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- EMDB-18157: Beta-galactosidase from Bacillus circulans conformational state 2 -

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Open data


ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-18157
TitleBeta-galactosidase from Bacillus circulans conformational state 2
Map data
Sample
  • Complex: Terciary complex of beta-galactosidase isoform A
    • Protein or peptide: beta-galactosidase
KeywordsGalactosidase / Transgalactosylation / Izoform A / Enzyme / HYDROLASE
Function / homology
Function and homology information


beta-galactosidase activity / carbohydrate metabolic process
Similarity search - Function
: / Beta-galactosidase-like, Galactose-binding domain / Bacterial Ig-like domain / Bacterial Ig-like domain (group 4) / Glycoside hydrolase family 2, domain 5 / Glycoside hydrolase family 2 C-terminal domain 5 / Domain of unknown function DUF4982 / Domain of unknown function (DUF4982) / : / Invasin/intimin cell-adhesion fragments ...: / Beta-galactosidase-like, Galactose-binding domain / Bacterial Ig-like domain / Bacterial Ig-like domain (group 4) / Glycoside hydrolase family 2, domain 5 / Glycoside hydrolase family 2 C-terminal domain 5 / Domain of unknown function DUF4982 / Domain of unknown function (DUF4982) / : / Invasin/intimin cell-adhesion fragments / Glycoside hydrolase, family 2 / Glycosyl hydrolases family 2, sugar binding domain / Glycoside hydrolase family 2, catalytic domain / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2, TIM barrel domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycosyl hydrolases family 2 / Beta-Galactosidase/glucuronidase domain superfamily / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesNiallia circulans subsp. circulans (bacteria) / Niallia circulans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsKascakova B / Hovorkova M / Petraskova L / Novacek J / Pinkas D / Gardian Z / Kren V / Bojarova P / Kuta Smatanova I
Funding support Czech Republic, 1 items
OrganizationGrant numberCountry
Grant Agency of the Czech Republic Czech Republic
CitationJournal: Structure / Year: 2024
Title: The variable structural flexibility of the Bacillus circulans β-galactosidase isoforms determines their unique functionalities.
Authors: Michaela Hovorková / Barbora Kaščáková / Lucie Petrásková / Petra Havlíčková / Jiří Nováček / Daniel Pinkas / Zdenko Gardian / Vladimír Křen / Pavla Bojarová / Ivana Kutá Smatanová /
Abstract: β-Galactosidase from Bacillus circulans ATCC 31382 (BgaD) is a biotechnologically important enzyme for the synthesis of β-galactooligosaccharides (GOS). Among its four isoforms, isoform A (BgaD-A) ...β-Galactosidase from Bacillus circulans ATCC 31382 (BgaD) is a biotechnologically important enzyme for the synthesis of β-galactooligosaccharides (GOS). Among its four isoforms, isoform A (BgaD-A) has distinct synthetic properties. Here, we present cryoelectron microscopy (cryo-EM) structures of BgaD-A and compare them with the known X-ray crystal structure of isoform D (BgaD-D), revealing substantial structural divergences between the two isoforms. In contrast to BgaD-D, BgaD-A features a flexible Big-4 domain and another enigmatic domain. The newly identified flexible region in BgaD-A is termed as "barrier domain 8," and serves as a barricade, obstructing the access of longer oligosaccharide substrates into the active site of BgaD-A. The transgalactosylation reactions catalyzed by both isoforms revealed that BgaD-A has a higher selectivity than BgaD-D in the earlier stages of the reaction and is prevailingly directed to shorter galactooligosaccharides. This study improves our understanding of the structural determinants governing β-galactosidase catalysis, with implications for tailored GOS production.
History
DepositionAug 7, 2023-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: PDBe / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_18157.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.818 Å
Density
Contour LevelBy AUTHOR: 0.143
Minimum - Maximum-1.1245815 - 1.8656473
Average (Standard dev.)-0.00079199334 (±0.043779712)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 261.76 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Terciary complex of beta-galactosidase isoform A

EntireName: Terciary complex of beta-galactosidase isoform A
Components
  • Complex: Terciary complex of beta-galactosidase isoform A
    • Protein or peptide: beta-galactosidase

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Supramolecule #1: Terciary complex of beta-galactosidase isoform A

SupramoleculeName: Terciary complex of beta-galactosidase isoform A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Niallia circulans subsp. circulans (bacteria)

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Macromolecule #1: beta-galactosidase

MacromoleculeName: beta-galactosidase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Niallia circulans (bacteria)
Molecular weightTheoretical: 192.584391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKAISCVFL ISALILSSFQ VPVQGQAMSK TTSAAGNSVS YDGERRVNFN ENWRFQRETN GSIAGAQNPG FDDSSWRKLN LPHDWSIEL DFNKNSLATH EGGYLDGGIG WYRKTFTIPE SMKGKRISLD FDGVYMNSTT YLNGEVLGTY PFGYNAFSYD I SDKLYKDG ...String:
MKKAISCVFL ISALILSSFQ VPVQGQAMSK TTSAAGNSVS YDGERRVNFN ENWRFQRETN GSIAGAQNPG FDDSSWRKLN LPHDWSIEL DFNKNSLATH EGGYLDGGIG WYRKTFTIPE SMKGKRISLD FDGVYMNSTT YLNGEVLGTY PFGYNAFSYD I SDKLYKDG RANVLVVKVN NTQPSSRWYS GSGIYRNVYL TVTDPIHVAR YGTFVTTPNL EKSIKEDRAD VNIKTKISND AA EAKQVKI KSTIYDGAGN TVQTVETEEK TAAAGTVTPF EQNTVIKQPK LWSIDKPYRY NLVTEVIVGG QTVDTYETKF GVR YFKFDE NEGFSLNGEY MKLHGVSMHH DLGALGAATN ARGVERQMQI MKDMGVNAIR VTHNPASPEL LEAANKLGLF IIEE AFDSW AQSKKPYDYG RFFNAWAEHD IKEMVDRGKN EPAIIMWSIG NEIYDTTNAA GVETARNLVG WVKEIDTTRP TTIGE DKTR GDKVNVTPIN SYIKEIFNIV DVVGLNYSEN NYDGYHKQNP SWKLYGSETS SATRSRGVYT HPYQYNQSTK YADLQQ SSY DNDYVGWGRT AEDAWKYDRD LKHIAGQFIW TGFDYIGEPT PYYNSYPAKS SYFGAVDTAG FPKDIFYYYQ SQWKKEP MV HLLPHWNWKE GEKVRVLAYT NASKVELVLN GESLGEKNYD NKQTSWGAPY KETKDGKTYL EWAVPFKPGK LEAVAKDE N GKVIARDQVV TAGEPASVRL TADRKVVKAD GTDLSFITAD IVDSKGIVVP DADHLITFNV TGQGELAGVD NGNASSVER YKDNKRKAFS GKALAIVQSS KLSGKITVHA SVAGLSSDST SVFTVTPADH DKKIVAGIDD VNLTVDVNEA PKLPSEIKVY YSDESAAAK NVTWDEVDPK QYSTVGEFTV EGSVEGTSLK AKAFVIVKGI VAVKPYSTAT KVGVQPVLPE KATLLYSDGT T KGATVTWD EIPEDKLAKE GRFTVEGSVE GTDLKANVYV RVTNEVKSVN IMLQEQGSAY PKLEATFTNP ADNLQHLNDG IK SYTNNPV NRWTNWTRTP RDAGDSITVN FGKKHVINNL DLFVFTDSGT VVPEKAEVQY WDGTAWKDVE NLTQPSPYVV EKN ELTFDA VATEKLKFHL TPSVKGKFLA LTEAEVYADQ IVMGETAKLQ SITVNGKALE GFDHAKKNYE LVLPYGSELP KIEA AAADN ATVTILPAFS YPGTAKLFVT SEDGKVTTEY SIGVSTEEPK LVSAELSADK TNVMEDDIID LKVIGLFESK EKIDV TDSQ PTYEFDQQII KIEGNKLYAL ETGNVKVKVT VTYKGVSVTT PALEFTIAKN PAPKYITSLE PVTVVVKKGE APELPA TVV AHYNRGIPRD VKVKWERINP SKYQQLGEFT VSGMVEGTDI KAQAKVIVKG AVAVEDIRMA VLLKQMPQLP GKVTVYY SD GAEEQRAVKW EEIPQEELEN VGEFKVKGDV NGVKLKATAT IRVTDEVGGE QNISRAKNGY EYPKAEASFT NNGPGSSD R IEAINDDVIS YEANPHNRWT NWQPVPRAGD WVSITFGDYE PTEYDVDSME IHWFADHGTS YPERFQIEYK SGDSWKEVT SLKSDPASPA LGKANVYSFD RVKTSAIRVK MTAQAGKSLA ITELKVFSKW PKAGTEPEVT DIKVGGKSIL EDFEQKGDHY EVTIDAGDA NVMPKINVKA KDQTSITIVP AVTSPSTAKV IAKSEDGKKV KVYSIHYK

UniProtKB: Beta galactosidase

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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

Concentration0.18 mg/mL
BufferpH: 6 / Component - Concentration: 0.18 mg/ml / Component - Name: sodium phosphate / Details: 50 mM sodium phosphate buffer pH 6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average exposure time: 5.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 265100
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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