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- PDB-8yee: Cryo-EM structure of in vitro reconstituted Light-harvesting comp... -

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Basic information

Entry
Database: PDB / ID: 8yee
TitleCryo-EM structure of in vitro reconstituted Light-harvesting complex II trimer
ComponentsChlorophyll a-b binding protein, chloroplastic
KeywordsPHOTOSYNTHESIS / Complex / light-harvesting / membrane protein
Function / homology
Function and homology information


photosynthesis, light harvesting in photosystem I / photosystem I / photosystem II / chlorophyll binding / chloroplast thylakoid membrane / response to light stimulus / metal ion binding
Similarity search - Function
Chlorophyll A-B binding protein, plant and chromista / Chlorophyll A-B binding protein / Chlorophyll A-B binding protein
Similarity search - Domain/homology
: / CHLOROPHYLL B / CHLOROPHYLL A / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / Chem-NEX / Chlorophyll a-b binding protein, chloroplastic
Similarity search - Component
Biological speciesSpinacia oleracea (spinach)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsSeki, S. / Miyata, T. / Tanaka, H. / Namba, K. / Kurisu, G. / Fujii, R.
Funding support Japan, 5items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)K23KJ1834 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121003 Japan
Japan Science and TechnologyJPMJFS2138 Japan
Japan Science and TechnologyJPMJCR20E1 Japan
Japan Society for the Promotion of Science (JSPS)23H04958 Japan
CitationJournal: PNAS Nexus / Year: 2024
Title: Structure-based validation of recombinant light-harvesting complex II.
Authors: Soichiro Seki / Tomoko Miyata / Naoko Norioka / Hideaki Tanaka / Genji Kurisu / Keiichi Namba / Ritsuko Fujii /
Abstract: Light-harvesting complex II (LHCII) captures sunlight and dissipates excess energy to drive photosynthesis. To elucidate this mechanism, the individual optical properties of pigments in the LHCII ...Light-harvesting complex II (LHCII) captures sunlight and dissipates excess energy to drive photosynthesis. To elucidate this mechanism, the individual optical properties of pigments in the LHCII protein must be identified. In vitro reconstitution with apoproteins synthesized by and pigment-lipid mixtures from natural sources is an effective approach; however, the local environment surrounding each pigment within reconstituted LHCII (rLHCII) has only been indirectly estimated using spectroscopic and biochemical methods. Here, we used cryo-electron microscopy to determine the 3D structure of the rLHCII trimer and found that rLHCII exhibited a structure that was virtually identical to that of native LHCII, with a few exceptions: some C-terminal amino acids were not visible, likely due to aggregation of the His-tags; a carotenoid at the V1 site was not visible; and at site 614 showed mixed occupancy by both chlorophyll and molecules. Our observations confirmed the applicability of the in vitro reconstitution technique.
History
DepositionFeb 22, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / em_admin ...chem_comp / em_admin / entity / pdbx_entity_nonpoly / pdbx_struct_conn_angle / struct_conn
Item: _chem_comp.name / _chem_comp.pdbx_synonyms ..._chem_comp.name / _chem_comp.pdbx_synonyms / _em_admin.last_update / _entity.pdbx_description / _pdbx_entity_nonpoly.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Chlorophyll a-b binding protein, chloroplastic
B: Chlorophyll a-b binding protein, chloroplastic
C: Chlorophyll a-b binding protein, chloroplastic
hetero molecules


Theoretical massNumber of molelcules
Total (without water)118,53057
Polymers73,3673
Non-polymers45,16354
Water2,270126
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 3 molecules ABC

#1: Protein Chlorophyll a-b binding protein, chloroplastic / Light-harvesting complex II / LHCII type I CAB / LHCP


Mass: 24455.611 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Spinacia oleracea (spinach) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P12333

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Non-polymers , 6 types, 180 molecules

#2: Chemical
ChemComp-CHL / CHLOROPHYLL B


Mass: 907.472 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: C55H70MgN4O6 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical...
ChemComp-CLA / CHLOROPHYLL A


Mass: 893.489 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C55H72MgN4O5 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-A1LXP / Lutein / (3R,3'R,6'R)-beta,epsilon-Carotene-3,3'-diol / (1~{R})-3,5,5-trimethyl-4-[(1~{E},3~{E},5~{E},7~{E},9~{E},11~{E},13~{E},15~{E},17~{E})-3,7,12,16-tetramethyl-18-[(1~{R},4~{R})-2,6,6-trimethyl-4-oxidanyl-cyclohex-2-en-1-yl]octadeca-1,3,5,7,9,11,13,15,17-nonaenyl]cyclohex-3-en-1-ol


Mass: 568.871 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C40H56O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-NEX / (1R,3R)-6-{(3E,5E,7E,9E,11E,13E,15E,17E)-18-[(1S,4R,6R)-4-HYDROXY-2,2,6-TRIMETHYL-7-OXABICYCLO[4.1.0]HEPT-1-YL]-3,7,12,16-TETRAMETHYLOCTADECA-1,3,5,7,9,11,13,15,17-NONAENYLIDENE}-1,5,5-TRIMETHYLCYCLOHEXANE-1,3-DIOL / (3S,5R,6R,3'S,5'R,6'S)-5',6'-EPOXY-6,7-DIDEHYDRO- 5,6,5',6'-TETRAHYDRO-BETA,BETA-CAROTENE-3,5,3'-TRIOL / 9'-CIS-NEOXANTHIN


Mass: 600.870 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C40H56O4
#6: Chemical ChemComp-LHG / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE


Mass: 722.970 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C38H75O10P / Comment: phospholipid*YM
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 126 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: In vitro reconstituted Light-harvesting complex II / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: Spinacia oleracea (spinach)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 0.03 % n-Dodexyl-alpha-D-maltoside
Buffer componentConc.: 25 mM / Name: HEPES / Formula: C8H18N2O4S
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingAverage exposure time: 3 sec. / Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6145
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2SerialEM4.1.0betaimage acquisition
4cryoSPARC4.2.1CTF correction
7UCSF Chimera1.17.1model fitting
9Coot0.9.4.1model refinement
10PHENIX1.19.2model refinement
11cryoSPARC4.2.1initial Euler assignment
12cryoSPARC4.2.1final Euler assignment
14cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4734387
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 179318 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 1RWT

1rwt


Pdb chain-ID: B / Accession code: 1RWT / Chain residue range: 14-229 / Pdb chain residue range: 14-229 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0098142
ELECTRON MICROSCOPYf_angle_d1.00911709
ELECTRON MICROSCOPYf_dihedral_angle_d7.9293237
ELECTRON MICROSCOPYf_chiral_restr0.048933
ELECTRON MICROSCOPYf_plane_restr0.0062778

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