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- EMDB-10109: Structure of beta-Galactosidase from Thermotoga maritima -

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Basic information

Entry
Database: EMDB / ID: EMD-10109
TitleStructure of beta-Galactosidase from Thermotoga maritima
Map dataMap from Relion3 Refine3D after postprocessing using the deposited mask and automatic filtering/sharpening, and sharpened with LocScale
Sample
  • Complex: Map of beta-Galactosidase from Thermotoga maritima
    • Protein or peptide: Beta-galactosidase
  • Ligand: MAGNESIUM ION
  • Ligand: water
Function / homology
Function and homology information


lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding
Similarity search - Function
Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site / Glycosyl hydrolases family 2 signature 1. / Glycoside hydrolase, family 2 ...Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site / Glycosyl hydrolases family 2 signature 1. / Glycoside hydrolase, family 2 / Glycoside hydrolase family 2, catalytic domain / Glycosyl hydrolases family 2, TIM barrel domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2 / Glycosyl hydrolases family 2, sugar binding domain / Beta-Galactosidase/glucuronidase domain superfamily / Glycoside hydrolase-type carbohydrate-binding / Galactose mutarotase-like domain superfamily / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesThermotoga maritima MSB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.0 Å
AuthorsMiguez-Amil S / Jimenez-Ortega E / Ramirez Escudero M / Sanz-Aparicio J / Fernandez-Leiro R
CitationJournal: ACS Chem Biol / Year: 2020
Title: The cryo-EM Structure of β-Galactosidase: Quaternary Structure Guides Protein Engineering.
Authors: Samuel Míguez Amil / Elena Jiménez-Ortega / Mercedes Ramírez-Escudero / David Talens-Perales / Julia Marín-Navarro / Julio Polaina / Julia Sanz-Aparicio / Rafael Fernandez-Leiro /
Abstract: Lactose intolerance is a common digestive disorder that affects a large proportion of the adult human population. The severity of the symptoms is highly variable, depending on the susceptibility to ...Lactose intolerance is a common digestive disorder that affects a large proportion of the adult human population. The severity of the symptoms is highly variable, depending on the susceptibility to the sugar and the amount digested. For that reason, enzymes that can be used for the production of lactose-free milk and milk derivatives have acquired singular biotechnological importance. One such case is β-galactosidase (TmLac). Here, we report the cryo-EM structure of TmLac at 2.0 Å resolution. The protein features a newly solved domain at its C-terminus, characteristic of the genus , which promotes a peculiar octameric arrangement. We have assessed the constraints imposed by the quaternary protein structure on the construction of hybrid versions of this GH2 enzyme. Carbohydrate binding modules (CBM) from the CBM2 and CBM9 families have been added at either the amino or carboxy terminus, and the structural and functional effects of such modifications have been analyzed. The results provide a basis for the rational design of hybrid enzymes that can be efficiently attached to different solid supports.
History
DepositionJul 4, 2019-
Header (metadata) releaseJul 17, 2019-
Map releaseMar 18, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6s6z
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6s6z
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10109.png

Downloads & links

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Map

FileDownload / File: emd_10109.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap from Relion3 Refine3D after postprocessing using the deposited mask and automatic filtering/sharpening, and sharpened with LocScale
Voxel sizeX=Y=Z: 0.67 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.21666068 - 1.0043567
Average (Standard dev.)0.0063757556 (±0.041183587)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 343.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.670.670.67
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z343.040343.040343.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.2171.0040.006

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Supplemental data

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Mask #1

Fileemd_10109_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from Relion3

Fileemd_10109_half_map_1.map
AnnotationHalf map 2 from Relion3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from Relion3

Fileemd_10109_half_map_2.map
AnnotationHalf map 2 from Relion3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Map of beta-Galactosidase from Thermotoga maritima

EntireName: Map of beta-Galactosidase from Thermotoga maritima
Components
  • Complex: Map of beta-Galactosidase from Thermotoga maritima
    • Protein or peptide: Beta-galactosidase
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Map of beta-Galactosidase from Thermotoga maritima

SupramoleculeName: Map of beta-Galactosidase from Thermotoga maritima / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Map from Relion3: Refined 3D after postprocessing using the deposited mask and automatic filtering/sharpening. Sharpened by LocScale
Source (natural)Organism: Thermotoga maritima MSB8 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: XL1-Blue / Recombinant plasmid: pRARE2
Molecular weightTheoretical: 1.02 MDa

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Macromolecule #1: Beta-galactosidase

MacromoleculeName: Beta-galactosidase / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: beta-galactosidase
Source (natural)Organism: Thermotoga maritima MSB8 (bacteria)
Molecular weightTheoretical: 127.653539 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: PYEWENPQLV SEGTEKSHAS FIPYLDPFSG EWEYPEEFIS LNGNWRFLFA KNPFEVPEDF FSEKFDDSNW DEIEVPSNWE MKGYGKPIY TNVVYPFEPN PPFVPKDDNP TGVYRRWIEI PEDWFKKEIF LHFEGVRSFF YLWVNGKKIG FSKDSCTPAE F RLTDVLRP ...String:
PYEWENPQLV SEGTEKSHAS FIPYLDPFSG EWEYPEEFIS LNGNWRFLFA KNPFEVPEDF FSEKFDDSNW DEIEVPSNWE MKGYGKPIY TNVVYPFEPN PPFVPKDDNP TGVYRRWIEI PEDWFKKEIF LHFEGVRSFF YLWVNGKKIG FSKDSCTPAE F RLTDVLRP GKNLITVEVL KWSDGSYLED QDMWWFAGIY RDVYLYALPK FHIRDVFVRT DLDENYRNGK IFLDVEMRNL GE EEEKDLE VTLITPDGDE KTLVKETVKP EDRVLSFAFD VKDPKKWSAE TPHLYVLKLK LGEDEKKVNF GFRKIEIKDG TLL FNGKPL YIKGVNRHEF DPDRGHAVTV ERMIQDIKLM KQHNINTVRT SHYPNQTKWY DLCDYFGLYV IDEANIESHG IDWD PEVTL ANRWEWEKAH FDRIKRMVER DKNHPSIIFW SLGNEAGDGV NFEKAALWIK KRDNTRLIHY EGTTRRGESY YVDVF SLMY PKMDILLEYA SKKREKPFIM CEYAHAMGNS VGNLKDYWDV IEKYPYLHGG CIWDWVDQGI RKKDENGREF WAYGGD FGD TPNDGNFCIN GVVLPDRTPE PELYEVKKVY QNVKIRQVSK DTYEVENRYL FTNLEMFDGA WKIRKDGEVI EEKTFKI FA EPGEKRLLKI PLPEMDDSEY FLEISFSLSE DTPWAEKGHV VAWEQFLLKA PAFEKKSISD GVSLREDGKH LTVEAKDT V YVFSKLTGLL EQILHRRKKI LKSPVVPNFW RVPTDNDIGN RMPQRLAIWK RASKERKLFK MHWKKEENRV SVHSVFQLP GNSWVYTTYT VFGNGDVLVD LSLIPAEDVP EIPRIGFQFT VPEEFGTVEW YGRGPHETYW DRKESGLFAR YRKAVGEMMH RYVRPQETG NRSDVRWFAL SDGETKLFVS GMPQIDFSVW PFSMEDLERV QHISELPERD FVTVNVDFRQ MGLGGDDSWG A MPHLEYRL LPKPYRFSFR MRISEEIPSW RVLAAIPETL HVEMSSEDVI REGDTLRVKF SLLNDTPLSK EKQVVLFVDG NE YSVRRVV IPPFKKEELV FKVEGLKKGE HLIHTNLNTR KTIYVR

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 1384 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration0.03 mg/mL
BufferpH: 6
Component:
ConcentrationFormulaName
50.0 mMNaH2PO4Sodium Phosphate Buffer
50.0 mMNaClSodium chlorideSodium Chloryde
2.0 mMC4H10O2S2Dithiothreitol
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 30.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.6 µm / Calibrated defocus min: 0.4 µm / Calibrated magnification: 20895 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling 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 / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Number real images: 3600 / Average exposure time: 25.0 sec. / Average electron dose: 35.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 229079
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Details: Standard CTF correction inside RELION's reconstruction
Startup modelType of model: INSILICO MODEL / In silico model: SGD in relion3.0
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: Standard CTF correction inside RELION's reconstruction
Number images used: 154632
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER
Output model

PDB-6s6z:
Structure of beta-Galactosidase from Thermotoga maritima

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