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- EMDB-27701: Focused map (monomer A) for Arabidopsis SPY in complex with GDP-fucose -

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Basic information

Entry
Database: EMDB / ID: EMD-27701
TitleFocused map (monomer A) for Arabidopsis SPY in complex with GDP-fucose
Map data
Sample
  • Complex: SPY GDP-fucose complex
    • Protein or peptide: SPY GDP-fucose complex
KeywordsO-Fucosyltransferase / PLANT PROTEIN
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKumar S / Zhou Y / Dillard L / Borgnia MJ / Bartesaghi A / Zhou P
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: 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
DepositionJul 25, 2022-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateJun 7, 2023-
Current statusJun 7, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27701.png

Downloads & links

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Map

FileDownload / File: emd_27701.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 320 pix.
= 345.6 Å		1.08 Å/pix.
x 320 pix.
= 345.6 Å		1.08 Å/pix.
x 320 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.56
Minimum - Maximum-6.078192 - 11.843108000000001
Average (Standard dev.)-0.0002246 (±0.051259764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27701_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27701_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27701_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SPY GDP-fucose complex

EntireName: SPY GDP-fucose complex
Components
  • Complex: SPY GDP-fucose complex
    • Protein or peptide: SPY GDP-fucose complex

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Supramolecule #1: SPY GDP-fucose complex

SupramoleculeName: SPY GDP-fucose complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: SPY GDP-fucose complex

MacromoleculeName: SPY GDP-fucose complex / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
SequenceString: MVGLEDDTER ERSPVVENGF SNGSRSSSSS AGVLSPSRKV TQGNDTLSYA NILRARNKFA DALALYEAML EKDSKNVEAH IGKGICLQTQ NKGNLAFDCF SEAIRLDPHN ACALTHCGIL HKEEGRLVEA AESYQKALMA DASYKPAAEC LAIVLTDLGT SLKLAGNTQE ...String:
MVGLEDDTER ERSPVVENGF SNGSRSSSSS AGVLSPSRKV TQGNDTLSYA NILRARNKFA DALALYEAML EKDSKNVEAH IGKGICLQTQ NKGNLAFDCF SEAIRLDPHN ACALTHCGIL HKEEGRLVEA AESYQKALMA DASYKPAAEC LAIVLTDLGT SLKLAGNTQE GIQKYYEALK IDPHYAPAYY NLGVVYSEMM QYDNALSCYE KAALERPMYA EAYCNMGVIY KNRGDLEMAI TCYERCLAVS PNFEIAKNNM AIALTDLGTK VKLEGDVTQG VAYYKKALYY NWHYADAMYN LGVAYGEMLK FDMAIVFYEL AFHFNPHCAE ACNNLGVLYK DRDNLDKAVE CYQMALSIKP NFAQSLNNLG VVYTVQGKMD AAASMIEKAI LANPTYAEAF NNLGVLYRDA GNITMAIDAY EECLKIDPDS RNAGQNRLLA MNYINEGLDD KLFEAHRDWG WRFTRLHPQY TSWDNLKDPE RPITIGYISP DFFTHSVSYF IEAPLTHHDY TKYKVVVYSA VVKADAKTYR FRDKVLKKGG VWKDIYGIDE KKIASMVRED KIDILVELTG HTANNKLGTM ACRPAPVQVT WIGYPNTTGL PTVDYRITDS LADPPDTKQK QVEELVRLPD CFLCYTPSPE AGPVCPTPAL SNGFVTFGSF NNLAKITPKV LQVWARILCA VPNSRLVVKC KPFCCDSIRQ RFLTTLEQLG LESKRVDLLP LILFNHDHMQ AYSLMDISLD TFPYAGTTTT CESLYMGVPC VTMAGSVHAH NVGVSLLTKV GLGHLVAKNE DEYVQLSVDL ASDVTALSKL RMSLRDLMAG SPVCNGPSFA VGLESAYRNM WKKYCKGEVP SLRRMEMLQK EVHDDPLISK DLGPSRVSVT GEATPSLKAN GSAPVPSSLP TQSPQLSKRM DSTSGGSENL YFQGGSHHHH HHHHHHGGWS HPQFEK

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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

Concentration0.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloride
2.0 mMC4H10O2S21,4-dithiothreitol
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 289.15 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average electron dose: 52.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 182947
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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