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Yorodumi- EMDB-27702: Focused map (monomer B) for Arabidopsis SPY in complex with GDP-fucose -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-27702 | |||||||||
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Title | Focused map (monomer B) for Arabidopsis SPY in complex with GDP-fucose | |||||||||
Map data | ||||||||||
Sample |
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Keywords | O-Fucosyltransferase / PLANT PROTEIN | |||||||||
Biological species | Arabidopsis thaliana (thale cress) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Kumar S / Zhou Y / Lucas D / Borgnia MJ / Bartesaghi A / Zhou P | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nat Commun / Year: 2023 Title: Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY. Authors: Shivesh Kumar / Yan Wang / Ye Zhou / Lucas Dillard / Fay-Wei Li / Carly A Sciandra / Ning Sui / Rodolfo Zentella / Emily Zahn / Jeffrey Shabanowitz / Donald F Hunt / Mario J Borgnia / ...Authors: Shivesh Kumar / Yan Wang / Ye Zhou / Lucas Dillard / Fay-Wei Li / Carly A Sciandra / Ning Sui / Rodolfo Zentella / Emily Zahn / Jeffrey Shabanowitz / Donald F Hunt / Mario J Borgnia / Alberto Bartesaghi / Tai-Ping Sun / Pei Zhou / Abstract: SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is ...SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is distinct from ER-localized POFUTs and contains N-terminal tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain resembling the O-linked-N-acetylglucosamine (GlcNAc) transferases (OGTs). However, the structural feature that determines the distinct enzymatic selectivity of SPY remains unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of SPY and its complex with GDP-fucose, revealing distinct active-site features enabling GDP-fucose instead of UDP-GlcNAc binding. SPY forms an antiparallel dimer instead of the X-shaped dimer in human OGT, and its catalytic domain interconverts among multiple conformations. Analysis of mass spectrometry, co-IP, fucosylation activity, and cryo-EM data further demonstrates that the N-terminal disordered peptide in SPY contains trans auto-fucosylation sites and inhibits the POFUT activity, whereas TPRs 1-5 dynamically regulate SPY activity by interfering with protein substrate binding. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_27702.map.gz | 118.2 MB | EMDB map data format | |
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Header (meta data) | emd-27702-v30.xml emd-27702.xml | 15.8 KB 15.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_27702_fsc.xml | 10.5 KB | Display | FSC data file |
Images | emd_27702.png | 47.2 KB | ||
Masks | emd_27702_msk_1.map | 125 MB | Mask map | |
Others | emd_27702_half_map_1.map.gz emd_27702_half_map_2.map.gz | 115.9 MB 115.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-27702 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-27702 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_27702.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_27702_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_27702_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_27702_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : SPY GPD-complex
Entire | Name: SPY GPD-complex |
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Components |
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-Supramolecule #1: SPY GPD-complex
Supramolecule | Name: SPY GPD-complex / type: cell / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.8 mg/mL | ||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 40 / Pretreatment - Type: PLASMA CLEANING | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 289.15 K / Instrument: LEICA EM GP |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000 |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average electron dose: 52.4 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |