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- EMDB-39192: Cryo-EM structure of in vitro reconstituted Light-harvesting comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-39192
TitleCryo-EM structure of in vitro reconstituted Light-harvesting complex II trimer
Map data
Sample
  • Complex: In vitro reconstituted Light-harvesting complex II
    • Protein or peptide: Chlorophyll a-b binding protein, chloroplastic
  • Ligand: CHLOROPHYLL B
  • Ligand: CHLOROPHYLL A
  • Ligand: Lutein
  • Ligand: (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
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
  • Ligand: water
KeywordsComplex / light-harvesting / membrane protein / PHOTOSYNTHESIS
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 a-b binding protein, chloroplastic
Similarity search - Component
Biological speciesSpinacia oleracea (spinach)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsSeki S / Miyata T / Tanaka H / Namba K / Kurisu G / Fujii R
Funding support Japan, 5 items
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, 2024-
Header (metadata) releaseNov 20, 2024-
Map releaseNov 20, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_39192.png

Downloads & links

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Map

FileDownload / File: emd_39192.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 256 pix.
= 223.488 Å		0.87 Å/pix.
x 256 pix.
= 223.488 Å		0.87 Å/pix.
x 256 pix.
= 223.488 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.873 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.6004252 - 2.652308
Average (Standard dev.)0.0016281516 (±0.065791026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 223.488 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_39192_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_39192_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : In vitro reconstituted Light-harvesting complex II

EntireName: In vitro reconstituted Light-harvesting complex II
Components
  • Complex: In vitro reconstituted Light-harvesting complex II
    • Protein or peptide: Chlorophyll a-b binding protein, chloroplastic
  • Ligand: CHLOROPHYLL B
  • Ligand: CHLOROPHYLL A
  • Ligand: Lutein
  • Ligand: (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
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
  • Ligand: water

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Supramolecule #1: In vitro reconstituted Light-harvesting complex II

SupramoleculeName: In vitro reconstituted Light-harvesting complex II / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Spinacia oleracea (spinach)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: Chlorophyll a-b binding protein, chloroplastic

MacromoleculeName: Chlorophyll a-b binding protein, chloroplastic / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Spinacia oleracea (spinach)
Molecular weightTheoretical: 24.455611 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SPWYGPDRVK YLGPFSGESP SYLTGEFPGD YGWDTAGLSA DPETFAKNRE LEVIHCRWAM LGALGCVFPE LLARNGVKFG EAVWFKAGS QIFSEGGLDY LGNPSLVHAQ SILAIWACQV ILMGAVEGYR IAGGPLGEVV DPLYPGGSFD PLGLADDPEA F AELKVKEI ...String:
SPWYGPDRVK YLGPFSGESP SYLTGEFPGD YGWDTAGLSA DPETFAKNRE LEVIHCRWAM LGALGCVFPE LLARNGVKFG EAVWFKAGS QIFSEGGLDY LGNPSLVHAQ SILAIWACQV ILMGAVEGYR IAGGPLGEVV DPLYPGGSFD PLGLADDPEA F AELKVKEI KNGRLAMFSM FGFFVQAIVT GKGPLENLAD HLADPVNNNA WNFATNFVPG KHHHHHH

UniProtKB: Chlorophyll a-b binding protein, chloroplastic

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Macromolecule #2: CHLOROPHYLL B

MacromoleculeName: CHLOROPHYLL B / type: ligand / ID: 2 / Number of copies: 18 / Formula: CHL
Molecular weightTheoretical: 907.472 Da
Chemical component information

ChemComp-CHL:
CHLOROPHYLL B

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Macromolecule #3: CHLOROPHYLL A

MacromoleculeName: CHLOROPHYLL A / type: ligand / ID: 3 / Number of copies: 24 / Formula: CLA
Molecular weightTheoretical: 893.489 Da
Chemical component information

ChemComp-CLA:
CHLOROPHYLL A

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Macromolecule #4: Lutein

MacromoleculeName: Lutein / type: ligand / ID: 4 / Number of copies: 6 / Formula: A1LXP
Molecular weightTheoretical: 568.871 Da

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Macromolecule #5: (1R,3R)-6-{(3E,5E,7E,9E,11E,13E,15E,17E)-18-[(1S,4R,6R)-4-HYDROXY...

MacromoleculeName: (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
type: ligand / ID: 5 / Number of copies: 3 / Formula: NEX
Molecular weightTheoretical: 600.87 Da
Chemical component information

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

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Macromolecule #6: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

MacromoleculeName: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / type: ligand / ID: 6 / Number of copies: 3 / Formula: LHG
Molecular weightTheoretical: 722.97 Da
Chemical component information

ChemComp-LHG:
1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / phospholipid*YM

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 126 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5.0 mg/mL
BufferpH: 7.5 / Component - Concentration: 25.0 mM / Component - Formula: C8H18N2O4S / Component - Name: HEPES / Details: 0.03 % n-Dodexyl-alpha-D-maltoside
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Pressure: 10.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6145 / Average exposure time: 3.0 sec. / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 4734387
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 179318
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1rwt


Chain - Chain ID: B / Chain - Residue range: 14-229 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-8yee:
Cryo-EM structure of in vitro reconstituted Light-harvesting complex II trimer

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