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- EMDB-32571: Cryo-EM structure of GH31 alpha-1,3-glucosidase from Lactococcus ... -

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Basic information

Entry
Database: EMDB / ID: EMD-32571
TitleCryo-EM structure of GH31 alpha-1,3-glucosidase from Lactococcus lactis subsp. cremoris
Map data
Sample
  • Complex: LlGH31_u1 hexamer
    • Protein or peptide: LlGH31_u1
Function / homology
Function and homology information


Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / hydrolase activity, hydrolyzing O-glycosyl compounds / carbohydrate metabolic process
Similarity search - Function
: / Glycosyl hydrolase family 31 C-terminal domain / Glycoside hydrolase family 31 / Glycosyl hydrolases family 31 TIM-barrel domain / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Biological speciesLactococcus lactis subsp. cremoris MG1363 (lactic acid bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsIkegaya M / Moriya T / Adachi N / Kawasaki M / Park EY / Miyazaki T
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21am0101071 Japan
CitationJournal: J Biol Chem / Year: 2022
Title: Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides.
Authors: Marina Ikegaya / Toshio Moriya / Naruhiko Adachi / Masato Kawasaki / Enoch Y Park / Takatsugu Miyazaki /
Abstract: Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is ...Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The k/K values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.
History
DepositionJan 12, 2022-
Header (metadata) releaseMar 30, 2022-
Map releaseMar 30, 2022-
UpdateMay 11, 2022-
Current statusMay 11, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32571.png

Downloads & links

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Map

FileDownload / File: emd_32571.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.11023122 - 0.22144265
Average (Standard dev.)-0.00012851362 (±0.0056734174)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 450.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32571_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_32571_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32571_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : LlGH31_u1 hexamer

EntireName: LlGH31_u1 hexamer
Components
  • Complex: LlGH31_u1 hexamer
    • Protein or peptide: LlGH31_u1

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Supramolecule #1: LlGH31_u1 hexamer

SupramoleculeName: LlGH31_u1 hexamer / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all / Details: homo hexamer
Source (natural)Organism: Lactococcus lactis subsp. cremoris MG1363 (lactic acid bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET28a
Molecular weightTheoretical: 516 KDa

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Macromolecule #1: LlGH31_u1

MacromoleculeName: LlGH31_u1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ec: 3.2.1.84
Source (natural)Organism: Lactococcus lactis subsp. cremoris MG1363 (lactic acid bacteria)
Strain: MG1363
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MVSELESKYM NNNIIKFDKA RFTVLTEHLI RIEYSETGEF EERMTQMVQN REFSEVNFDI IEKEETIEII TSTVHLYYNG GEFTNASLFA DVKFNFSVYS NRWYFGEKSD GNLKGTTRTL DMIDGECPLE DGIMSKNGFA VLADKGKVLT ...String:
MGSSHHHHHH SSGLVPRGSH MVSELESKYM NNNIIKFDKA RFTVLTEHLI RIEYSETGEF EERMTQMVQN REFSEVNFDI IEKEETIEII TSTVHLYYNG GEFTNASLFA DVKFNFSVYS NRWYFGEKSD GNLKGTTRTL DMIDGECPLE DGIMSKNGFA VLADKGKVLT EVGDIAGNSV STIDLYLFAY GRDYRQALKD FYQLTGNTPK LPRFALGNWW SRYYDYSDKS YLALMDKFTD KKVPLSVSVI DMDWHKVSEV PSRFGSGWTG YSWNKKLFPN PENFIDELHQ RKLKVTLNDH PADGIRAFED PYPQVAQTLD LNTELEEAAK FDFDNLKFRK AYFEEVHGPL EKEGVDFWWI DWQQGAISKS GVDPLWLLNH YQYQNAQKKH KNNIILSRYA GPGSHRYPLG FSGDSVISWA SLDFQPYFTS TASNIGYTWW SHDIGGHMQG YKDAELSLRW LQFGVFSPIN RLHSSKSEFT SKEPWHFDAV IEQSMIDFLQ LRHQLIPYLY SANLITASEG RALVEPLYYE YPMEEEAYQH RNQYLFGEQL MVAPITEKMN SLLQMGSVEV WFPEGTWYDF FSGQPYDGKV SLKVYREITE MPVFAKAGAI IPLDKNPLKK EEIPSEIIWK IFPGADGEYL LLEEDNETKA EFVNGIFTVT SKKESSRKHT IIYGEHEIVS AKRGEFSIDL NGKEENFDWN FSTALFRRLD IAEISYEQKD EILQQLSLIE EHEKQVAFIK TNENQELQNS LFELLYSGK

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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

Concentration0.58 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
300.0 mMNaClSodium chloridesodium chloride
20.0 mMNaH2PO4sodium dihydrogen phosphate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: The grid was washed by acetone prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 5 seconds (blot force 15).
DetailsThis sample was mono-disperse.

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 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: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 120000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 995 / Average exposure time: 65.95 sec. / Average electron dose: 50.0 e/Å2

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Image processing

Particle selectionNumber selected: 267294
CTF correctionSoftware - Name: Gctf (ver. 1.18) / Software - details: GCTF_v1.18_sm30-7a5_cu10.1
Startup modelType of model: OTHER
Details: An ab initio model was generated using RELION3.1.2's own implementation of Stochastic Gradient Descent (SGD) algorithm and low-pass filtered to 30 A for use as an initial model for 3D classification.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.2)
Final 3D classificationNumber classes: 2 / Avg.num./class: 31669 / Software - Name: RELION (ver. 3.1.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.2)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.2) / Number images used: 44606
FSC plot (resolution estimation)

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