Tunah hian†Tun dinhmunah chuan: OX11 0DE, UK, Diamond Building, Harwell Science leh Innovation Park, Dietcote, Oxfordshire, UK, Diamond Light Source Co., Ltd., Electronic Biological Imaging Center-ah te a awm a.
Reaction center light-harvesting complex 1 (RC-LH1) hi purple phototrophic bacteria-a photosynthetic component bulpui ber a ni. Rhodopseudomonas palustris atanga RC-LH1 complex atanga cryo-electron microscopy structure pahnih kan rawn luh tir a. RC-LH114-W complex-a 2.65-Å resolution structure hi RC vel subunit LH1 loop 14 awmin, chu chu protein W-in a titawp a, protein-W tel lo complex erawh chu RC-in a hual vel RC composition kimchang tak a ni thung. Subunit 16 LH1 loop khar a ni. Heng structure hrang hrangte tehkhin hian RC-LH1 complex-a quinone dynamics chungchang hriatna a pe a, RC QB site-a quinone binding-a conformational changes a hmaa hriat lawk loh te, auxiliary quinone binding site awmna hmun te, RC-a Pass-a puitu te pawh a tel a ni. W protein structure danglam tak hian LH1 loop khar a veng a, chu chuan quinone/quinolone exchange ti chakna tur channel a siam a ni.
Photosynthesis hmanga chakna pek hian leia nunna zawng zawng deuhthaw a chhawm nung thei a, solar biotechnology atan pawh theihna nasa tak a nei bawk. Global photosynthesis a tihhmasawn rual hian purple phototrophic bacteria te hian energy mode leh metabolic capabilities hrang hrang an lantir bawk. Photosynthesis an pumpelh thei a, thimah heterotrophic bacteria angin an thang lian thei a, nitrogen leh carbon dioxide an fix thei a, hydrogen an siam thei a, aromatic compound an tichhe thei bawk (1-3). Heng thilte atana chakna pe tur chuan êng hi rang tak leh tha taka chemical energy-ah a chantir a ngai a ni. Hetiang kalphung hi light-trapping antenna complex hian light a absorb a, trapped energy chu reaction center (RC) ah a transfer atang hian a intan a, chu chuan charge separation a intan ta a ni (4 – 7). Purple phototrophic bacteria-a photosynthesis unit bulpui ber chu type 2 RC atanga siam a ni a, light-harvesting complex 1 (LH1) in a hual vel a, RC-LH1 core complex a siam a ni. LH1 hi curved αβ heterodimers array atanga siam a ni a, chungte chuan bacterial chlorophyll (BChl) a molecule pahnih leh carotenoid pakhat emaw pahnih emaw a binding vek a ni (8-12). LH1 antenna awlsam ber chu closed loop-a RC (9-13) vel αβ heterodimer 16 emaw 17 emaw a awm a, mahse core complex dang ah chuan transmembrane peptide te chuan a chhehvel LH1 chhunzawm zel chu a titawp a, Chu chuan RC leh cytochrome bc1 complex inkara quinol/quinone diffusion a tichak a ni (11, 13-15-ah a awm). Purple phototrophic plant Rhodopseudomonas (Rps.) hi model organism a ni a, photosynthesis puitu energy leh electron transfer a hrethiam thei a ni. Crystal structure hmasa ber chu Rps. Palustris RC-LH1 complex model chu RC a ni a, heterodimeric LH1 loop 15 in a hual vel a, chungte chu protein hriat loh “Protein W” tih hian a titawp a ni (14). Chumi hnuah protein-W chu RPA4402 tia hriat a ni a, hei hi uncharacterized 10.5kDa protein a ni a, predicted transmembrane helices (TMH) pathum a nei a ni (16). RC-L, M (pufL, pufM) leh LH1α, β (pufA, pufB) subunit encoding genes atana hman nomenclature nena inmil turin rpa4402 gene encoding protein W chu pufW tia thlak kan rawt a ni. Ngaihven awm tak chu protein-W hi RC-LH1 10% vel ah chauh a awm a, hei hian Rps. palustris hian RC-LH1 complex hrang hrang pahnih a siam chhuak thin. Hetah hian core complex pahnih high-resolution cryo-EM (cryo-EM) structure kan report a, pakhatah hian protein W leh 14 αβ heterodimers a awm a, pakhatah chuan protein W a awm lo a, closed 16 Heterodimer LH1 loop a awm bawk. Kan structure hian Rps-a RC-LH1 complex hriatthiamna step change a entir a ni. palustris, a chhan chu variant tinte homogeneous population kan zirchian tawh avangin peptide tinte leh bound pigment leh a kaihhnawih lipid leh quinones tinte chiang taka assign theihna tur resolution tling kan nei a ni. Heng structure te hi khaikhin chuan tun thlenga RC-LH1 complex dang dangah hmuh tur la awm lo TMH protein-W pathum te hian quinone/quinolone inthlak danglamna ti chak turin quinone channel an siam chhuak tih a hriat theih. Conserved lipid leh quinone binding site engemaw zat hmuhchhuah a ni tawh a, quinone leh RC te inzawmkhawm hnuah conformational change thar kan hmuchhuak a, hei hi oxygenated phototrophic organisms photosystem II (PSII) RC atan a tha thei ang. Kan thil hmuhchhuahte hian purple phototrophic bacteria RC-LH1 core complex-a quinone/quinolone binding leh exchange kinetics chungchangah hriatna thar a pe a ni.
Rps.-a complex pahnih hmuhchhuah te chipchiar taka zirchian a awlsam theih nan. palustris, RC-LH1 tinte chu biochemical method hmangin kan isolate thin. Protein W-deficient complex (hetah hian ΔpufW tia sawi tur) chu pufW gene nei lo strain atang hian tihthianghlim a ni a (16), RC-LH1 complex pakhat chauh siam theih a ni. Protein W awmna complex hi strain pakhat atanga siam a ni. He strain-a protein W hi a C-terminus-ah 10x His tag hmanga siam danglam a ni a, chutiang chuan protein W-containing complex chu protein W tlachham tam zawk nen metal immobilize-in a tha thei ang bera inzawm theih a ni. Complex hi a tha zawngin a inthen a (16) Affinity Chromatography (IMAC).
Figure 1-a kan hmuh angin complex pahnihah hian sub-unit pathum RC (RC-L, RC-M leh RC-H) a awm a, LH1 antenna-in a hual vel a ni. Protein-W nei lo complex-a 2.80-A structure-ah hian αβ heterodimers 16 a lang a, RC vel chu closed LH1 loop a siam a, hetah hian RC-LH116 complex tia sawi a ni ang. Protein-W awmna complex 2.65Å structure hian 14-heterodimer LH1 a nei a, protein-W hian a titawp a, hetah hian RC-LH114-W tia sawi a ni ang.
(A leh B) Compound chunglam atanga a lan dan. (C leh D) Rod-a lang chhuak bonded pigments. (E leh F) Cytoplasmic surface atanga complex hmuhchhuah te hian cartoon-a tarlan peptide leh LH1 subunit te an nei a, protein-W gap atanga clockwise number pek an ni [Rba numbering nen a inmil. sphaeroides complex (13)) a ni. LH1-α tan chuan protein subunit rawng chu sen a ni a; LH1-β tan chuan protein subunit rawng chu blue a ni a; protein-W tan chuan protein chu a sen a; RC-H tan chuan cyan a ni a; RC-L tan chuan Orange a ni a; RC-M tan chuan magenta a ni. Cofactors te hi rod hmanga entir a ni a, green hian BChl leh BPh a molecule a entir a, purple hian carotenoid a entir a, yellow hian UQ10 molecule a entir bawk. (G leh H) RC-LH114-W complex (G) leh RC-LH116 complex (H) te equivalent region-a protein-W gap awm zat tihpun. Cofactors te hi space filling angin a lang a, chelated quinone chu blue in a lang bawk. Protein-W gap hi (G)-a blue dashed line hmanga tarlan a ni a, LH116 ring-a quinone/quinolol diffuse na hole te te chu (H)-a black dashed line hmanga tarlan a ni bawk.
Figure 1 (A leh B)-ah hian RC chu LH1αβ heterodimers open emaw closed emaw array-in a hual vel a, chungte chuan BChl pahnih leh carotenoid pakhat an binding vek a ni (Figure 1, C leh D). Tun hmaa zirchianna hrang hrangah Rps hi LH1 complex a ni tih hmuhchhuah a ni. Spirulina xanthin biosynthetic pathway-ah hian heng chi hrang hrangah hian carotenoids population mixed an awm a ( 17 ). Mahse, spiropyrroxanthin hi carotenoid lian ber a nih avangin a density pawh a lungawithlak hle. Chuvangin LH1 binding site zawng zawngah spiroxanthin model kan thlang ta a ni. Alpha leh beta polypeptide te hi TMH pakhat, membrane pawn lam bial tawi tak nei an ni (Figure 1, A, B, E, leh F). C-terminus-a residue 17 density hmuh tur awm lo mah se, complex pahnihah hian alpha polypeptide chu Met1 aṭangin Ala46-ah a inthen a ni. β polypeptide chu RC-LH116-ah Gly4 atanga Tyr52-ah tihhniam a ni a, RC-LH114-W-ah chuan Ser5 atanga Tyr52-ah tihhniam a ni bawk. N-terminal 3 emaw 4 emaw C-terminal residue 13 emaw density hmuh tur a awm lo (Figure S1). Wild-type strain atanga mixed RC-LH1 complex siam chu mass spectrometry analysis hmangin a awm lohna hmun chu heng peptide te heterologous cleavage avanga lo awm a ni tih hmuhchhuah a ni (Figure S1 leh S2). α-Met1 N-terminal formylation pawh hmuh a ni bawk (f). Analysis an neihah chuan α-peptide hi residue fMet1 atanga Asp42/Ala46/Ala47/Ala50 atanga siam a ni a, β-peptide hi residue Ser2 atanga Ala53 atanga siam a ni a, hei hi low-temperature EM density map nen a inmil tha hle.
α-His29 leh β-His36 te thawhhona chuan BChls te chu hmaichhanah a siam a; αβ heterodimer tin hian a ṭhenawmte nen an inzawm khawm a, RC The exciton coupled pigment array (Figure 1, C leh D) vel chu open loop (RC-LH114-W) emaw closed loop (RC-LH116) emaw an siam a ni. RC-LH114-W 877 nm band nen khaikhin chuan RC-LH116 880 nm absorption red shift chu 3 nm a ni (Figure 2A). Mahse, circular dichroism spectrum hi a inang tlang hle (Figure 2B), hei hian open leh closed loop inkarah danglamna chiang tak awm mahse, BChls local environment chu a inang hle tih a tilang a ni. Absorption redshift hi thermal motion tlahniam leh closed loop-a stability tihpun avanga lo awm a ni thei (18, 19), closed loop avanga pigment coupling inthlak danglam vang (20, 21), emaw, heng effect pahnih inzawmkhawm vang pawh a ni thei (11).
(A) Ultraviolet/visible/near-infrared absorption spectrum, a peak te chu an pigment inmil hmanga chhinchhiah a ni a, 775 nm-a BPh peak-ah normalized a ni. (B) Circular dichroism spectrum chu 805 nm-ah BChl absorbance-ah normalized a ni. (C leh D) RC-LH114-W complex (C) leh RC-LH116 complex (D) te time-resolved absorption spectra atanga ΔA spectra thlan chhuah te. Comparability tha zawk atan spectra zawng zawng chu 0.2 ps-ah −A ∆A-ah normalized vek a ni. (E) UQ2 concentration hrang hrang awmnaah irradiation hnua cytochrome c2 oxidation rate (raw data atan Figure S8 en rawh). (F) Light low, medium emaw high intensity light (10, 30 emaw 300μMm-2 s-1, a hnuaia mi ang hian) hnuaia cell-ah chuan purified complex-a protein W leh RC-L subunit te leh separated membrane ratio te. SDS-polyacrylamide gel electrophoresis leh immunoassay hmangin protein level teh rawh (raw data atan Figure S9 en rawh). RC-LH114-W complex tihthianghlim tawh nena khaikhin chuan ratio chu teh rawh. Complex-a RC-L leh protein-W te stoichiometric ratio chu 1:1 a ni.
RC-LH114-W (Figure 1, A, C, leh E)-a deformed αβ14 loop-a position 1-a BChls te hi RC-LH116-a BChls tlukpui aiin RC primary donor (P) nen 6.8Å in an inhnaih zawk (Figure. 1, B, D, leh F, leh Figure S3) mahse, complex pahnih transient absorption kinetics atanga a lan dan chuan RC-LH114-W leh RC-LH116 tan chuan LH1 atanga RC thlenga excitation energy transfer time constant chu 40 ±4 leh 44±3 ps a ni (Figure 2). , C leh D te, Figure S4 leh Table S2 te an ni). RC chhunga electronic transfer-ah pawh danglamna lian tham a awm lo (Figure S5 leh a kaihhnawih supplementary text). LH1 leh RC-P inkara energy transfer hun inzawmna hnai tak hi LH1 loop pahnih a BChl tam zawkte distance, angle leh potential energy inang vang a ni tih kan ring. Minimum distance thlen theihna tura LH1 energy pattern explore hi suboptimal site atanga RC-a direct energy transfer aiin a rang lo niin a lang. RC-LH114-W-a open-loop LH1 loop hian structural analysis atan temperature hniam takah thermal motion pawimawh lo tak a tawk thei bawk a, RC 1 awmna hmuna βBChls pigmentation distance atanga room temperature-ah αβ14 ring conformation sei zawk a awm bawk.
RC-LH116 complex-ah hian BChls 32 leh carotenoids 16 a awm a, a inrem dan zawng zawng hi Thermochromatium (Tch.) pidpidum [Protein Data Bank (PDB) ID 5Y5S] (9), Thiorhodovibrio (Trv.) 970 strain ( PDB ID 7C9R) (12) leh green atanga hmuh nen a inang a ni algae (Blc.viridis) (PDB ID 6ET5) (10) te chu a awm a. Alignment hnuah αβ heterodimers awmna hmunah deviation tlemte chauh hmuh a ni a, a bik takin 1-5, 15, leh 16-ah te (Figure S6). Protein-W awmna hian LH1 structure-ah nghawng lian tak a nei a ni. A TMH pathumte hi short loop hmanga inzawm a ni a, N-terminal chu complex lumen lam a ni a, C-terminal chu cytoplasmic lam a ni (Figure 1A leh 3, A to D). Protein-W hi a tam zawk chu hydrophobic a ni (Figure 3B), TMH2 leh TMH3 te hian LH1αβ-14 nen an inzawm a, transmembrane surface an siam a (Figure 3, B leh E to G). Interface hi transmembrane region-a Phe, Leu leh Val residue atanga siam a ni ber. Heng residue te hi hydrophobic amino acid leh αβ-14 pigment te nen an inzawm khawm a ni. Polar residue thenkhat pawh hian inzawmnaah hian an thawhhlawk hle a, chung zingah chuan complex cavity chunglama W-Thr68 leh β-Trp42 inkara hydrogen bond awm pawh a tel (Figure 3, F leh G). Cytoplasm chung lamah chuan Gln34 hi αβ-14 carotenoids keto group nen a inhnaih hle a. Chu bakah, n-dodecyl β-d-maltoside (β-DDM) molecule chu a resolve a, a hydrophobic tail chu protein-W leh αβ-14 inkar interface thlengin a inzar pharh a, lipid tail chu taksaah a awm thei bawk. Tin, protein W leh RCH te C-terminal resolution region te hi an inhnaih hle tih kan hmu bawk a, mahse specific interaction siam theihna scope chhungah erawh a awm lo (Figure 1, A leh E). Mahse, heng protein pahnih C-terminal amino acids chinfel lohah hian inzawmna a awm thei a, hei hian RC-LH114-W complex assembly chhunga protein-W recruitment neih theihna tur mechanism a siam thei a ni.
(A) Cartoon angin LH1αβ14 nena inzawmna lam hawi protein-W hian rod ang chi side chain (red) a nei a, electrostatic potential diagram hmun thenkhatah (transparent grey surface contour level 0.13 nei) a lang. (B) Protein-W chu hydrophobic colored surface hmanga entir a ni. Polar leh charged area te chu cyan-a tarlan a ni a, hydrophobic area te chu white-a tarlan a ni a, strongly hydrophobic area te chu orange-a tarlan a ni bawk. (C leh D) Cartoon-a protein-W tarlan, a orientation chu (A) (C)-a tarlan nen a inang a, 180° (D)-a inher a ni. Sequence-a position awm dan azirin, distinguishable residues te hian rainbow color scheme an hmang a, chutah chuan N-terminal chu blue a ni a, C-terminal chu red a ni. (E) (A)-a kan hmuh ang chiah hian protein-W chu a lang a, protein-W:LH1 inkar a residue te chu attached mark nei rod hmanga entir a ni. (F) Protein-W hi cartoon representation-a (E) leh LH1αβ14 nena khaikhin chuan 90°-in a inher a, bar representation-a interface residue-te nena khaikhin chuan a inher bawk. Beta polypeptide atanga overhanging residue te chu label a ni. Cofactor chu Figure 1-a rawng nena inmil bar angin tarlan a ni a, decomposed β-DDM chu gray-in a lang a, oxygen chu red-in a lang bawk. (G) (F)-a view hi 180°-a rotated a ni a, labeled alpha polypeptide-a residue langsar tak takte chu a awm.
Protein-W hian αβ heterodimer (Figure 1F-a 15-na) a thlak a, chu chuan loop khar a veng a, αβ heterodimer hmasa pathumte chu a tilt thei a ni. Film normal nena khaikhin chuan αβ-1 heterodimer hmasa ber inclination angle sang ber chu 25° atanga 29° a ni tih hmuhchhuah a ni (Figure 1, A leh E), hei hi RC A sharp contrast-LH116-a αβ-1 inclination 2° atanga 8° atanga siam a ni (Figure 1, B leh F). Heterodimer pahnihna leh pathumna te hi 12° atanga 22° leh 5° atanga 10° ah an inclined a ni. RC steric hindrance avang hian αβ-1 tilt hian αβ pair pahnihna (Figure 1F-a αβ 16-na nen a inmil) a huam lo va, chu chuan LH1 ring-ah gap chiang tak a siam a ni (Figure 1, A leh E) αβ heterodimer pahnih a awm loh avangin, BChl pali leh carotenoid pahnih hloh a nih rualin, carotenoid pakhat mah hi twisted αβ-1 subunit-ah a inzawm lo va, chu chuan LH114-W ring a siam a, chutah chuan carotenoid 13 Vegetarian leh BChls 28 a awm a ni. αβ1 atanga 7 region-a complex pahnih local resolution estimate chu LH1 loop dang zawng aiin a hniam zawk a, hei hian RC QB site bula LH1 subunit-a inherent plasticity a lantir thei a ni (Figure 4)
RC-LH114-W (A leh B) leh RC-LH116 (C leh D) thlalak hi Fig. 1. (B leh D)-a top view/side view (A leh B) (A leh C) leh cavity surface inang atanga kan hmuh a ni. Key rawng hrang hrang te chu dinglamah a lang.
Core complex dang pakhat chauh, stoichiometric ratio 1:14 nei chu Rhodococcus sphaeroides (Rba.) RC-LH1-PufX dimer (13) a ni. Mahse, protein W leh PufX te hian homology chiang tak an nei lo va, an LH1 structure hrang hrangah nghawng lian tak an nei a ni. PufX hi TMH pakhat, N-terminal cytoplasmic domain nei a ni a, RC-H subunit (13) cytoplasmic side nen Rps nena inmil position-ah an inzawm a ni. palustris LH116αβ-16 tih chu a ni. PufX hian RC-LH1 leh cytochrome bcl complex inkara quinone/quinolone inthlak danglamna tur channel a siam a, Rba zawng zawngah a awm vek a ni. sphaeroides core complex (13) a ni. Monomer-monomer interface chu Rba-ah awm mahse. Sphaeroides RC-LH1-PufX dimer hi RC-LH114-W-a protein W binding position-ah a awm a, PufX leh protein-W-in a thlen gap chu hmun inangah a awm bawk (Figure S7A). RC-LH114-W-a gap hi Pseudomonas rosea LH1-a hypothetical quinone channel (8) nen pawh a inmil a, hei hi protein W emaw PufX emaw nena inzawm lo peptide-te siam a ni (Figure S7B). Hei bakah hian quinone channel in Blc. γ subunit pakhat (7) paih chhuah avanga emerald green LH1 lo awm chu a awmna hmunah a awm ve tho (Figure S7C). Protein hrang hrangin mediated ni mahse, RC-LH1 complex-a hmun khata heng quinone/quinolol channel-te lo lang hi convergent evolution entir nan a lang a, hei hian protein W-in gap a siam chu quinone channel angin a thawk thei tih a tilang a ni.
LH114-W loop-a gap awm hian RC-LH114-W complex chhungril space leh bulk membrane inkarah continuous membrane region siam a phalsak a (Figure 1G), protein-a awm angin domain pahnih hi protein pore hmanga inzawm tir ai chuan. RC-LH116 complex hi closed Tch nen a inang a ni. Needle ang chi complex (22) (Figure 1H) a ni. Membrane kaltlanga quinone diffusion chu protein channel tawi tak hmanga diffusion aiin a rang zawk avangin open LH114-W loop hian closed LH116 loop aiin RC turnover chak zawk a phal thei a, RC chhunga quinone diffusion pawh a khap zawk thei bawk. Protein W hian RC hmanga quinones inthlak danglamna a nghawng em tih test nan ubiquinone 2 (UQ2) (natural UQ10 analogue, isoprene tail tawi zawk) concentration engemaw zatah cytochrome oxidation assay kan nei a (Figure 2E). Chelated quinone awmna hian apparent Michaelis constant dik taka hriat theihna a tikhawlo a (RC-LH114-W leh RC-LH116 te hi 0.2±0.1μM leh 0.5±0.2μM atan a tha a ni), mahse RC-LH114-W rate sang ber ( 4.6±0.2 e-RC-1 s-1) chu a ni RC-LH116 (3.6±0.1 e-RC-1 s-1) aiin 28±5% in a lian zawk.
A tirah chuan protein-W hi core complex 10% vel ah a awm tih kan chhut a (16); hetah hian low-light, medium-light, leh high-light growth cell-te occupancy rates chu 15±0.6%, 11±1% leh 0.9±0.5 a ni a, % a ni (Figure 2F). Mass spectrometry hmanga quantitative comparison-ah chuan histidine tag dah belh hian wild-type strain nena khaikhin chuan protein-W relative abundance a tihtlem lo tih hmuhchhuah a ni (P = 0.59), chuvangin heng level te hi modified protein-W artifact a ni lo (Figure S10). Mahse, RC-LH1 complex-a protein-W occupancy hniam tak hian RC thenkhat chu accelerated rate-in a flip thei a, chu chuan RC-LH116 complex-a quinone/quinolone exchange slow zawk chu a tiziaawm thei a ni. Light occupancy rate sang tak hi tun hnaia transcriptomics data nen a inmil lo tih kan hmu a, hei hian pufW gene expression chu light chak tak hnuaiah a pung tih a tilang (Figure S11) (23). PufW transcription leh RC-LH1 complex-a protein-W incorporation inthlauhna hi a buaithlak hle a, protein complex regulation a lantir thei bawk.
RC-LH114-W ah hian cardiolipin (CDL) 6, phosphatidylcholine (POPC) 7, phosphatidylglycerol (POPG) 1 leh β-DDM molecule 29 te chu allocate a, model a ni a, chutah chuan CDL 6, POPC 24, POPG 2 leh βDDM 12 te an model a ni. RC-LH116 (Figure 5, A leh B) te hi a ni. Heng structure pahnih ah hian CDL hi complex cytoplasmic side-ah a awm deuh ber a, POPC, POPG leh β-DDM te hi luminal side-ah a awm tam ber thung. RC-LH114-W complex-a αβ-1 to αβ-6 region-ah lipid leh detergent molecule pahnih an isolated a (Figure 5A), RC-LH116 equivalent region-ah panga an isolate bawk (Figure 5B ). Complex sir lehlamah lipid tam zawk hmuh a ni a, a bik takin CDL, RC leh αβ-7 inkara αβ-13 inkara pungkhawm (Figure 5, A leh B). Structurally resolved lipid leh detergent dangte chu LH1 ring pawnah an awm a, acyl chain resolve tha tak takte chu LH1 subunit inkarah a inzar pharh a, RC-LH114-W-ah chuan tentative-in β-DDM tia vuah a ni a, RC A mixture of β-DDM leh POPC-LH116-ah chuan β-DDM tia sawi a ni bawk. Kan structure-a chelating lipids leh detergents te dinhmun inang chiah hian physiologically relevant binding site an nih thu a tarlang (Figure S12A). Tch-a molecule tlukpui awmna hmun pawh hian inmilna tha tak a nei bawk. Gentle leh Trv. Strain 970 RC-LH1s (Figure S12, B to E) (9, 12) leh lipid head group-a hydrogen-bonding residues te hian sequence alignment-ah conservation tha tawk tak an nei a (Figure S13), hei hian RC (24) nena inzawm Conserved CDL, heng hmunte hi RC-LH1 complex-ah hian conserved a ni thei tih a tilang.
(A leh B) RC-LH114-W (A) leh RC-LH116 (B) peptide te chu cartoon hmanga entir a ni a, pigment te chu rod hmanga entir a ni a, Figure 1-a color scheme hmangin lipid te chu sen-in a lang a, detergent te chu gray-in a lang bawk. RC QA leh QB site-a UQ bound chu yellow a ni a, isolated UQ erawh blue a ni thung. (C leh D) (A) leh (B) te ngaihdan inang, lipids te chu paih chhuah a ni. (E to G) RC-LH116 atanga Q1(E), Q2(F) leh Q3(G) te enlarged view, side chain inthlunzawmna nei. Hydrogen bonds te hi black dashed line angin tarlan a ni.
RC-LH116-ah chuan charge separation process-a electron transfer-a tel RC QA leh QB UQ te pahnih hi an binding site-ah an chhe vek a ni. Mahse, RC-LH114-W-ah chuan QB quinone hi chinfel a la ni lo a, a hnuaiah hian chipchiar takin kan sawi dawn a ni. QA leh QB quinones bakah hian chelated UQ molecule pahnih (RC leh LH1 ring inkar a awm) te chu an head group resolve tha tak (Q1 leh Q2-a awm) angin RC-LH114-W structure-ah hian dah a ni. awl). Figure 5C-ah hian a lang). Q1-ah hian isoprene unit pahnih dah a ni a, density map hian Q2-a isoprene tail 10 kimchang chu a resolve a ni. RC-LH116 structure-ah hian chelated UQ10 molecule pathum (Q1 atanga Q3, Figure 5D) chu resolve a ni a, molecule zawng zawng hian a tail zawng zawngah density chiang tak an nei vek a (Figure 5, D to G). Structure pahnih ah hian Q1 leh Q2-a quinone head group awmna hmun hian inmilna tha tak a nei a (Figure S12F), RC nen chauh an inzawm a ni. Q1 hi RC-LH114-W W gap luhna bulah a awm a (Figure 1G leh 5, C, D leh E), Q2 hi QB binding site bulah a awm bawk (Figure 5, C, D) leh F). Conserved L-Trp143 leh L-Trp269 residue te hi Q1 leh Q2 te nen an inhnaih hle a, π-stacking interaction awm thei an pe a ni (Figure 5, E leh F, leh Figure S12). Q1 distal oxygen atanga 3.0 Å a hla L-Gln88 hian hydrogen bond chak tak a pe a (Figure 5E); he residue hi inzawmna hla ber tih loh chu RC zawng zawngah a humhim vek a ni (Figure S13). L-Ser91 hi RC dang tam zawkah chuan Thr aiah conservative takin a thlak a (Figure S13), Q1-a methyl oxygen atanga Angstrom 3.8 a hla a ni a, hydrogen bonds chak lo tak a pe thei bawk (Figure 5E). Q3 hian inzawmna bik a nei lo niin a lang a, mahse RC-M subunit leh LH1-α subunit 5 atanga 6 inkar hydrophobic region-ah a awm a ni (Figure 5, D leh G). Q1, Q2 leh Q3 emaw a bul hnaia chelated quinones te pawh Tch. Mi ngilnei, Trv. Strain 970 leh Blc. Iris structure (9, 10, 12) hian RC-LH1 complex-a auxiliary quinone binding site conserved tak a kawk a (Figure S12G). RC-LH116-a decomposed UQ pangate hi high performance liquid chromatography (HPLC) hmanga complex tinte 5.8±0.7 tehna nen a inmil tha hle a, RC-LH114-W-a decomposed UQ pathumte erawh chu The measured value of 6.2±0.3 (Fig. S14) aiin a hniam zawk thung.
Pseudo-symmetric L leh M polypeptide te hian TMH panga ve ve an nei a, heterodimer an siam a, chu chuan BChl dimer pakhat, BChl monomer pahnih, bacteriophage (BPh) monomer pahnih, leh non- Heme iron pakhat leh UQ10 molecule pakhat emaw pahnih emaw a thlunzawm a ni. Terminal ketone group-a hydrogen bonds awm leh Rps-a a khawlkhawm nia hriat hmangin carotenoids chu M-subunit-ah a insiam lut a, chu chu cis-3,4-dehydroorhodopin tih a ni. A chi hrang hrang (25). RC-H pawn lam membrane domain chu TMH pakhat hmangin membrane-ah anchor a ni. RC structure pum pui hi a inzawm chi (Rba ang chi) subunit pathum RC nen a inang a ni. sphaeroides (PDB ID: 3I4D) a awm bawk. BChl leh BPh te macrocycle te, carotenoid backbone leh non-heme iron te hi heng structure te resolution range chhungah hian an inzawm khawm a, chutiang bawkin QA site-a UQ10 head group leh RC-LH116-a QB quinone te pawh an inzawm tlat bawk (Figure S15).
QB site occupancy rate hrang hrang nei RC structure pahnih awm hian QB quinone binding nena inzawm conformational changes awm rengte zirchian theihna hun remchang thar a siam a ni. RC-LH116 complex-ah chuan QB quinone hi fully bound “proximal” position-ah a awm a (26), mahse RC-LH114-W inthenna hian QB quinone a nei lo. RC-LH114-W ah hian QB quinone a awm lo a, hei hi mak tak a ni a, a chhan chu complex hi active a ni a, structurally resolved QB quinone nei RC-LH116 complex aiin a active zawk a ni. LH1 ring pahnih hian quinones paruk vel chelate mahse, panga chu closed RC-LH116 ring-ah structurally resolved an ni a, open RC-LH114-W ring-ah chuan pathum chauh structurally limited an ni thung. He structural disorder pung hian RC-LH114-W QB sites te chu a thlak chak zawk a, complex chhunga quinone kinetics a chak zawk a, LH1 loop a pelh theihna a tipung thei bawk. Kan rawtna chu RC-LH114-W-a RC QB site-a UQ awm lohna hi complex complex zawk leh active zawk avanga lo awm a ni thei a, RC-LH114-W-a QB site chu UQ turnover-ah a frozen nghal bawk The specific stage (the entrance to the QB site has been closed) reflects the conformation of this activity.
QB tel lo chuan L-Phe217 chu UQ10 binding nena inmil lo position-a accompanying rotation a ni a, a chhan chu tail-a isoprene unit hmasa ber nen spatial collision a thlen dawn avangin (Figure 6A). Chu bakah, conformational changes langsar tak takte chu a lang chiang hle a, a bik takin helix de (TMH D leh E inkar loop-a helix tawi)-ah L-Phe217 chu QB binding pocket-ah a inthlak a, L-Tyr223 a rotation (Figure 6A ) M-Asp45 framework nena hydrogen bond tihchhiat leh QB binding site luhna khar nan (Figure 6B) a ni. Helix de pivot at his base, L-Ser209 Cα chu 0.33Å in a inthlak a, L-Val221Cα chu 3.52Å in a inthlak thung. TMH D leh E-ah hian danglamna hmuh tur a awm lo a, chu chu structure pahnihah hian superimposable a ni (Figure 6A). Kan hriat dan chuan hei hi natural RC-a QB site khar hmasa ber a ni. Complete (QB-bound) structure nena khaikhin chuan quinone a tihtlem hmain quinone chhunga a luh theih nan conformational change a ngai tih a hriat theih. L-Phe217 chu a inher a, quinone head group nen π-stacking interaction a siam a, helix chu pawn lamah a inthlak a, chu chuan L-Gly222 skeleton leh L-Tyr223 side chain te chu hydrogen bond network stable hydrogen bond structure neiin a siam thei ta a ni (Figure 6, A leh C).
(A) Hologram (L chain, orange/M chain, magenta) leh apo (gray) structure-a cartoon inzawmkhawm, chutah chuan key residue-te chu rod ang maia entir a ni. UQ10 hi yellow bar hmanga entir a ni. Dotted line hian structure pumpuiah hydrogen bonds lo awmte a tarlang a ni. (B leh C) Apolipoprotein leh ring structure pumpui surface representation, L-Phe217-a side chain oxygen chu blue-a leh L-Tyr223-a red-a tarlanna a ni. L subunit chu orange a ni a; M leh H subunit te hi a rawng a awm lo. (D leh E) Apolipoprotein (D) leh whole (E) RC QB site [color by (A) ve ve] leh Thermophilus thermophilus PSII (green, blue leh plastic quinone; PDB ID: 3WU2) Align (58).
Beisei loh takin LH1 nei lo QB-deficient RCs structure engemawzat awm mahse, he zirchiannaa conformational changes hmuhchhuah hi a hmain report a la ni lo. Chung zingah chuan Blc atanga QB depletion structure pawh a tel. viridis (PDB ID: 3PRC) (27), Tch.-ah hian a awm a, a rilru a hah lutuk chuan a rilru a buai em em a. tepidum (PDB ID: 1EYS) (28) leh Rba. sphaeroides (PDB ID: 1OGV) (29), chung zawng zawng chu an QB structure pumpui nen a inang vek tawh. 3PRC ngun taka enfiah chuan LDAO (Lauryl Dimethyl Amine Oxide) detergent molecule te chu QB position luhnaah an inzawm tih hmuhchhuah a ni a, hei hian closed conformation-a rearrangement a veng thei a ni. LDAO hi 1EYS emaw 1OGV emaw-ah hmun khatah a chhe lo nain, heng RC te hi detergent inang hmanga siam an nih avangin effect inang an nei thei a ni. A crystal structure chu Rba. Sphaeroides RC co-crystallized with cytochrome c2 (PDB ID: 1L9B) pawh hian QB site khar a nei niin a lang. Mahse, hetiang a nih chuan RC-M polypeptide N-terminal region (Q helix-a Tyr residue H bond hmanga QB binding site nena inzawm) chuan unnatural conformation a nei a, QB conformational change chu zirchian belh a ni lo (30 ). Thil thlamuanthlak tak chu RC-LH114-W structure-ah hian M polypeptide-a hetiang deformation hi kan hmu lo a, hei hi RC-LH116 RC-a N-terminal region nen a inang deuh ber a ni. Tin, hriat tur chu detergent-based LH1 antenna tihbo a nih hnuah PDB-a apolipoprotein RCs te chu a resolve a, chu chuan RC leh a chhehvel LH1 ring chhung lam inkar gap-a internal quinone pool leh lipids te chu a ti bo ta a ni (31, 32). RC hi a thawk chhunzawm zel a, a chhan chu cofactor zawng zawng a vawng reng a, decomposable QB quinone tih loh chu, a stable lo zawk a, a buatsaih lai hian a bo fo thin (33). Chu bakah, RC atanga LH1 leh natural cyclic lipids lakchhuah hian hnathawhnaah nghawng a nei thei tih hriat a ni a, chu chu charge-separated P+QB-state dam rei lutuk te hi a ni (31, 34, 35). Chuvangin, RC chhehvela local LH1 ring awmna hian QB site “closed” chu a vawng reng thei a, chu chuan QB bula local environment chu a humhim thei ang tih kan ngaihtuah a ni.
Apolipoprotein (QB quinone tel lo) leh a structure kimchang hian QB site turnover snapshot pahnih chauh ni mahse, thil thleng hrang hrang ni lovin, hydroquinone To inhibition hmanga rebinding ven nan binding hi gated theih a nih thu hriattirna a awm. Apolipoprotein QB site bula quinolol leh quinone inzawmna hi a danglam thei a, hei hian RC-in a hnawl phah a ni. Hun rei tak chhung chu conformational changes hian quinones binding leh reduction-ah hian chanvo a nei tih an lo sawi tawh a. Frozen RC-te hian dark adaptation hnua quinones an tihtlem theihna chu a tlahniam (36); X-ray crystallography atanga a lan dan chuan he chhiatna hi QB quinones te chu active proximal position atanga 4.5 Å vel a “distal” conformation-a an tang vang a ni (26) , 37). He distal binding conformation hi apolipoprotein leh full ring structure inkara intermediate state snapshot a ni tih kan rawt a, hei hian quinone nena inzawmna hmasa ber leh QB site hawn dan a zui a ni.
Phototrophic bacteria thenkhat leh cyanobacteria, algae leh thlai chi hrang hrang PSII complex-a hmuh type II RC hian structural leh functional conservation a nei a (38). Figure 6 (D leh E)-a structural alignment kan hmuh hian PSII RCs leh bacterial RC complex-a QB site inang lo tak chu a sawi uar hle. He tehkhin thu hi hun rei tak chhung chu quinone binding leh reduction system inzawm tlat zirchianna atana model a ni tawh a ni. Tun hmaa thuchhuah hrang hrangah chuan conformational changes hi PSII reduction of quinones nen a inzawm tih an sawi (39, 40). Chuvangin, RC evolutionary conservation ngaihtuah chuan, he binding mechanism hi a hmaa hmuh ngai loh hi oxygenated phototrophic plant-a PSII RC QB site-ah pawh hman theih a ni thei bawk.
Rps ΔpufW (unlabeled pufW deletion) leh PufW-His (C-terminal 10x His-tagged protein-W natural pufW locus atanga express) te hi a awm a. palustris CGA009 hi kan hnathawh hmasaah khan kan sawi tawh a (16). Heng strain leh isogenic wild-type parent te hi freezer atang hian PYE (5 g liter -1 ve ve) (LB-a -80 °C-a dah, 50% ( w/v) glycerol) protein, yeast extract leh succinate awmna) agar [1.5% (w/v)] plate-a cell tlemte streak hmangin an la chhuak a ni. Plate chu anaerobic condition-ah room temperature-ah thimah zan khat chhung an incubate a, chutah chuan OSRAM 116-W halogen bulbs (RS Components, UK)-in a pek êng dum (~50 μmolm-2 s-1) hmangin ni 3 atanga ni 5 chhung colony pakhat a lo chhuah thlengin an eng a ni. Colony pakhat hmangin M22+ medium (41) 0.1% (w/v) casamino acids (hetah hian M22 tia sawi tur) supplemented 10 ml inoculate a ni. Culture chu oxygen hniam condition hnuaiah thimah 34°C-ah 180 rpm-a darkar 48 chhung shaking-in an tipung a, chutah chuan culture 70 ml chu chutiang bawka darkar 24 chhung inoculate a ni. Semi-aerobic culture volume 1 ml hmangin 30 ml universal screw-top transparent glass bottle-ah M22 medium 30 ml inoculate a ni a, sterile magnetic force Stirring rod hmangin darkar 48 chhung agitation (~50μmolm-2 s-1) hmangin irradiated a ni. Tichuan culture 30 ml chu chutiang bawka culture litre 1 vel nen inoculate a ni a, chu chu ~200 μmolm-2 s-1-a eng culture litre 9 vel darkar 72 chhung inoculate nan hman a ni. Cell te chu 7132 RCF-ah minute 30 chhung centrifugation hmangin lakkhawm a ni a, ~10 ml 20 mM tris-HCl (pH 8.0)-ah resuspended leh a ni a, a mamawh thlengin -20°C-ah dah a ni.
Thawing hnuah deoxyribonuclease I (Merck, UK), lysozyme (Merck, UK) leh Roche holoenzyme protease inhibitor tablet pahnih (Merck, UK) crystal thenkhat chu resuspended cell-ah dah la. French pressure cell 20,000 psi (Aminco, USA)-ah chuan vawi 8 atanga vawi 12 vel an tibuai a. Cell chhe lo leh insoluble debris te chu 18,500 RCF-ah minute 15 chhung 4°C-ah centrifugation hmanga lakchhuah a nih hnuah, membrane chu pigmented lysate atang hian 113,000 RCF-ah darkar 2 chhung 43,000°C-ah centrifugation hmangin precipitate a ni. Soluble fraction chu paih la, colored membrane chu 20 mM tris-HCl (pH 8.0) 100 atanga 200 ml-ah resuspend la, aggregate hmuh theih loh thlengin homogenize rawh. Suspended membrane chu 20 mM tris-HCl (pH 8.0) (Anatrace, USA) 2% (w/v) β-DDM awmnaah darkar 1 chhung thimah 4°C-ah zawi zawiin an incubate a. Tichuan 70°C-ah centrifuge-in RCF 150,000 chu 4°C-ah darkar 1 chhung dissolve la, residual insolubles chu paih chhuak rawh.
ΔpufW strain atanga solubilizing membrane chu 50 ml DEAE Sepharose ion exchange column-ah binding buffer [20 mM tris-HCl (pH 8.0) 0.03% (w / v) β-DDM awmna column volume pathum (CV) hmangin an hnawih a. Column chu CV binding buffer pahnih hmangin silfai la, chutah chuan 50 mM NaCl awmna binding buffer pahnih hmangin silfai ang che. RC-LH116 complex chu 1.75 CV-ah linear gradient 150 to 300 mM NaCl (in binding buffer) hmangin eluted a ni a, a bak binding complex chu 0.5 CV-ah 300 mM NaCl awmna binding buffer hmangin eluted a ni. Absorption spectrum 250 leh 1000 nm inkar chu la khawm la, absorbance ratio (A880/A280) 1 aia tam nei fraction chu 880 atanga 280 nm-ah dah la, binding buffer-ah vawi hnih dilute la, chutiang bawk chuan DEAE column On purification-ah hmang leh rawh. A880/A280 ratio 1.7 aia sang leh A880/A805 ratio 3.0 aia sang nei fraction te chu dilute la, ion exchange round thumna chu ti la, A880/A280 ratio 2.2 aia sang leh A880/A805 ratio 5.0 aia sang nei fraction te chu vawng reng rawh. Complex a then thianghlim tawh chu Amicon 100,000 molecular weight cut-off (MWCO) centrifugal filter (Merck, UK)-ah ~2 ml thleng concentrate a ni a, Superdex 200 16/600 size exclusion column (GE Healthcare, US) 200 mM NaCl buffer awmnaah load a ni a, chutah chuan buffer khatah 1.5 CV-ah eluted a ni. Size exclusion fraction-a absorption spectra te chu la khawm la, absorption spectra te chu A880/A280 ratio 2.4 leh A880/A805 ratio 5.8 atanga 100 A880 aia tam neiin concentrate la, cryo-TEM grid buatsaih emaw dahkhawmna atan hmang nghal rawh A tul hma chu -80°C ah dah rawh.
PufW-His strain atanga solubilizing membrane chu IMAC buffer ( GE Healthcare)-ah 20 ml HisPrep FF Ni-NTA Sepharose column (20 mM tris-HCl (pH 8.0) 200 mM NaCl leh 0.03% (w/w) awmna)-ah hnawih a ni. v) β-DDM hmanga siam a ni]. Column chu IMAC buffer CV panga hmangin silfai a ni a, chutah chuan IMAC buffer CV panga 10 mM histidine awmna hmangin silfai a ni. Core complex chu column atang hian IMAC buffer panga hmangin 100 mM histidine awmna hmangin eluted a ni. RC-LH114-W complex awmna fraction chu Amicon 100,000 MWCO filter (Merck, UK) hmanga thuam stirred tank-ah ~10 ml thleng concentrate a ni a, binding buffer hmangin vawi 20 diluted a ni a, chutah chuan 25 ml-ah dah a ni DEAE Sepharose column-ah chuan buffer-a bound CV pali hman hmasak a ni. Column chu CV binding buffer pali hmangin silfai la, chutah chuan CV pariat-ah complex chu linear gradient 0 atanga 100 mM NaCl (binding buffer-ah)-ah elute la, a bak pali chu 100 mM binding buffer awmnaah elute rawh The residual complexes eluted on the sodium chloride combined with the A880/A280 ratio higher than 2.4 and the A880/A805 ratio 4.6 aia sang chu Amicon 100,000 MWCO centrifugal filter-ah ~2 ml-ah concentrate a ni a, 1.5 CV IMAC-in a hmain Buffer equilibrated Superdex 200 16/600 size exclusion column-ah eluted a ni a, chutah chuan buffer khatah 1.5 CV-ah eluted a ni. Size-exclusion fractions absorption spectra te chu la khawm la, absorption spectra te chu A880/A280 ratio 2.1 aia sang leh A880/A805 ratio 4.6 atanga 100 A880 aia tam neiin concentrate la, chu chu frozen TEM grid siamna atan hman nghal emaw, a tul hma chuan -80°C-ah dah emaw a ni.
Leica EM GP immersion freezer hmangin low temperature TEM grids siam a ni. Complex chu IMAC buffer-ah A880 of 50-ah diluted a ni a, chutah chuan 5μl chu glow-discharged QUANTIFOIL 1.2/1.3 carbon-coated copper mesh (Agar Scientific, UK)-ah load a ni. Grid chu 20°C leh 60% relative humidity-ah s 30 chhung incubate la, s 3 chhung blot dry la, liquid ethane-ah -176°C-ah quench rawh.
RC-LH114-W complex data hi eBIC (Electronic Bioimaging Center) (British Diamond Light Source) ah Titan Krios microscope hmangin record a ni a, hei hian accelerating voltage 300kV-ah hna a thawk a, nominal magnification 130,000× leh energy of- Choose a gap of 20 eV. Gatan 968 GIF Quantum K2 peak detector nei hmangin data lakkhawm nan counting mode-a thlalak record a ni. Calibrate pixel size chu 1.048Å a ni a, dose rate chu 3.83 e-Å-2s-1 a ni. Second 11 chhungin movie chu a khawlkhawm a, hmun 40-ah a then a. Carbon-coated area hmangin microscope chu refocus la, chutah chuan hole khatah movie pathum la khawm rawh. A vaiin movie 3130 an khawlkhawm a, defocus value chu -1 leh -3μm inkar a ni.
RC-LH116 complex atana data chu microscope ang chiah hmangin Asterbury Biostructure Laboratory (University of Leeds, UK)-ah lakkhawm a ni. Data hi counting mode-ah magnification 130 k-a lakkhawm a ni a, pixel size chu 1.065 Å-ah calibrate niin dose 4.6 e-Å-2s-1 a ni. He movie hi second 12 chhunga record a ni a, part 48-ah ṭhen a ni. A vaiin film 3359 lakkhawm a ni a, defocus value chu -1 leh -3μm inkar a ni.
Data processing zawng zawng hi Relion 3.0 pipeline (42) hmanga tih a ni. Dose weighting hmanga beam motion siamthat nan Motioncorr 2 (43) hmang la, chutah chuan CTFFIND 4.1 (44) hmangin CTF (contrast transfer function) parameter chu teh rawh. Heng initial processing stages hnua photomicrographs tlangpui chu Figure 2. S16-ah hian tarlan a ni. Automatic selection template hi 250-pixel frame-a particle 1000 pixel 250 vel manual-a thlan chhuah hmanga siam a ni a, reference two-dimensional (2D) classification nei lo, chu chuan sample contamination nena inmil emaw, hriat theih tur characteristic nei lo emaw classification-te chu hnawl a ni. Tichuan, microphotograph zawng zawngah automatic selection an ti a, RC-LH114-W chu particle 849,359 a ni a, RC-LH116 complex chu particle 476,547 a ni. Particle thlan chhuah zawng zawngte hian non-reference 2D classification round hnih an paltlang vek a, run tin hnuah carbon area, sample contamination, feature langsar lo emaw, strongly overlapping particle emaw nena inmil particle te chu hnawl a ni a, chu chuan 772,033 (90.9%) leh 359,678 (75.5%) ) Particle te chu 3D classification of RC-LH114-W leh RC-LH116 te an ni. A tir lama 3D reference model chu stochastic gradient descent method hmanga siam a ni. Initial model chu reference atan hmangin, particle thlan chhuah te chu 3D-ah category paliah then a ni. He category-a model hi reference atan hmangin category lian ber a particle-te chu 3D refining ti la, chutah chuan initial 15Å low-pass filter hmangin solvent area chu khuh la, soft edges pixels 6 dah la, pixels te chu post-process la, top detector-a Gatan K2 peak Modulation transfer function siamthat nan. RC-LH114-W dataset tan chuan he model hmasa ber hi mask kil hrang hranga density chak tak (UCSF Chimera-a core complex density atanga inzawm lo) tihbo hmangin siam danglam a ni. Model lo chhuak (RC-LH114-W leh RC-LH116 te resolution chu 3.91 leh 4.16 Å a ni) te chu 3D classification round hnihna atan reference atan hman a ni. Particle hmante hi initial 3D class-ah group-ah dah an ni a, neighborhood nena inzawmna nghet tak an nei lo. Overlap emaw, structural features langsar tak tak awm lohna emaw. 3D classification round hnihna hnuah resolution sang ber category thlan a ni [RC-LH114-W tan chuan category pakhat chu particle 377,703 (44.5%) a ni a, RC-LH116 tan chuan category pahnih a awm a, a vaiin particle 260,752 (54.7%) , A tir lama rotation hnua danglamna tlemte nena align a nih chauhvin an inang vek a ni]. Particle thlan chhuah te chu 400-pixel box-ah lakchhuah leh niin 3D refining hmangin tihthianghlim a ni. Solvent mask hi a tir lama 15Å low-pass filter hmanga siam a ni a, 3 pixel map expansion leh 3 pixel soft mask hmanga siam a ni. Per-particle CTF refinement hmangin per-particle motion correction leh per-particle CTF refinement round hnihna hmangin step tin hnuah 3D refinement, solvent masking leh post-processing te chu a chhuak texture tihthianghlim lehzual nan tih a ni. FSC (Fourier Shell Correlation Coefficient) cut-off value 0.143 hmangin RC-LH114-W leh RC-LH116 model hnuhnung ber resolution chu 2.65 leh 2.80Å a ni. Model hnuhnung ber FSC curve chu Figure 2. S17-ah hian kan hmu a.
Protein sequence zawng zawng hi UniProtKB: LH1-β (PufB; UniProt ID: Q6N9L5) atanga download vek a ni a; LH1-α (PufA; UniProtID: Q6N9L4) te chu a lo chhuak a; RC-L (PufL; UniProt ID: O83005) te chu a chhuak a; RC-M (PufM; UniProt ID: A0A4Z7) leh a chhuahna tur hmun leh a hmanna tur hmun; RC-H (PuhA; UniProt ID: A0A4Z9) te chuan an ziak a; Protein-W (PufW; UniProt ID: Q6N1K3) hmanga siam a ni. SWISS-MODEL (45) hmangin RC homology model siam a ni a, chutah chuan RC-L, RC-M leh RC-H te protein sequence leh Rba crystal structure te a awm a ni. sphaeroides RC chu template atan hman a ni (PDB ID: 5LSE) (46). UCSF Chimera-a “fit map” tool hmangin generated model chu map (47)-ah fit turin, protein structure siam that nan, leh cofactor [4×BChl a (monomer library residue hming = BCL), 2×BPh a (BPH), UQ10 chi khat emaw chi hnih (U10), non-heme iron (Fe) pakhat leh 3,4-dihydrohexacarbonylcholine pakhat emaw cofactor rawh (QAK)] hmangin Coot (48) hmangin add rawh. QAK hi monomer library-ah a awm loh avangin PHENIX-a eLBOW tool hmangin parameterized a ni (49).
A dawtah chuan LH1 subunit siam a ni. A tirah chuan PHENIX (49)-a automatic construction tool hmangin map leh LH1-α leh LH1-β protein sequence te chu input atan hmangin LH1 sequence thenkhat chu automatic-in an siam a ni. LH1 subunit tling ber thlang la, extract la, Coot-ah load la, a chhunga sequence awm lo chu manual-in dah la, BCl pahnih a (BCL) leh spirilloxanthin (CRT) dah hmain structure pumpui chu manual-in refine rawh [Rps kaihhnawih angin LH1 complex density leh carotenoid awm zat hriat tawh angin. A chi hrang hrang (17)]. LH1 subunit kimchang chu copy la, UCSF Chimera “Docking Map Tool” hmangin LH1 density awmna non-model area kianga dock la, chutah chuan Coot-ah refine rawh LH1 subunit zawng zawng model a nih hma loh chuan tih leh tur a ni. RC-LH114-W structure atan chuan Coot-a unallocated density lakchhuah hmangin USCF Chimera map-a non-protein components la awm atang hian protein chu segmented a ni a, Autobuild tool hmangin initial model siam a ni a, subunit dang (protein-W) Modeling siam nan hman a ni. PHENIX (49)-ah chuan. Coot (48)-a model lo chhuakah sequence awm lo awm zawng zawng chu dah la, chutah chuan subunit pumpui chu manual-in refine rawh. Unallocated density la awm chu lipids (PDB monomer library ID of CDL = CDL, POPC = 6PL leh POPG = PGT), β-DDM detergent (LMT) leh UQ10 molecule (U10) te inzawmkhawm nen a inmil a ni. Model statistics leh fit-a visual quality tihchangtlun belh theih a nih loh hma chuan initial model kimchang tak chu ti famkim turin Coot (48)-a PHENIX optimization (49) leh manual optimization hmangin. A tawp berah chuan LocScale (50) hmangin local map chu sharpen la, chutah chuan unallocated density modeling leh automatic leh manual optimization cycle dang engemaw zat ti rawh.
Peptide hrang hrang, cofactors leh lipid leh quinones dangte chu an density hrang hrang chhunga docked te chu Figure 1 leh 2. S18 atanga S23 ah hian tarlan a ni. Model hnuhnung ber statistical information chu Table S1-ah hian tarlan a ni.
A danglamna a awm loh chuan UV/Vis/NIR absorption spectra te chu Cary60 spectrophotometer (Agilent, USA) hmangin 250 nm atanga 1000 nm inkar 1 nm inkar leh integration time 0.1s ah lakkhawm a ni.
Sample chu quartz cuvette-ah 2 mm path A880 of 1-ah dilute la, absorption spectrum 400 leh 1000 nm inkar chu la khawm rawh. Circular dichroic spectra te hi Jasco 810 spectropolarimeter (Jasco, Japan) ah 400 nm leh 950 nm inkar 1 nm inkar ah scan rate 20 nm min-1 ah lakkhawm a ni.
Molar extinction coefficient chu core complex chu A880 50 vel a dilute hmanga teh a ni a, 10μl volume chu 990μl binding buffer emaw methanol emaw-ah dilute la, BChl degradation tih tlem nan absorption spectrum chu khawlkhawm nghal rawh. Methanol sample tina BChl awm zat chu 771 nm-a extinction coefficient 54.8 mM-1 cm-1 hmanga chhut a ni a, extinction coefficient chu chhut a ni (51). BChl concentration tehna chu 32 (RC-LH114-W) emaw 36 (RC-LH116) emaw-ah then la, core complex concentration chu hriat theih a ni a, chu chu buffer Extinction coefficient-a sample lakkhawm ang chiah absorption spectrum hriat nan hman a ni. tlukpui. Sample tin atan vawi thum tehna nawn leh a ni a, BChl Qy maximum-a absorbance average chu chhut nan hman a ni. RC-LH114-W extinction coefficient 878 nm-a teh chu 3280±140 mM-1 cm-1 a ni a, RC-LH116 extinction coefficient 880 nm-a teh chu 3800±30 mM-1 cm-1 a ni thung.
UQ10 chu (52)-a hman dan angin quantified a ni. A tawi zawngin, reverse phase HPLC (RP-HPLC) chu Agilent 1200 HPLC system hmangin an ti a. RC-LH116 emaw RC-LH114-W emaw 0.02 nmol vel chu 0.02% (w/v) ferric chloride awmna 50:50 methanol:chloroform 50μl-ah hmin la, pre-equilibrated Beckman Coulter Ultrasphere ODS 4.6 mm chu HPLC-a 40°C-ah 1 ml-1 min-1-ah inject rawh solvent (80:20 methanol:2-propanol) chu ×25 cm column-ah dah a ni. HPLC solvent-ah isocratic elution hmangin 275 nm (UQ10), 450 nm (carotenoids) leh 780 nm (BChl)-ah darkar 1 chhung absorbance enfiah rawh. 275 nm chromatogram-a minute 25.5 chhunga peak chu integrated a ni a, chutah chuan detectable compound dang a awm lo. Integrated area hmang hian UQ10 extracted molar amount chu pure standards 0 atanga 5.8 nmol thlenga injection atanga chhut calibration curve hmangin chhut a ni (Figure S14). Sample tin hi replicate thum-ah an zirchiang a, reported error chu average SD nen a inmil a ni.
RC-LH1 complex awmna solution, Qy absorption sang ber 0.1 nei chu 30 μM reduced horse heart cytochrome c2 (Merck, UK) leh 0 to 50 μMUQ2 (Merck, UK) hmangin siam a ni. UQ2 concentration khatah 1-ml sample pathum siam a ni a, 4°C-ah thimah zan khat incubate a ni a, chu chuan teh hmain thimah a insiamrem kim theih nan. Solution chu OLIS RSM1000 modular spectrophotometer-ah load a ni a, 300 nm flame/500 line grating, 1.24 mm inlet, 0.12 mm middle leh 0.6 mm outlet slits hmanga thuam a ni. Excitation light awm loh nan sample phototube leh reference photomultiplier tube luhnaah 600 nm long pass filter dah a ni. Absorbance chu 550 nm-ah enfiah a ni a, integration time chu 0.15 s a ni. Excitation light hi 880 nm M880F2 LED (Light Emitting Diode) (Thorlabs Ltd., UK) atanga fiber optic cable kaltlangin DC2200 controller (Thorlabs Ltd., UK) kaltlangin 90% intensity-in a chhuak a, light source-ah angle 90° of. Measuring beam chu mirror nen a inhmachhawn a, a tir lama sample-in a la lut lo light eng pawh chu a rawn let leh thei a ni. 50 s chhunga eng a chhuah hma s 10-ah absorbance enfiah thin ang che. Tichuan, quinolol hian a takin cytochrome c23 + a tihhniam dan tehna atan thimah s 60 chhung absorbance chu enfiah leh a ni (raw data atan Figure S8 en rawh).
Data chu 0.5 atanga 10 s chhunga linear initial rate fit (UQ2 concentration a zirin) hmanga process a ni a, UQ2 concentration tinah sample pathumte rates chu average a ni. Extinction coefficient hrang hranga RC-LH1 concentration chhut chu rate chu catalytic efficiency-ah chantir nan hman a ni a, Origin Pro 2019 (OriginLab, USA)-ah plot a ni a, Michaelis-Menten model-ah fit a ni a, Km leh Kcat value langsar tak takte chu hriat theih a ni.
Transient absorption tehna atan RC-LH1 sample chu IMAC buffer-ah 50 mM sodium ascorbate (Merck, USA) leh 0.4 mM Terbutin (Merck, USA) awmnaah ~2μM-ah diluted a ni. Ascorbic acid hi sacrificial electron donor atan hman a ni a, tert-butaclofen hi QB inhibitor atan hman a ni a, chu chuan main RC donor chu a tlahniam zel (chu chu photooxidized a ni lo) a, chu chu measurement process chhung zawngin a ni. Sample 3 ml vel chu custom rotating cell (0.1 m in diameter, 350 RPM)-ah 2 mm optical path sei zawngah dah a ni a, chu chuan laser path-a sample chuan excitation pulse inkara dark adaptation atan hun a nei tling tih enfiah a ni. ~100-fs laser pulse hmangin Ti: Sapphire laser system (Spectra Physics, USA) chu amplify la, sample chu 880 nm-ah 1 kHz (NIR tan 20 nJ emaw Vis tan 100 nJ emaw) repetition rate-in excite rawh. Data lakkhawm hmain sample chu excitation light-ah minute 30 vel dah hmasa phawt ang che. Exposure hian QA inactivation a thlen ang (QA vawi khat emaw vawi hnih emaw tihtlem a ni thei). Mahse he process hi reversible a ni tih hre reng ang che, a chhan chu hun rei tak dark adaptation hnuah RC hi zawi zawiin QA activity-ah a kir leh dawn a ni. Helios spectrometer (Ultrafast Systems, USA) hmangin transient spectra tehna atan delay time -10 atanga 7000 ps a awm a ni. Surface Xplorer software (Ultrafast Systems, USA) hmangin data set te chu ungroup la, chutah chuan merge leh standardize rawh. CarpetView software package (Light Conversion Ltd., Lithuania) hmangin decay nena inzawm differential spectra hmuh nan combined data set hmang la, a nih loh leh Origin (OriginLab, USA)-a single-wavelength spectral evolution fit turin instrument response nena exponent tam tak convolve tu function hmang bawk ang che.
A chunga kan sawi tawh ang khan (53) RC leh peripheral LH2 antenna pahnih nei lo LH1 complex awmna photosynthetic film siam a ni. Membrane chu 20 mM tris (pH 8.0)-ah diluted a ni a, chutah chuan 2 mm optical path nei quartz cuvette-ah load a ni. 30nJ laser pulse hmangin sample chu 540 nm-ah excite a ni a, delay time chu -10 atanga 7000 ps a ni. Rps.-a tarlan angin data set chu process rawh. pal sample a ni.
Membrane chu 150,000 RCF-ah darkar 2 chhung 4°C-ah centrifugation hmangin pellet a ni a, chutah chuan 880 nm-a a absorbance chu 20 mM tris-HCl (pH 8.0) leh 200 mM NaCl-ah resuspended a ni. Membrane chu 2% (w/v) β-DDM-ah darkar 1 chhung darkar 1 chhung 4°C-a thimah zawi zawiin hrual la, a hmin ang. Sample chu 100 mM triethylammonium carbonate (pH 8.0) (TEAB; Merck, UK)-ah protein concentration 2.5 mg ml-1 (Bio-Rad analysis)-ah diluted a ni. A hmaa kan tihchhuah tawh dan (54) atang khan processing dang an ti leh a, protein 50 μg chu TEAB 1% (w/v) sodium laurate (Merck, UK) awmna TEAB zawng zawng 50 μl-ah dilution atanga tan a ni. 60 s chhung sonication hnuah, 5 mM tris (2-carboxyethyl) phosphine (Merck, UK) 37 ° C-ah minute 30 chhung tihtlem a ni. S-alkylation atan chuan sample chu 10 mM methyl S-methylthiomethanesulfonate (Merck, UK) nen incubate la, 200 mM isopropanol stock solution atang chuan room temperature-ah minute 10 chhung dah la. Proteolytic digestion chu trypsin/endoproteinase Lys-C mixture (Promega UK) 2 μg dahin 37°C-ah darkar 3 chhung an incubate a. Laurate surfactant chu ethyl acetate 50 μl leh 10 μl 10% (v/v) LC grade trifluoroacetic acid (TFA; Thermo Fisher Scientific, UK) dahin 60 s chhung vortexing hmangin lakchhuah a ni. Phase separation chu 15,700 RCF-ah minute 5 chhung centrifugation hmangin promote a ni. Manufacturer protocol ang chuan C18 spin column (Thermo Fisher Scientific, UK) hmangin peptide awmna lower phase chu uluk takin aspirate leh desalt a ni. Vacuum centrifugation hmanga a vawt hnuah sample chu 0.5% TFA leh 3% acetonitrile-ah a hmin a, a hmaa kan sawi tawh system parameters detailed hmangin nanoflow RP chromatography coupled with mass spectrometry hmangin 500 ng chu an zirchiang a ni.
Protein hriat theihna leh quantification atan MaxQuant v.1.5.3.30 (56) hmangin Rps. palustris proteome database (www.uniprot.org/proteomes/UP000001426) ah hian a awm a. Mass spectrometry proteomics data hi ProteomeXchange Alliance-ah PRIDE partner repository (http://proteomecentral.proteomexchange.org) kaltlangin dataset identifier PXD020402 hnuaiah dah a ni.
RPLC hmanga electrospray ionization mass spectrometry hmanga zirchianna atan chuan RC-LH1 complex chu wild-type Rps atanga siam a ni. A hmaa kan lo tih tawh angin (16) palustris cell-a protein concentration siam chu 20 mM Hepes (pH 7.8), 100 mM NaCl leh 0.03% (w/v) β- (Bio-Rad analysis) ) DDM-ah 2 mg ml-1 a ​​ni. Manufacturer protocol angin 2D purification kit (GE Healthcare, USA) hmangin precipitation method hmangin protein 10 μg extract la, chu precipitate chu 20 μl 60% (v / v) formic acid (FA), 20% (v / v) Acetonitrile leh 20% (v/v) water-ah hmin rawh. Microliter panga chu RPLC (Dionex RSLC) hmangin mass spectrometry (Maxis UHR-TOF, Bruker) nena inzawm hmangin an zirchiang a. 60°C leh 100μlmin -1-a inthen nan MabPac 1.2×100 mm column (Thermo Fisher Scientific, UK) hmang la, gradient chu 85% (v / v) solvent A [0.1% (v / v) FA leh 0.02% (V/v) TFA aqueous solution] atanga 85%(v/v) solvent B thleng a ni [0.1%(v/v) FA leh 90%(v/v) acetonitrile TFA-ah 0.02%(v/v)] Standard electrospray ionization source leh default parameters hmangin minute 60 aia rei hmangin mass spectrometer chuan 100 atanga 2750 m/z (mass-to-charge ratio) a hmu thei a ni. ExPASy bioinformatics resource portal FindPept tool (https://web.expasy.org/findpept/) hmangin mass spectrum chu complex chhunga subunit hrang hrangah map rawh.
Cell te chu darkar 72 chhung NF-low (10μMm-2 s-1), medium (30μMm-2 s-1) emaw high (300μMm-2 s-1) light hnuaiah darkar 72 chhung an enkawl a. M22 medium (M22 medium ah chuan ammonium sulfate paih a, sodium succinate chu sodium acetate hmanga thlak a ni) chu 100 ml screw-top bottle (23) ah dah a ni. 30-s cycle panga chhungin 0.1 micron glass beads chu volume ratio 1:1-ah beaded niin cells te chu lyse a ni a, ice-ah minute 5 chhung chill a ni. Insoluble matter, unbroken cells leh glass beads te chu benchtop microcentrifuge-ah 16,000 RCF-ah minute 10 chhung centrifugation hmangin lakchhuah a ni. Membrane chu Ti 70.1 rotor-ah RCF 100,000 hmangin 20 mM tris-HCl (pH 8.0)-ah 40/15% (w/w) sucrose gradient hmangin darkar 10 chhung an inthen a.
Kan hnathawh hmasa lama kan sawi tawh ang khan PufW-a His tag immunodetection (16). A tawi zawngin, purified core complex (11.8 nM) emaw, 2x SDS loading buffer (Merck, UK)-a RC concentration inang (oxidation hmanga reduced difference spectrum subtracting leh stained gel-a load matching hmanga teh) emaw chu vawi hnih diluted a ni. Protein te hi replica 12% bis-tris NuPage gel (Thermo Fisher Scientific, UK) hmangin an inthen a. Gel pakhat chu Coomassie Brilliant Blue (Bio-Rad, UK) hmangin stain a ni a, RC-L subunit load leh visualize a ni. Gel pahnihna-a protein chu immunoassay atan methanol-activated polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, UK)-ah dah a ni. PVDF membrane chu 50 mM tris-HCl (pH 7.6), 150 mM NaCl, 0.2% (v / v) Tween-20 leh 5% (w / v) skimmed milk powder-ah block a ni a, chutah chuan anti-His primary antibody (in Dilute the antibody buffer [50 mM tris-HCl (pH 7.6)) nen incubate a ni a, antibody buffer [50 mM tris-HCl (pH 7.6)-ah chuan incubate a ni. 150 mM NaCl leh 0.05% (v/v) Tween-20] chu 1:1000 A190-114A, Bethyl Laboratories, USA)-ah darkar 4 chhung dah a ni. Antibody buffer-a minute 5 chhung vawi 3 silfai hnuah, membrane chu horseradish peroxidase (Sigma-Aldrich, UK) anti-mouse secondary antibody (antibody buffer-a 1:10,000-a diluted) nen WESTAR ETA C 2.0 chemiluminescence substrate hmangin detection (antibody buffer-a vawi 3 sil hnu minute 5 hnuah) incubate a ni (Cyanagen, Italy) leh Amersham Imager 600 (GE Healthcare, UK) te a ni.
Stained gel emaw immunoassay lane tinte intensity distribution drawing hmangin, peak hnuaia area chu integrate a, RC-L (stained gel) leh Protein-W (immunoassay) intensity ratio chhut a, ImageJ (57) ah chuan Image chu process rawh. Heng ratio te hi RC-LH114-W sample thianghlim takah RC-L leh protein-W ratio chu 1:1 a nih anga ngaihin molar ratio ah an chantir a, chutiang chuan data set pumpui chu normalize a ni.
He thuziak atana supplementary materials hriat duh chuan http://advances.sciencemag.org/cgi/content/full/7/3/eabe2631/DC1 ah hian en theih a ni
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David J. K. 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 Kirmaier, Andrew Hitchcock, C. Neil Hunter te an ni
Reaction center-a light trap 1 complex-a high-resolution structure hian quinone dynamics chungchangah hriatna thar a pe a ni.
David J. K. 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 Kirmaier, Andrew Hitchcock, C. Neil Hunter te an ni
Reaction center-a light trap 1 complex-a high-resolution structure hian quinone dynamics chungchangah hriatna thar a pe a ni.
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