​​um lonak cu a har deuhmi le a cawlcang deuhmi a harmi ruangah a si kho men tiah ruahnak kan cheuh, cun RC-LH114-W i QB hmun cu UQ thlennak ah a lum colh cang.
QB tel loin, L-Phe217 he aa pehtlaimi UQ10 pehtlaihnak he aa tlak lomi dirhmun ah aa mer, zeicahtiah a hngawng i a hmasa bik isoprene unit he hmunhma i pah a chuahter lai (Figure 6A). Cun, a fiangmi a tlangpi thlennak hna cu a fiang ko, a hlei in helix de (TMH D le E karlak i a ummi helix tawite) cu L-Phe217 cu QB pehtlaihnak hmun ah aa thlen i L-Tyr223 aa mernak (Figure 6A) M-A p5 he hydrogen pehtlaihnak hrawh awk le QB pehtlaihnak frame kha khar awkah hmun (Hmanthlak 6B). Helix de cu a hram ah aa mer, L-Ser209 i Cα cu 0.33Å in aa thlen, L-Val221Cα cu 3.52Å in aa thlen. TMH D le E ah hmuh khawhmi thlennak a um lo, cu hna cu an pahnih in an i fonh khawh (Figure 6A). Kan theih khawh tawk ahcun, hi cu QB hmun a khartu natural RC chung i a hmasa bik dirhmun a si. A tlingmi (QB-bound) sining he tahchunh tikah quinone a zor hlanah, quinone chungah a luh khawh nakhnga a sining thlennak a herh ti a langhter. L-Phe217 cu quinone lu phu he π-stacking pehtlaihnak ser awkah aa mer, cun helix cu lenglei ah aa mer, cu nih cun L-Gly222 ruhro le L-Tyr223 a kam hri kha a fekmi hydrogen pehtlaihnak sining he hydrogen pehtlaihnak network a ser khawh (Figure 6, A le C).
(A) Hologram (L chain, orange/M chain, magenta) le apo (gray) sining aa tlukmi cartoon, cu ah cun a biapimi a tangmi hna cu fung bantuk langhternak in an langhter. UQ10 cu a senmi hri in a langhter. Dot line nih cun a dihlak in a chuakmi hydrogen pehtlaihnak kha a langhter. (B le C) Apolipoprotein le a dihlak in a ummi a lenglei langhternak, L-Phe217 i a kam oxygen kha a sen in a langhter i L-Tyr223 cu a sen in a langhter. L subunit cu a sen; M le H subunits cu rong an ngei lo. (D le E) Apolipoprotein (D) le a dihlak (E) RC QB hmun [color by (A) respectively] le Thermophilus thermophilus PSII (a sen, a sen plastic quinone he; PDB ID: 3WU2) Align (58).
Ruah lo piin, LH1 tel loin QB-tlawmmi RCs sining zeimawzat a um ko nain, hi hlathlainak ah hmuhmi sining thlennak hna cu a hlan ah report an rak tuah bal lo. Cu hna ah cun Blc in QB tlawmternak sining aa tel. viridis (PDB ID: 3PRC) (27), Tch. tepidum (PDB ID: 1EYS) (28) le Rba. sphaeroides (PDB ID: 1OGV) (29), an dihlak in an QB dirhmun he aa khat dih. 3PRC kha naih tein zohfelnak nih LDAO (Lauryl Dimethyl Amine Oxide) thil sawhnak molecule pawl cu QB dirhmun luhnak ah an i pehtlaih ti a langhter, cu nih cun kharmi dirhmun ah remh ṭhannak kha a kham khawh. LDAO cu 1EYS asiloah 1OGV ah hmun khat te ah a rawk lo nain, hi RC pawl cu thil thianhnak sii pakhat hmangin timhtuahnak an ngei i cucaah aa khatmi thil an chuahter khawh. Rba i a crystal sining. Sphaeroides RC cu cytochrome c2 (PDB ID: 1L9B) he aa fonhmi zong nih a kharmi QB hmun a ngei ko rua tiah a lang. Sihmanhsehlaw, hi kongah, RC-M polypeptide N-terminal region (Q helix cung i Tyr residue H bond hmangin QB pehtlaihnak hmun he aa pehtlaimi) nih a phungning lomi dirhmun a lak, cun QB dirhmun thlennak cu a dang tein hlathlainak tuah a si ti lo (30 ). Lungdaihnak a pemi cu RC-LH114-W sining ah M polypeptide hi bantuk thlennak kan hmu lo, cucu RC-LH116 RC N-terminal region he aa khat deuh. Cun, thil sawhnak sii-hram bunhmi LH1 antenna hloh hnu ah, PDB chung i apolipoprotein RCs pawl cu an remh hna, cu nih cun RC le a pawngkam LH1 ring chunglei karlak i a ummi chunglei quinone pools le lipids pawl kha a hloh hna ti zong kha hngalh a herh (31, 32). RC cu a rian a ttuan peng ko, zeicahtiah a rawk khomi QB quinone dah ti lo ahcun, a fekmi a si lo i timhtuahnak tuah lio ah a tlau tawn (33). Cun, RC chungin LH1 le natural cyclic lipids chuahnak nih riantuannak ah hnahnawhnak a pek khawh ti zong theih a si, cu hna cu charge-separated P+QB-state nunchung caan tawiternak tibantuk an si (31, 34, 35). Cucaah, RC pawngkam ah a ummi LH1 ring a ummi nih “kharmi” QB hmun kha a kilven khawh lai tiah kan ruah, cucaah QB pawng i a ummi pawngkam kha a kilven khawh.
Apolipoprotein (QB quinone tel loin) le a tlingmi sining nih QB hmunhma thlennak hmanthlak pahnih lawng a langhter ko nain, thil cangmi a zulmi si loin, hydroquinone nih a peh ṭhanmi khamnak caah pehtlaihnak kha khar khawh a si ti langhternak a um. Apolipoprotein QB hmun pawng i quinolol le quinone i pehtlaihnak cu aa dang kho, cu nih cun RC nih a hlawtnak a chuahter. Quinones i pehtlaihnak le zorternak ah a sining thlennak nih hmunhma a lak tiah caan saupi chung an rak ruah cang. Muihnak i remh hnu ah a kikmi RCs nih quinones zorternak an tuah khawhnak cu a rawk (36); X-ray crystallography nih a langhter ning ahcun hi rawhnak hi QB quinones cu a riantuanmi a naihnak hmun in 4.5 Å hrawng a hlatnak “a hlatnak” dirhmun ah an i hren caah a si (26) , 37). Hi a hlatnak pehtlaihnak sining cu apolipoprotein le a tlingmi ring sining karlak i a karlak dirhmun hmanthlak a si tiah ruahnak kan cheuh, cucu quinone he a hramthawk pehtlaihnak le QB hmun onnak a zul.
Phototrophic bacteria cheukhat le cyanobacteria, algae le thingram hna i PSII complex ah hmuhmi type II RC cu a sining le a riantuan ning kilvennak a ngei (38). Figure 6 (D le E) ah langhtermi sining tlukruannak nih PSII RCs le bacterial RC complex QB hmun karlak i aa lawhnak kha a langhter. Hi tahchunhnak hi quinone pehtlaihnak le zorternak aa pehtlaimi system hna hlathlainak caah zohchunh awk pakhat a si cang. A hlan chuahmi hna nih cun sining thlennak cu quinones PSII zorternak he aa pehtlaimi a si tiah an rak ti (39, 40). Cucaah, RC thlennak kilvennak kong ruah tikah, hi hlan ah hmuh bal lomi pehtlaihnak lam hi oxygen a ngeimi phototrophic thingram hna ah PSII RC QB hmun zongah hman khawh a si.
Rps ΔpufW (unlabeled pufW deletion) le PufW-His (C-terminal 10x His-tagged protein-W expressed from the natural pufW locus) strains. palustris CGA009 cu kan riantuannak hlan (16) ah kan rak langhter cang. Hi phun hna le isogenic wild-type nu le pa cu PYE (5 g liter -1 cio) (LB ah -80 °C ah chiahmi, 50% ( w/v) glycerol aa telmi) protein, yeast extract le succinate) agar [1.5%/v) plate cung ah cell tlawmte an thlet hna i freezer chungin an hmuh hna. Cu plate cu anaerobic dirhmun tangah khaan lumnak ah zankhuadei an chiah, cun OSRAM 116-W halogen bulbs (RS Components, UK) nih a pekmi ceunak rang (~50 μmolm-2 s-1) in ni 3 in ni 5 chung colony pakhat a chuah hlan tiang an ceuter. Colony pakhat lawng kha M22+ medium (41) 10 ml 0.1% (w/v) casamino acids (hi hnu ah M22 tiah auh a si lai) he cawhmi thlopnak caah hman a si. Cu cinmi cu 34°C ah muilak ah oxygen tlawmte lawng a ummi dirhmun ah suimilam 48 chung 180 rpm in hninh in cin a si, cun cinmi 70 ml cu cu bantuk thil sining tangah suimilam 24 chung an cin. A volume 1 ml a ngeimi semi-aerobic culture cu 30 ml universal screw-top transparent glass bottle chungah 30 ml M22 medium kha thlopbulnak caah hman a si i suimilam 48 chung sterile magnetic force Stirring rod in hninhnak (~50μmolm-2 s-1) in a ceuter. Cun 30 ml cinmi cu cu bantuk thil sining tangah cinmi liter 1 hrawng he an thlop, cu hnu ah ~200 μmolm-2 s-1 ah suimilam 72 chung ceutermi cinmi liter 9 hrawng thlopbulnak caah hman a si. Cell pawl cu 7132 RCF ah minutes 30 chung centrifugation in an lak hna, ~10 ml 20 mM tris-HCl (pH 8.0) ah an hnim ṭhan hna, cun a herh hlan tiang -20°C ah an chiah hna.
A lum hnu ah, deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) le Roche holoenzyme protease inhibitor tablet pahnih (Merck, UK) hna kha a tthawnmi cells ah chap. 20,000 psi French pressure cell (Aminco, USA) ah, cell pawl cu voi 8 in voi 12 tiang an hrawh hna. A rawk lomi cells le a cawh kho lomi thilri pawl kha 18,500 RCF ah 4°C ah minutes 15 chung centrifugation in lak dih hnu ah, 113,000 RCF ah 43,000°C ah suimilam 2 chung centrifugation in pigmented lysate chungin membrane cu an hlonh. A cawhmi tthen kha hlonh law rong a ngeimi membrane kha 100 in 200 ml 20 mM tris-HCl (pH 8.0) ah hnim ṭhan law hmuh khawhmi fonhmi a um ti lo tiang aa khatmi siter. A hrenmi membrane cu 20 mM tris-HCl (pH 8.0) (Anatrace, USA) ah 2% (w/v) β-DDM aa telmi ah suimilam 1 chung 4°C ah nem tein cawh in an chiah. Cun 70°C ah centrifuge tuah law 150,000 RCF kha 4°C ah suimilam 1 chung cawh law a tangmi a cawh lomi thil pawl kha hloh.
ΔpufW phun in a chuakmi a thletmi membrane cu 50 ml DEAE Sepharose ion thlennak column ah column volume pathum (CV) binding buffer [20 mM tris-HCl (pH 8.0) aa telmi 0.03% (w / v) β-DDM] he hman a si. CV pehtlaihnak buffer pahnih in column kha tawl, cun 50 mM NaCl aa telmi pehtlaihnak buffer pahnih in column kha tawl. RC-LH116 complex cu 1.75 CV ah 150 in 300 mM NaCl (binding buffer ah) linear gradient in eluted a si, cun a tangmi binding complex cu 0.5 CV ah 300 mM NaCl aa telmi binding buffer in eluted a si. 250 le 1000 nm karlak ah laknak spectrum kha lak, laknak ratio (A880/A280) 1 nak tam a ngeimi tthen kha 880 in 280 nm ah chiah, pehtlaihnak buffer ah voihnih cawh, cun DEAE column On purification ah mah phunglam kha hmang ṭhan. A880/A280 ratio 1.7 nak sang deuh le A880/A805 ratio 3.0 nak sang deuh a ngeimi tthencheunak kha cawh, ion thlennak a voithumnak tuah, cun A880/A280 ratio 2.2 nak sang deuh le A880/A805 ratio 5.0 nak sang deuh a ngeimi tthencheunak kha chiah. A cheuchum in thianh cangmi complex cu Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK) ah ~2 ml tiang an chiah, cun Superdex 200 16/600 size exclusion column (GE Healthcare, US) ah 200 mM NaCl aa telmi ah an chiah, cun cu buffer ah cun 15 mM buffer ah an thlet. CV. Size exclusion fraction i a laknak spectra kha lak law, A880/A280 ratio 2.4 cung le A880/A805 ratio 5.8 in 100 A880 cung a laknak spectra kha fonh law, cryo-TEM grid timhtuahnak asiloah chiahnak caah hman colh hna A herh tiang -8°C ah chiah.
PufW-His phun in a chuakmi a thletmi membrane cu 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) 200 mM NaCl le 0.03% (w/w) aa telmi) ah IMAC buffer (GE Healthcare) ah hman a si. v) β-DDM]. Column cu IMAC buffer CV panga in tawl a si, cun 10 mM histidine aa telmi IMAC buffer CV panga in tawl a si. A hrampi complex cu 100 mM histidine aa telmi IMAC buffer panga he column chungin an chuah. RC-LH114-W complex aa telmi tthen cu Amicon 100,000 MWCO filter (Merck, UK) he aa pehtlaimi cawhmi tank ah ~10 ml tiang an cawh, binding buffer in voi 20 an cawh, cun 25 ml ah an chap. Column cu CV pehtlaihnak buffer pali in tawl, cun 0 in 100 mM NaCl (pehtlaihnak buffer chungah) linear gradient ah CV pariat cung ah complex kha thlet, cun a tangmi CV pali cu 100 mM pehtlaihnak buffer aa tel. A880/A805 ratio 4.6 nak tam deuh cu Amicon 100,000 MWCO centrifugal filter ah ~2 ml tiang an cawh hna i, 1.5 CV IMAC in a hlankan in an khahter hna. A ngan-a tel lomi tthencheunak hna i an laknak spectra kha lak law A880/A280 ratio 2.1 cung le A880/A805 ratio 4.6 in 100 A880 cung ah laknak spectra kha fonh, cu hna cu a lummi TEM grid timhtuahnak caah hman colh an si asiloah a herh tiang -8°C ah chiah an si.
Leica EM GP immersion freezer cu a niammi TEM grids timhtuahnak caah hman a si. Cu complex cu IMAC buffer ah A880 of 50 tiang an cawh, cun 5μl cu a thar in a ceumi QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK) ah an chiah. Grid cu 20°C le 60% tlukruannak ah 30 s chung chiah, cun 3 s chung a roter, cun -176°C ah ti ethane ah hnim.
RC-LH114-W complex data cu eBIC (Electronic Bioimaging Center) (British Diamond Light Source) ah Titan Krios microscope he an khumh, mah microscope cu 300kV a rangmi voltage in rian a ttuan, 130,000× a nganternak le 200V a tlawmmi thazaang he. K2 peak detector he Gatan 968 GIF Quantum cu data laknak caah relnak mode ah hmanthlak pawl khumh awkah hman a si. Calibrated pixel size cu 1.048Å a si, cun dose rate cu 3.83 e-Å-2s-1 a si. Second 11 chungah zukcawl cu ka lak i tthen 40 ah ka ṭhen. Microscope kha zoh ṭhan awkah carbon-coated hmun kha hmang law, cun khur pakhat ah zukcawl pathum kha lak. A dihlak ah, zukcawl 3130 an lak hna, -1 le -3μm karlak ah defocus man he.
RC-LH116 complex caah data cu Asterbury Biostructure Laboratory (University of Leeds, UK) ah mah microscope pakhat hmangin an lak. Data cu 130 k in relnak mode in an lak, cun pixel ngan le hme cu 1.065 Å ah 4.6 e-Å-2s-1 dose in an tah. Cu zukcawl cu second 12 chung ah record an tuah i tthen 48 ah an tthen. A dihlak ah, zukcawl 3359 an lak hna, -1 le -3μm karlak ah defocus man he.
Data thlennak vialte cu Relion 3.0 pipeline (42) ah tuah a si. Dose weighting in beam cawlcanghnak remh awkah Motioncorr 2 (43) hmang, cun CTF (thleidannak thlennak riantuannak) parameter tuaktan awkah CTFFIND 4.1 (44) hmang. Hi a hramthawk riantuannak hnu ah a tlangpi in hmanmi hmanthlak hme tete cu hmanthlak 2 ah hmuh khawh a si. S16. Mah tein thimnak template cu 250-pixel frame chung i 1000 particles chungin 250 pixels hrawng kha kut in thimnak in sermi a si i, zohchunh awk pahnih (2D) thleidannak a um lo, cucaah sample thurhnawmhnak a tlinhmi asiloah hmuh khawhmi sining a ngei lomi thleidannak hna kha hlawt a si. Cun, hmanthlak hmete vialte ah amah tein thimnak tuah a si, cun RC-LH114-W cu 849,359 particles a si, cun RC-LH116 complex cu 476,547 particles a si. Thimmi particle vialte nih non-reference 2D thleidannak voihnih an tuah dih cang, cun an kal dih hnu ah, carbon area a tongmi particle, sample thurhnawmhnak, a fiangmi sining a um lomi asiloah aa tlukmi particle hna cu an hlawt hna, cu nih cun 772,033 (90.9%) le 359,678 (75.5%) particle hna cu 3D thleidannak caah hman an si. RC-LH114-W le RC-LH116 ciocio. A hramthawk 3D zohchunh awk model cu stochastic gradient descent method hmangin sermi a si. A hramthawk model kha zohchunh awk ah hmangin, thimcia mi particle pawl cu 3D ah phun li ah tthen an si. Hi phun chung i model kha zohchunh awk ah hman in, a ngan bikmi phun chung i a ummi thil hme tete cungah 3D thlennak tuah, cun a hramthawk 15Å low-pass filter kha solvent area kha khuh awkah hmang, a nemmi tthen 6 pixels chap, cun pixel pawl kha Gatan K2 peak Modulation transfer function of the top detector remh awkah post-process tuah. RC-LH114-W dataset caah, hi a hramthawk model cu hmaihuh tthennak (UCSF Chimera ah a hrampi complex density he aa pehtlaimi) ah a tthawngmi density kha hloh in remh a si. A chuakmi model hna (RC-LH114-W le RC-LH116 hna i an resolution cu 3.91 le 4.16 Å, ciocio an si) cu 3D thleidannak a voihnihnak caah zohchunh awk ah hman an si. A hmanmi thil hme tete cu a hramthawk 3D phun ah an i fonh i pawngkam he pehtlaihnak fek an ngei lo. Aa tlukmi asiloah a fiangmi dirhmun sining a um lo. 3D thleidannak a voihnihnak hnu ah, a sang bikmi tthencheunak a ngeimi phun kha thim a si [RC-LH114-W caah, phunkhat cu 377,703 particles (44.5%) a si, RC-LH116 caah, phun hnih a um, a dihlak ah 260,752 particles (54.7% W), an hnu ah aa khatmi lawng an si tikah an i khat. aa dannak hmete he a hramthawk i mernak]. Thimmi thil hme tete cu 400-pixel bawm chungin an chuah ṭhan hna i 3D thlennak in an thianh hna. Solvent mask cu a hramthawk 15Å low-pass filter, 3 pixel map kauhnak le 3 pixel soft mask hmangin sermi a si. Particle pakhat cio CTF remhnak, particle pakhat cio cawlcanghnak remhnak le particle pakhat cio CTF remhnak a voihnihnak hmangin, 3D remhnak, solvent masking le post-processing cu step pakhat hnu pakhat in a chuakmi texture kha remhnak caah tuah a si. FSC (Fourier Shell Correlation Coefficient) cut-off value 0.143 hmangin, RC-LH114-W le RC-LH116 a donghnak model hna i an resolution cu 2.65 le 2.80Å an si. A donghnak model i FSC curve cu hmanthlak 2 ah hmuh khawh a si. S17.
Protein zulhin vialte cu UniProtKB in download an si: 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). SWISS-MODEL (45) cu RC i aa lawhnak model sernak caah hman a si, cu ah cun RC-L, RC-M le RC-H i protein zulhin le Rba i a ummi crystal sining aa tel. sphaeroides RC cu zohchunh awk ah hman a si (PDB ID: 5LSE) (46). UCSF Chimera chung i “fit map” thilri kha hmangin sermi model kha map (47) he aa tlakter, protein sining kha tthanchoter, cun cofactor [4×BChl a (monomer library residue name = BCL), 2×BPh a (BPH), UQ10 (U10) phun khat asiloah pahnih, heme a si lomi thir phunkhat (Fe) le 34 hexa-hydrocaryline (QAK)] Coot (48) hmangin chap. QAK cu monomer library ah a um lo caah, PHENIX (49) chung i eLBOW thilri hmangin parameterized tuah a si.
A hnu ah, LH1 subunit cu ser a si. A hramthawk ahcun, PHENIX (49) chung i amah tein sernak thilri cu map le LH1-α le LH1-β protein zulhin hna kha input ah hmangin LH1 zulhphung cheukhat kha amah tein sernak caah hman a si. A tling bikmi LH1 subunit kha thim law, chuah law Coot chungah khumh, a chung i a tlaumi sequence kha kut in chap, cun BCls pahnih a (BCL) le spirilloxanthin (CRT) [a pehtlaimi Rps ningin LH1 complex a tlawm le tam le theihmi carotenoid a ummi ningin] na chap hlanah a dihlak in a sining kha kut in remh. Phun (17)]. LH1 subunit dihlak kha copy tuah law, UCSF Chimera “Docking Map Tool” kha hmang law LH1 density a pawng i a ummi model a si lomi hmun ah dock tuah, cun Coot ah remh ṭhan; LH1 subunits vialte model an tuah dih hlan tiang tuah tthan. RC-LH114-W sining caah, Coot chung i aa tthencheu lomi tlawmtam chuah in, USCF Chimera map chung i a tangmi non-protein thilri hna chungin protein cu tthen a si i Autobuild thilri cu a hramthawk model sernak caah hman a si, cun a tangmi subunits (protein-W) Modeling sernak caah hman a si. PHENIX (49) ah. Coot (48) chung i a chuakmi model ah a tlaumi zulhphung pawl kha chap, cun subunit dihlak kha kut in remh. A tangmi tthencheu lomi tlawmtam cu lipids (PDB monomer library ID of CDL = CDL, POPC = 6PL le POPG = PGT), β-DDM detergent (LMT) le UQ10 molecules (U10) fonhmi he aa tlak. Coot (48) ah PHENIX tthatnak (49) le kut in tthatnak kha hmang law model zatlangbu le aa tlakmi hmuh khawhmi tthatnak kha tthanchoter khawh a si ti lo hlan tiang a tlingmi a hramthawk model kha tlamtling tein tuah. A donghnak ah, LocScale (50) hmangin khuachung map kha ttha deuh in ser, cun a dang cycle zeimawzat kha tthen lomi density model tuahnak le mah tein le kut in tthaternak tuah.
Peptides, cofactors le a dang lipids le quinones pawl cu anmah le an density chung ah aa hngatmi hna cu hmanthlak 1 le 2 ah hmuh khawh an si. S18 in S23 tiang. A donghnak model i zatlang konglam cu Table S1 ah langhter a si.
A dang tein langhter a si lo ahcun, UV/Vis/NIR laknak spectra cu Cary60 spectrophotometer (Agilent, USA) ah 250 nm in 1000 nm tiang 1 nm karlak le 0.1s fonh caan ah an lak.
Sample cu quartz cuvette ah 2 mm lam in A880 of 1 tiang cawh law, 400 le 1000 nm karlak ah laknak spectrum kha lak. A tlongmi dichroic spectra cu Jasco 810 spectropolarimeter (Jasco, Japan) ah 1 nm karlak ah 400 nm le 950 nm karlak ah 20 nm min-1 scan rate in an lak hna.
Molar hloh khawhnak coefficient cu core complex kha A880 50 hrawng ah tlawmternak in tuak a si. 10μl volume kha 990μl binding buffer asiloah methanol ah tlawmter law, BChl rawhnak tlawmternak caah lak khawhnak spectrum kha lak colh. Methanol sample pakhat cio i BChl dat cu 771 nm 54.8 mM-1 cm-1 ah a ummi hlohnak tlukruannak in tuak a si, cun hlohnak tlukruannak cu tuak a si (51). Tahmi BChl tlawmtam kha 32 (RC-LH114-W) asiloah 36 (RC-LH116) in tthen law a hrampi complex tlawmtam kha hngalh khawh a si, cucu buffer Extinction coefficient ah lakmi sample pakhat i a laknak spectrum hngalh khawhnak caah hman a si. aa ruang in. Sample pakhat cio caah tahnak voithum tuah a si, cun BChl Qy a tam bik a tlangpi in a lakmi kha tuaktannak caah hman a si. 878 nm ah tahmi RC-LH114-W a thihnak tlukruannak cu 3280±140 mM-1 cm-1 a si, cu lio ah 880 nm ah tahmi RC-LH116 a thihnak tlukruannak cu 3800±30 mM-1 cm-1 a si.
UQ10 cu (52) chung i hmanmi lam ning in tah a si. A tawinak in, Agilent 1200 HPLC system hmangin a hnulei HPLC (RP-HPLC) tuah a si. RC-LH116 asiloah RC-LH114-W 0.02 nmol hrawng kha 50μl 50:50 methanol:chloroform ah 0.02% (w/v) ferric chloride aa telmi ah cawh law, a hlankan in aa tlukmi Beckman Coulter Ultrasphere ODS 4 mml kha 1-1-m ah cawh. 40°C ah HPLC cawhnuk (80:20 methanol:2-propanol) ah ×25 cm column ah chiah. Suimilam 1 chung 275 nm (UQ10), 450 nm (carotenoids) le 780 nm (BChl) ah hnahnawhnak zohfel awkah HPLC cawhmi ah isocratic elution tuah. Minute 25.5 ah 275 nm chromatogram chung i a sangbik cu aa fonh, cu ah cun hmuh khawhmi thilri dang aa tel lo. Aa fonhmi hmun cu 0 in 5.8 nmol tiang a thiangmi tahfung thunnak in tuakmi calibration curve he tahchunh in chuahmi UQ10 molar zat tuaknak caah hman a si (Figure S14). Sample pakhat cio cu a let thum in hlathlainak tuah a si, cun report tuahmi palhnak cu a tlangpi SD he aa tlak.
RC-LH1 complex aa telmi a tam bik Qy 0.1 a ngeimi cawhnuk cu 30 μM tlawmtermi rang lungthin cytochrome c2 (Merck, UK) le 0 in 50 μMUQ2 (Merck, UK) he timhtuahnak a si. UQ2 tlawmtam cio ah 1-ml sample pathum an timhtuah i tah hlan ah muihnak ah tling tein i remh khawhnak ding caah 4°C ah muilak ah zankhuadei an chiah hna. A phichuak cu OLIS RSM1000 modular spectrophotometer ah 300 nm mei/500 line grating, 1.24 mm luhnak, 0.12 mm laifang le 0.6 mm chuahnak tthennak he an chiah. Excitation ceunak hrial khawhnak ding caah sample phototube le reference photomultiplier tube luhnak ah 600 nm saumi filter chiah a si. A laknak cu 550 nm ah 0.15 s fonh caan he zohfel a si. Excitation ceunak cu 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) in DC2200 controller (Thorlabs Ltd., UK) hmangin 90% thazaang in fiber optic cable hmangin a chuak i ceunak hram ah 90° angle in a chuak. Tahnak ceunak cu thlalang he aa ralchanh i a hramthawk ah sample nih a lak lomi ceunak paohpaoh kha a kirter. s 50 ceunak hlan ah 10 s ah a laknak kha zohfel. Cun, quinolol nih cytochrome c23 + kha amah tein a zorter khawhnak ding caah muilak ah 60 s chung a laknak kha zohfel tthan a si (raw data caah Figure S8 zoh).
Data cu 0.5 in 10 s chung ah linear initial rate (UQ2 tlawmtam cungah aa hngat) chiah in le UQ2 tlawmtam pakhat cio ah sample pathum hna i an zat kha a tlangpi in tuak in tuah a si. RC-LH1 tlawmtam cu aa pehtlaimi hloh khawhnak tlukruannak in tuakmi cu a tlawmtam kha catalytic tthatnak ah thlennak caah hman a si, Origin Pro 2019 (OriginLab, USA) ah suaimi a si, cun a langmi Km le Kcat man hngalh khawhnak ding caah Michaelis-Menten model ah aa tlakmi a si.
A caan tawite chung laknak tahnak caah, RC-LH1 sample cu 50 mM sodium ascorbate (Merck, USA) le 0.4 mM Terbutin (Merck, USA) aa telmi IMAC buffer ah ~2μM tiang cawh a si. Ascorbic acid cu raithawinak electron petu ah hman a si, cun tert-butaclofen cu QB khamnak ah hman a si i tahnak tuahnak chung vialte ah RC petu bik cu a zor (cucu, photooxidized a si lo) a um khawh nakhnga caah a si. Laser lam chung i sample nih excitation pulse karlak ah muihnak thlennak caah caan a ngeih khawh nakhnga 2 mm optical path saunak he aa thlengmi cell (0.1 m hrawng a kauhnak, 350 RPM) ah sample 3 ml hrawng an chap. Ti: Sapphire laser system (Spectra Physics, USA) kha 880 nm ah 1 kHz (NIR caah 20 nJ asiloah Vis caah 100 nJ) in sample kha thawhternak caah ~100-fs laser pulse hmang. Data lak hlanah, sample kha minutes 30 hrawng lungthawhnak ceunak ah chiah. Langhternak nih QA riantuan khawh lonak a chuahter lai (QA voikhat voihnih a zorter men lai). Asinain hi thil kalning hi thlen khawh a si ti kha philh hlah, zeicahtiah caan saupi muihnak he aa remh hnu ah, RC cu QA cawlcanghnak ah zaangfah tein a kir than lai. Helios spectrometer (Ultrafast Systems, USA) cu -10 in 7000 ps tiang a tlaimi caan tahnak caah hman a si. Surface Xplorer software (Ultrafast Systems, USA) hmangin data pawl kha tthen, cun fonh law tlukruannak tuah. CarpetView software package (Light Conversion Ltd., Lithuania) kha hmang law, rawhnak he aa pehtlaimi aa dangmi spectra hmuh khawhnak ding caah fonhmi data set kha hmang, asiloah Origin (OriginLab, USA) ah single-wavelength spectral evolution he aa tlakmi thilri lehnak he exponent tampi aa fonhmi function kha hmang.
A cunglei ah kan chim cang bantuk in (53), RC le peripheral LH2 antenna pahnih a ngei lomi LH1 complex aa telmi photosynthetic film cu an timhtuah. Membrane cu 20 mM tris (pH 8.0) ah cawh a si i 2 mm optical path he quartz cuvette chungah chiah a si. 30nJ laser pulse cu 540 nm ah -10 in 7000 ps tiang a tlaimi caan he sample kha thawhternak caah hman a si. Rps caah langhtermi bantukin data set kha tuah. pal zohchunh awk.
Membrane cu 150,000 RCF ah 4°C ah suimilam 2 chung centrifugation in an thlet, cun 880 nm ah a lak khawhnak cu 20 mM tris-HCl (pH 8.0) le 200 mM NaCl ah an hnim ṭhan. 4°C ah muilak ah suimilam 1 chung 2% (w/v) β-DDM kha zaangfah tein cawh in membrane kha cawh. Sample cu 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK) ah 2.5 mg ml-1 (Bio-Rad hlathlainak) protein tlawmtam tiang cawh a si. A hlan ah chuahmi lam (54) in a dang riantuannak cu tuah a si, 50 μg protein kha 1% (w/v) sodium laurate (Merck, UK) aa telmi 50 μl TEAB ah cawh in thawk a si. s chung sonication tuah hnu ah, 5 mM tris(2-carboxyethyl)phosphine (Merck, UK) in 37°C ah minutes 30 chung a zorter. S-alkylation caah, sample cu 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) he cawh law 200 mM isopropanol stock solution in khaan lumternak ah minutes 10 chung cawh. Proteolytic rawlchuannak cu 2 μg trypsin/endoproteinase Lys-C cawhmi (Promega UK) cawh in tuah a si i 37°C ah suimilam 3 chung chiah a si. Laurate surfactant cu 50 μl ethyl acetate le 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) cawh in 60 s chung vortexing in chuah a si. Phase tthennak cu 15,700 RCF ah minutes 5 chung centrifugation in a thanchoter. Sertu phunglam ningin, C18 spin column (Thermo Fisher Scientific, UK) cu peptide aa telmi a niam deuhmi kha ralring tein hnuh le chiti hlonh awkah hman a si. Vacuum centrifugation in a hnih hnu ah, sample cu 0.5% TFA le 3% acetonitrile ah cawh a si, cun 500 ng cu a hlan ah a fiangmi system parameters hmangin nanoflow RP chromatography he mass spectrometry he fonh in hlathlainak tuah a si.
Rps kawlnak caah protein hngalh khawhnak le zat tuak khawhnak caah MaxQuant v.1.5.3.30 (56) kha hmang. palustris proteome database (www.uniprot.org/proteomes/UP000001426). Mass spectrometry proteomics data cu PRIDE partner repository (http://proteomecentral.proteomexchange.org) hmangin ProteomeXchange Alliance ah dataset identifier PXD020402 tangah chiah a si.
RPLC le electrospray ionization mass spectrometry he hlathlainak caah, RC-LH1 complex cu wild-type Rps in timhtuahnak a si. A hlan ah chuahmi lam (16) hmangin, palustris cells ah chuahmi protein tlawmtam cu 20 mM Hepes (pH 7.8), 100 mM NaCl le 0.03% (w/v) β- (Bio-Rad hlathlainak) ) DDM ah 2 mg ml-1 a ​​si. Sertu phunglam ningin, 2D thianhnak thilri (GE Healthcare, USA) hmangin 10 μg protein kha thletnak lam in chuah law, thletnak kha 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile le 20% (v/v) ti ah cawh. Microliter panga cu RPLC (Dionex RSLC) he mass spectrometry (Maxis UHR-TOF, Bruker) he fonh in an hlathlai hna. MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) kha 60°C le 100μlmin -1 ah tthennak caah hmang, 85% (v / v) solvent A [0.1% (v / v) FA le 0.02% (V/v) TFA aqueous solution] to solvent(v / v) 85% (v / v) [0.1%(v/v) FA le 0.02%(v/v) in 90%(v/v) acetonitrile TFA] A tlangpi in electrospray ionization source le default parameters hmangin minutes 60 nak tam, mass spectrometer nih 100 in 2750 m/z (mass-ratio-charge) a hmuh. ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/) bawmhnak in, mass spectrum kha complex chung i a ummi subunits ah map tuah.
Cell pawl cu 100 ml NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) asiloah high (300μMm-2 s-1) ceunak tangah suimilam 72 chung an ṭhanter hna. M22 medium (M22 medium ah ammonium sulfate aa tel lo i sodium succinate cu sodium acetate in aa thleng) cu 100 ml screw-top bottle (23) ah a um. 30-s cycle panga ah, 0.1 micron glass beads cu 1:1 volume ratio in cells pawl lyse tuahnak ding caah beaded an tuah hna i minutes 5 chung tikhor cungah an chiah hna. A cawh kho lomi thil, a rawk lomi cells le thlalang hnah hna cu 16,000 RCF ah minutes 10 chung benchtop microcentrifuge ah centrifugation in an lak hna. Membrane cu Ti 70.1 rotor ah 100,000 RCF in 20 mM tris-HCl (pH 8.0) he 40/15% (w/w) sucrose gradient he suimilam 10 chung tthen a si.
Kan riantuannak hlun ah kan rak langhter cang bantukin, PufW (16) cung i His tag i ralrinnak hmuh khawhnak. A tawinak in, thianh cangmi core complex (11.8 nM) asiloah RC (a zormi thleidannak spectrum kha oxidation in lak i a thurhnawmmi gel cung i a ritmi he aa tlakmi) aa khatmi aa telmi membrane cu 2x SDS loading buffer (Merck, UK) ah voihnih cawhmi a si. Protein pawl cu 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK) ah an i tthen. RC-L subunit kha khumh le hmuh khawhnak ding caah Coomassie Brilliant Blue (Bio-Rad, UK) in gel cu an thuh. A pahnihnak gel cung i a ummi protein cu immunoassay caah methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK) ah thlen a si. PVDF membrane cu 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 le 5% (w / v) skimmed milk powder ah an khar, cun anti-His primary antibody (Dilute the antibody buffer [pH 7.6 mM-HM) he an cawh. 7.6), 150 mM NaCl le 0.05% (v/v) Tween-20] 1:1000 A190-114A, Bethyl Laboratories, USA) ah suimilam 4 chung. Antibody buffer ah minutes 5 chung voi 3 tawl hnu ah, membrane cu horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (antibody buffer ah 1:10,000 in cawhmi) he fonh a si. chemiluminescence substrate (Cyanagen, Italy) le Amersham Imager 600 (GE Healthcare, UK).
A thurhnawmmi gel asiloah immunoassay lam pakhat cio i a tthawnnak tthencheunak suai in, a sangbik tang i a ummi hmun kha fonh in RC-L (a thurhnawmmi gel) le Protein-W (immunoassay) tthawnnak zat kha ImageJ (57) ah tuaktan in. Hmanthlak kha thlen. Hi zat hna cu RC-LH114-W sample thianghlim ah RC-L le protein-W zat cu 1:1 a si tiah ruah in le cu ning cun data set dihlak kha a tlarimi ah ser in molar zat ah thlen an si.
Hi capar caah bawmtu thilri caah, http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1 zoh
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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, Andrew C. Necoil Necoilter.
Reaction center chung i ceunak tlaih 1 complex i a ttha ngaimi dirhmun nih quinone thlennak kongah hmuhnak thar a pek.
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, Andrew C. Necoil Necoilter.
Reaction center chung i ceunak tlaih 1 complex i a ttha ngaimi dirhmun nih quinone thlennak kongah hmuhnak thar a pek.
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