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- EMDB-27655: Intermediate resolution structure of barley (1,3;1,4)-beta-glucan... -

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Basic information

Entry
Database: EMDB / ID: EMD-27655
TitleIntermediate resolution structure of barley (1,3;1,4)-beta-glucan synthase CslF6.
Map data
Sample
  • Organelle or cellular component: Barley cellulose synthase-like F6
    • Protein or peptide: Cellulose synthase-like CslF6
Keywordsglucan / glycosyltransferase / cellulose / barley / TRANSFERASE
Function / homologyplant-type cell wall organization or biogenesis / Cellulose synthase / Cellulose synthase / cellulose synthase (UDP-forming) activity / cellulose biosynthetic process / Nucleotide-diphospho-sugar transferases / membrane => GO:0016020 / Cellulose synthase-like CslF6
Function and homology information
Biological speciesHordeum vulgare (barley)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsHo R / Purushotham P / Zimmer J
Funding support United States, 1 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)CE1101007 United States
CitationJournal: Sci Adv / Year: 2022
Title: Mechanism of mixed-linkage glucan biosynthesis by barley cellulose synthase-like CslF6 (1,3;1,4)-β-glucan synthase.
Authors: Pallinti Purushotham / Ruoya Ho / Long Yu / Geoffrey B Fincher / Vincent Bulone / Jochen Zimmer /
Abstract: Mixed-linkage (1,3;1,4)-β-glucans, which are widely distributed in cell walls of the grasses, are linear glucose polymers containing predominantly (1,4)-β-linked glucosyl units interspersed with ...Mixed-linkage (1,3;1,4)-β-glucans, which are widely distributed in cell walls of the grasses, are linear glucose polymers containing predominantly (1,4)-β-linked glucosyl units interspersed with single (1,3)-β-linked glucosyl units. Their distribution in cereal grains and unique structures are important determinants of dietary fibers that are beneficial to human health. We demonstrate that the barley cellulose synthase-like CslF6 enzyme is sufficient to synthesize a high-molecular weight (1,3;1,4)-β-glucan in vitro. Biochemical and cryo-electron microscopy analyses suggest that CslF6 functions as a monomer. A conserved "switch motif" at the entrance of the enzyme's transmembrane channel is critical to generate (1,3)-linkages. There, a single-point mutation markedly reduces (1,3)-linkage formation, resulting in the synthesis of cellulosic polysaccharides. Our results suggest that CslF6 monitors the orientation of the nascent polysaccharide's second or third glucosyl unit. Register-dependent interactions with these glucosyl residues reposition the polymer's terminal glucosyl unit to form either a (1,3)- or (1,4)-β-linkage.
History
DepositionJul 19, 2022-
Header (metadata) releaseNov 30, 2022-
Map releaseNov 30, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_27655.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 6.453
Minimum - Maximum-15.062754999999999 - 34.456659999999999
Average (Standard dev.)-0.000000000002949 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27655_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_27655_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Barley cellulose synthase-like F6

EntireName: Barley cellulose synthase-like F6
Components
  • Organelle or cellular component: Barley cellulose synthase-like F6
    • Protein or peptide: Cellulose synthase-like CslF6

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Supramolecule #1: Barley cellulose synthase-like F6

SupramoleculeName: Barley cellulose synthase-like F6 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Hordeum vulgare (barley)

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Macromolecule #1: Cellulose synthase-like CslF6

MacromoleculeName: Cellulose synthase-like CslF6 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Hordeum vulgare (barley)
Molecular weightTheoretical: 105.193797 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAPAVAGGGR VRSNEPVAAA AAAPAASGKP CVCGFQVCAC TGSAAVASAA SSLDMDIVAM GQIGAVNDES WVGVELGEDG ETDESGAAV DDRPVFRTEK IKGVLLHPYR VLIFVRLIAF TLFVIWRISH KNPDAMWLWV TSICGEFWFG FSWLLDQLPK L NPINRVPD ...String:
MAPAVAGGGR VRSNEPVAAA AAAPAASGKP CVCGFQVCAC TGSAAVASAA SSLDMDIVAM GQIGAVNDES WVGVELGEDG ETDESGAAV DDRPVFRTEK IKGVLLHPYR VLIFVRLIAF TLFVIWRISH KNPDAMWLWV TSICGEFWFG FSWLLDQLPK L NPINRVPD LAVLRQRFDR PDGTSTLPGL DIFVTTADPI KEPILSTANS VLSILAADYP VDRNTCYVSD DSGMLLTYEA LA ESSKFAT LWVPFCRKHG IEPRGPESYF ELKSHPYMGR AQDEFVNDRR RVRKEYDEFK ARINSLEHDI KQRNDGYNAA IAH SQGVPR PTWMADGTQW EGTWVDASEN HRRGDHAGIV LVLLNHPSHR RQTGPPASAD NPLDLSGVDV RLPMLVYVSR EKRP GHDHQ KKAGAMNALT RASALLSNSP FILNLDCDHY INNSQALRAG ICFMVGRDSD TVAFVQFPQR FEGVDPTDLY ANHNR IFFD GTLRALDGMQ GPIYVGTGCL FRRITVYGFD PPRINVGGPC FPRLAGLFAK TKYEKPGLEM TTAKAKAAPV PAKGKH GFL PLPKKTYGKS DAFVDTIPRA SHPSPYAAAA EGIVADEATI VEAVNVTAAA FEKKTGWGKE IGWVYDTVTE DVVTGYR MH IKGWRSRYCS IYPHAFIGTA PINLTERLFQ VLRWSTGSLE IFFSKNNPLF GSTYLHPLQR VAYINITTYP FTAIFLIF Y TTVPALSFVT GHFIVQRPTT MFYVYLGIVL STLLVIAVLE VKWAGVTVFE WFRNGQFWMT ASCSAYLAAV CQVLTKVIF RRDISFKLTS KLPSGDEKKD PYADLYVVRW TPLMITPIII IFVNIIGSAV AFAKVLDGEW THWLKVAGGV FFNFWVLFHL YPFAKGILG KHGKTPVVVL VWWAFTFVIT AVLYINIPHM HTSGGKHTTV HGHHGKKLVD TGLYGWLH

UniProtKB: Cellulose synthase-like CslF6

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: AlphaFold2 predicted structure
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 80347
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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