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- EMDB-14123: Cryo-EM structure of catalytically active Spinacia oleracea cytoc... -

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Basic information

Entry
Database: EMDB / ID: EMD-14123
TitleCryo-EM structure of catalytically active Spinacia oleracea cytochrome b6f in complex with endogenous plastoquinones at 2.7 A resolution
Map data
Sample
  • Complex: Cytochrome b6f complex
    • Protein or peptide: x 8 types
  • Ligand: x 9 types
Function / homology
Function and homology information


plastoquinol--plastocyanin reductase activity / cytochrome b6f complex / plastoquinol-plastocyanin reductase / electron transporter, transferring electrons within cytochrome b6/f complex of photosystem II activity / cytochrome complex assembly / : / mitochondrial respiratory chain complex III / photosynthetic electron transport chain / mitochondrial electron transport, ubiquinol to cytochrome c / chloroplast thylakoid membrane ...plastoquinol--plastocyanin reductase activity / cytochrome b6f complex / plastoquinol-plastocyanin reductase / electron transporter, transferring electrons within cytochrome b6/f complex of photosystem II activity / cytochrome complex assembly / : / mitochondrial respiratory chain complex III / photosynthetic electron transport chain / mitochondrial electron transport, ubiquinol to cytochrome c / chloroplast thylakoid membrane / electron transporter, transferring electrons within the cyclic electron transport pathway of photosynthesis activity / photosynthesis / respiratory electron transport chain / proton transmembrane transport / 2 iron, 2 sulfur cluster binding / electron transfer activity / membrane => GO:0016020 / iron ion binding / heme binding / metal ion binding
Similarity search - Function
Cytochrome B6-F complex subunit VI (PetL) / Cytochrome b6-f complex subunit 6 / Cytochrome b6-f complex iron-sulfur subunit / Cytochrome b6/f complex, subunit 5 / Cytochrome b6-f complex, subunit 8 / Cytochrome b6/f complex, subunit IV / PetM of cytochrome b6/f complex subunit 7 / Cytochrome b6, PetB / Cytochrome b6/f complex, subunit 5 superfamily / Cytochrome b6-f complex, subunit 8 superfamily ...Cytochrome B6-F complex subunit VI (PetL) / Cytochrome b6-f complex subunit 6 / Cytochrome b6-f complex iron-sulfur subunit / Cytochrome b6/f complex, subunit 5 / Cytochrome b6-f complex, subunit 8 / Cytochrome b6/f complex, subunit IV / PetM of cytochrome b6/f complex subunit 7 / Cytochrome b6, PetB / Cytochrome b6/f complex, subunit 5 superfamily / Cytochrome b6-f complex, subunit 8 superfamily / Cytochrome B6-F complex subunit 5 / PetN / PetM family of cytochrome b6f complex subunit 7 / Cytochrome f transmembrane anchor / Cytochrome f / Cytochrome f large domain / Cytochrome f large domain superfamily / Apocytochrome F, C-terminal / Apocytochrome F, N-terminal / Cytochrome f family profile. / : / : / Cytochrome b/b6, C-terminal / Cytochrome b(C-terminal)/b6/petD / Cytochrome b/b6 C-terminal region profile. / Cytochrome b/b6, C-terminal domain superfamily / Cytochrome b/b6/petB / Rieske iron-sulphur protein, C-terminal / Cytochrome b/b6, N-terminal / Cytochrome b/b6-like domain superfamily / Cytochrome b/b6 N-terminal region profile. / Di-haem cytochrome, transmembrane / Rieske iron-sulphur protein / Rudiment single hybrid motif / Rieske [2Fe-2S] iron-sulphur domain / Rieske [2Fe-2S] domain / Rieske [2Fe-2S] iron-sulfur domain profile. / Rieske [2Fe-2S] iron-sulphur domain superfamily
Similarity search - Domain/homology
Cytochrome b6 / Cytochrome b6-f complex subunit 4 / Cytochrome b6-f complex iron-sulfur subunit, chloroplastic / Cytochrome f / Cytochrome b6-f complex subunit 8 / Cytochrome b6-f complex subunit 5 / Cytochrome b6-f complex subunit 7 / Cytochrome b6-f complex subunit 6
Similarity search - Component
Biological speciesSpinacia oleracea (spinach) / spinach (spinach)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsSarewicz M / Szwalec M / Indyka P / Rawski M / Pintscher S / Pietras R / Mielecki B / Jaciuk M / Glatt S / Osyczka A
Funding support Poland, 2 items
OrganizationGrant numberCountry
Foundation for Polish SciencePOIR.04.04.00-00-5B54/17-00 Poland
Foundation for Polish ScienceTEAM TECH CORE FACILITY/2017-4/6 Poland
CitationJournal: Sci Adv / Year: 2023
Title: High-resolution cryo-EM structures of plant cytochrome bf at work.
Authors: Marcin Sarewicz / Mateusz Szwalec / Sebastian Pintscher / Paulina Indyka / Michał Rawski / Rafał Pietras / Bohun Mielecki / Łukasz Koziej / Marcin Jaciuk / Sebastian Glatt / Artur Osyczka /
Abstract: Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome bf (Cyt bf) is known as one of the key steps of photosynthesis, but the ...Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome bf (Cyt bf) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Q) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt bf homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Q in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt bf in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.
History
DepositionJan 11, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateJan 25, 2023-
Current statusJan 25, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14123.png

Downloads & links

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Map

FileDownload / File: emd_14123.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.29
Minimum - Maximum-0.958162 - 2.2018747
Average (Standard dev.)0.00018867545 (±0.039735388)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 344.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cytochrome b6f complex

EntireName: Cytochrome b6f complex
Components
  • Complex: Cytochrome b6f complex
    • Protein or peptide: Cytochrome b6Cytochrome b
    • Protein or peptide: Cytochrome b6-f complex subunit 4
    • Protein or peptide: Cytochrome f
    • Protein or peptide: Cytochrome b6-f complex iron-sulfur subunit, chloroplastic
    • Protein or peptide: Cytochrome b6-f complex subunit 6
    • Protein or peptide: Cytochrome b6-f complex subunit 7
    • Protein or peptide: Cytochrome b6-f complex subunit 5
    • Protein or peptide: Cytochrome b6-f complex subunit 8
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: HEME C
  • Ligand: CHLOROPHYLL A
  • Ligand: 2,3-DIMETHYL-5-(3,7,11,15,19,23,27,31,35-NONAMETHYL-2,6,10,14,18,22,26,30,34-HEXATRIACONTANONAENYL-2,5-CYCLOHEXADIENE-1,4-DIONE-2,3-DIMETHYL-5-SOLANESYL-1,4-BENZOQUINONE
  • Ligand: UNDECYL-MALTOSIDE
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
  • Ligand: 1,2-DI-O-ACYL-3-O-[6-DEOXY-6-SULFO-ALPHA-D-GLUCOPYRANOSYL]-SN-GLYCEROL
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: BETA-CAROTENEΒ-Carotene

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Supramolecule #1: Cytochrome b6f complex

SupramoleculeName: Cytochrome b6f complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Spinacia oleracea (spinach) / Organ: Leaves / Organelle: Chloroplasts / Location in cell: thylakoids
Molecular weightTheoretical: 220 KDa

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Macromolecule #1: Cytochrome b6

MacromoleculeName: Cytochrome b6 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: spinach (spinach)
Molecular weightTheoretical: 24.186504 KDa
SequenceString: MSKVYDWFEE RLEIQAIADD ITSKYVPPHV NIFYCLGGIT LTCFLVQVAT GFAMTFYYRP TVTDAFASVQ YIMTEVNFGW LIRSVHRWS ASMMVLMMIL HVFRVYLTGG FKKPRELTWV TGVVLGVLTA SFGVTGYSLP WDQIGYWAVK IVTGVPDAIP V IGSPLVEL ...String:
MSKVYDWFEE RLEIQAIADD ITSKYVPPHV NIFYCLGGIT LTCFLVQVAT GFAMTFYYRP TVTDAFASVQ YIMTEVNFGW LIRSVHRWS ASMMVLMMIL HVFRVYLTGG FKKPRELTWV TGVVLGVLTA SFGVTGYSLP WDQIGYWAVK IVTGVPDAIP V IGSPLVEL LRGSASVGQS TLTRFYSLHT FVLPLLTAVF MLMHFLMIRK QGISGPL

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Macromolecule #2: Cytochrome b6-f complex subunit 4

MacromoleculeName: Cytochrome b6-f complex subunit 4 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: spinach (spinach)
Molecular weightTheoretical: 17.456662 KDa
SequenceString:
MGVTKKPDLN DPVLRAKLAK GMGHNYYGEP AWPNDLLYIF PVVILGTIAC NVGLAVLEPS MIGEPADPFA TPLEILPEWY FFPVFQILR TVPNKLLGVL LMASVPAGLL TVPFLENVNK FQNPFRRPVA TTVFLVGTVV ALWLGIGATL PIDKSLTLGL F

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Macromolecule #3: Cytochrome f

MacromoleculeName: Cytochrome f / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: spinach (spinach)
Molecular weightTheoretical: 35.355664 KDa
SequenceString: MQTINTFSWI KEQITRSISI SLILYIITRS SIANAYPIFA QQGYENPREA TGRIVCANCH LANKPVDIEV PQAVLPDTVF EAVVRIPYD MQLKQVLANG KKGGLNVGAV LILPEGFELA PPDRISPEMK EKMGNLSFQS YRPNKQNILV IGPVPGQKYS E ITFPILAP ...String:
MQTINTFSWI KEQITRSISI SLILYIITRS SIANAYPIFA QQGYENPREA TGRIVCANCH LANKPVDIEV PQAVLPDTVF EAVVRIPYD MQLKQVLANG KKGGLNVGAV LILPEGFELA PPDRISPEMK EKMGNLSFQS YRPNKQNILV IGPVPGQKYS E ITFPILAP DPATKKDVHF LKYPIYVGGN RGRGQIYPDG SKSNNTVYNS TATGIVKKIV RKEKGGYEIN IADASDGREV VD IIPRGPE LLVSEGESIK LDQPLTSNPN VGGFGQGDAE VVLQDPLRIQ GLLFFFASVI LAQIFLVLKK KQFEKVQLSE MNF

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Macromolecule #4: Cytochrome b6-f complex iron-sulfur subunit, chloroplastic

MacromoleculeName: Cytochrome b6-f complex iron-sulfur subunit, chloroplastic
type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO / EC number: plastoquinol-plastocyanin reductase
Source (natural)Organism: spinach (spinach)
Molecular weightTheoretical: 24.347898 KDa
SequenceString: MASFTLSSAT PSQLCSSKNG MFAPSLALAK AGRVNVLISK ERIRGMKLTC QATSIPADNV PDMQKRETLN LLLLGALSLP TGYMLLPYA SFFVPPGGGA GTGGTIAKDA LGNDVIAAEW LKTHAPGDRT LTQGLKGDPT YLVVESDKTL ATFGINAVCT H LGCVVPFN ...String:
MASFTLSSAT PSQLCSSKNG MFAPSLALAK AGRVNVLISK ERIRGMKLTC QATSIPADNV PDMQKRETLN LLLLGALSLP TGYMLLPYA SFFVPPGGGA GTGGTIAKDA LGNDVIAAEW LKTHAPGDRT LTQGLKGDPT YLVVESDKTL ATFGINAVCT H LGCVVPFN AAENKFICPC HGSQYNNQGR VVRGPAPLSL ALAHCDVDDG KVVFVPWTET DFRTGEAPWW SA

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Macromolecule #5: Cytochrome b6-f complex subunit 6

MacromoleculeName: Cytochrome b6-f complex subunit 6 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: spinach (spinach)
Molecular weightTheoretical: 3.4522 KDa
SequenceString:
MFTLTSYFGF LLAALTITSA LFIGLNKIRL I

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Macromolecule #6: Cytochrome b6-f complex subunit 7

MacromoleculeName: Cytochrome b6-f complex subunit 7 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: spinach (spinach)
Molecular weightTheoretical: 3.774431 KDa
SequenceString:
NAAAEIFRIA AVMNGLTLVG VAIGFVLLRI EATVEE

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Macromolecule #7: Cytochrome b6-f complex subunit 5

MacromoleculeName: Cytochrome b6-f complex subunit 5 / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: spinach (spinach)
Molecular weightTheoretical: 4.171985 KDa
SequenceString:
MIEVFLFGIV LGLIPITLAG LFVTAYLQYR RGDQLDL

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Macromolecule #8: Cytochrome b6-f complex subunit 8

MacromoleculeName: Cytochrome b6-f complex subunit 8 / type: protein_or_peptide / ID: 8 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: spinach (spinach)
Molecular weightTheoretical: 3.170809 KDa
SequenceString:
MDIVSLAWAA LMVVFTFSLS LVVWGRSGL

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Macromolecule #9: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 9 / Number of copies: 4 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

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Macromolecule #10: HEME C

MacromoleculeName: HEME C / type: ligand / ID: 10 / Number of copies: 4 / Formula: HEC
Molecular weightTheoretical: 618.503 Da
Chemical component information

ChemComp-HEC:
HEME C / Heme C

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Macromolecule #11: CHLOROPHYLL A

MacromoleculeName: CHLOROPHYLL A / type: ligand / ID: 11 / Number of copies: 2 / Formula: CLA
Molecular weightTheoretical: 893.489 Da
Chemical component information

ChemComp-CLA:
CHLOROPHYLL A / Chlorophyll a

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Macromolecule #12: 2,3-DIMETHYL-5-(3,7,11,15,19,23,27,31,35-NONAMETHYL-2,6,10,14,18,...

MacromoleculeName: 2,3-DIMETHYL-5-(3,7,11,15,19,23,27,31,35-NONAMETHYL-2,6,10,14,18,22,26,30,34-HEXATRIACONTANONAENYL-2,5-CYCLOHEXADIENE-1,4-DIONE-2,3-DIMETHYL-5-SOLANESYL-1,4-BENZOQUINONE
type: ligand / ID: 12 / Number of copies: 7 / Formula: PL9
Molecular weightTheoretical: 749.201 Da
Chemical component information

ChemComp-PL9:
2,3-DIMETHYL-5-(3,7,11,15,19,23,27,31,35-NONAMETHYL-2,6,10,14,18,22,26,30,34-HEXATRIACONTANONAENYL-2,5-CYCLOHEXADIENE-1,4-DIONE-2,3-DIMETHYL-5-SOLANESYL-1,4-BENZOQUINONE / Plastoquinone

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Macromolecule #13: UNDECYL-MALTOSIDE

MacromoleculeName: UNDECYL-MALTOSIDE / type: ligand / ID: 13 / Number of copies: 10 / Formula: UMQ
Molecular weightTheoretical: 496.589 Da
Chemical component information

ChemComp-UMQ:
UNDECYL-MALTOSIDE / detergent*YM

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Macromolecule #14: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
type: ligand / ID: 14 / Number of copies: 4 / Formula: PGT
Molecular weightTheoretical: 751.023 Da
Chemical component information

ChemComp-PGT:
(1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / phospholipid*YM / Phosphatidylglycerol

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Macromolecule #15: 1,2-DI-O-ACYL-3-O-[6-DEOXY-6-SULFO-ALPHA-D-GLUCOPYRANOSYL]-SN-GLYCEROL

MacromoleculeName: 1,2-DI-O-ACYL-3-O-[6-DEOXY-6-SULFO-ALPHA-D-GLUCOPYRANOSYL]-SN-GLYCEROL
type: ligand / ID: 15 / Number of copies: 2 / Formula: SQD
Molecular weightTheoretical: 795.116 Da
Chemical component information

ChemComp-SQD:
1,2-DI-O-ACYL-3-O-[6-DEOXY-6-SULFO-ALPHA-D-GLUCOPYRANOSYL]-SN-GLYCEROL

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Macromolecule #16: FE2/S2 (INORGANIC) CLUSTER

MacromoleculeName: FE2/S2 (INORGANIC) CLUSTER / type: ligand / ID: 16 / Number of copies: 2 / Formula: FES
Molecular weightTheoretical: 175.82 Da
Chemical component information

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER / Iron–sulfur cluster

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Macromolecule #17: BETA-CAROTENE

MacromoleculeName: BETA-CAROTENE / type: ligand / ID: 17 / Number of copies: 2 / Formula: BCR
Molecular weightTheoretical: 536.873 Da
Chemical component information

ChemComp-BCR:
BETA-CAROTENE / Β-Carotene

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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

Concentration4.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mM2-Amino-2-(hydroxymethyl)propane-1,3-diolTris
15.0 mMNaClSodium chloridesodium chloride
0.7 mM(2R,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-4,5-dihydroxy-2-(hydroxymethyl)-6-undecoxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triolundecyl maltoside
2.6 mM(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-octoxyoxane-3,4,5-trioloctyl glucoside
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 7784 / Average exposure time: 1.82 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 177412
Details: given number of particles is after template picker and 2D cleaning
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6rqf

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.1) / Software - details: Ab-Initio Reconstruction / Details: Ab-Initio Reconstruction
Final 3D classificationNumber classes: 2 / Avg.num./class: 80000 / Software - Name: cryoSPARC (ver. v3.3.1) / Software - details: Heterogeneous Refinement
Details: asymmetric division of particles, 97k vs 43k, with a structural difference, refinement box 200
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v3.3.1) / Software - details: Heterogeneous Refinement
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.1) / Software - details: Non-Uniform Refinement / Details: Batchsize snrfactor tuned to 300 / Number images used: 97597
Details20 eV slit, fully tuned before the experiment
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7qrm:
Cryo-EM structure of catalytically active Spinacia oleracea cytochrome b6f in complex with endogenous plastoquinones at 2.7 A resolution

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