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- PDB-6sd0: Structure of beta-galactosidase from Thermotoga maritima. -

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Basic information

Entry
Database: PDB / ID: 6sd0
TitleStructure of beta-galactosidase from Thermotoga maritima.
ComponentsBeta-galactosidase
KeywordsHYDROLASE / BETA-GALACTOSIDASE / GLYCOSIDE HYDROLASE / HYDROLYSIS / GALACTOOLIGOSACCHARIDE / THERMOTOGA MARITIMA / THERMORESISTANT / LACTOSE / GLYCOSYL HYDROLASE 2 FAMILY. SUGAR BINDING PROTEIN
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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.7 Å
AuthorsJimenez-Ortega, E. / Ramirez-Escudero, M. / Sanz-Aparicio, J.
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 26, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Beta-galactosidase
B: Beta-galactosidase
C: Beta-galactosidase
D: Beta-galactosidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)511,2368
Polymers511,1394
Non-polymers974
Water543
1
A: Beta-galactosidase
C: Beta-galactosidase
hetero molecules

A: Beta-galactosidase
C: Beta-galactosidase
hetero molecules

A: Beta-galactosidase
C: Beta-galactosidase
hetero molecules

A: Beta-galactosidase
C: Beta-galactosidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,022,47216
Polymers1,022,2788
Non-polymers1948
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_655-y+1,x,z1
crystal symmetry operation2_665-x+1,-y+1,z1
crystal symmetry operation4_565y,-x+1,z1
2
B: Beta-galactosidase
D: Beta-galactosidase
hetero molecules

B: Beta-galactosidase
D: Beta-galactosidase
hetero molecules

B: Beta-galactosidase
D: Beta-galactosidase
hetero molecules

B: Beta-galactosidase
D: Beta-galactosidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,022,47216
Polymers1,022,2788
Non-polymers1948
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_575-x,-y+2,z1
crystal symmetry operation3_665-y+1,x+1,z1
crystal symmetry operation4_465y-1,-x+1,z1
Unit cell
Length a, b, c (Å)167.852, 167.852, 519.990
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number79
Space group name H-MI4
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
31C
41D

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1113A1 - 1084
2113B1 - 1084
3113C1 - 1084
4113D1 - 1084

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-0.544372, 0.838834, 0.004138), (0.838829, 0.544385, -0.003412), (-0.005115, 0.001614, -0.999986)-56.61729, -7.83474, -51.415089
3given(0.997958, -0.063515, 0.006724), (-0.063502, -0.99798, -0.002016), (0.006839, 0.001585, -0.999975)5.18696, 173.065903, 70.390839
4given(-0.597367, 0.801933, -0.007488), (-0.801963, -0.59737, 0.002018), (-0.002855, 0.007211, 0.99997)-51.403179, 184.249207, 121.059143

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Components

#1: Protein
Beta-galactosidase / Beta-gal / Lactase


Mass: 127784.742 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima MSB8 (bacteria) / Gene: lacZ, TM_1193 / Plasmid: pRARE2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q56307, beta-galactosidase
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.59 Å3/Da / Density % sol: 65.7 % / Description: Crystal bar
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: 12% PEG 3350, 0.1 M Bis-Tris propane pH 8.5, 0.2 M NaNO3, 2% MPD, 8 mM n-Nonyl-b-D-thiomaltoside. The crystals were enhanced by the microseeding technique.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALBA / Beamline: XALOC / Wavelength: 0.97947 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 4, 2014 / Details: KB focusing mirrors
RadiationMonochromator: Si(111) channel-cut, cryocooled / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97947 Å / Relative weight: 1
Reflection twin
Crystal-IDIDOperatorDomain-IDFraction
11H, K, L10.158
11-K, -H, -L20.842
ReflectionResolution: 3.7→40 Å / Num. all: 442617 / Num. obs: 76050 / % possible obs: 99.8 % / Redundancy: 5.8 % / CC1/2: 0.996 / Rmerge(I) obs: 0.165 / Rpim(I) all: 0.075 / Rrim(I) all: 0.182 / Net I/σ(I): 10.1
Reflection shellResolution: 3.7→3.78 Å / Redundancy: 6 % / Rmerge(I) obs: 1.74 / Num. unique obs: 26943 / CC1/2: 0.581 / Rpim(I) all: 0.771 / Rrim(I) all: 1.906 / % possible all: 99.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0238refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
Aimless7.0.071data scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1DP0

1dp0


Resolution: 3.7→40 Å / Cor.coef. Fo:Fc: 0.924 / Cor.coef. Fo:Fc free: 0.889 / SU B: 41.163 / SU ML: 0.623 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.161
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2855 3720 4.9 %RANDOM
Rwork0.2411 ---
obs0.2432 72328 99.78 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 276.79 Å2 / Biso mean: 146.58 Å2 / Biso min: 38.45 Å2
Baniso -1Baniso -2Baniso -3
1-38.67 Å20 Å20 Å2
2--38.67 Å20 Å2
3----77.34 Å2
Refinement stepCycle: final / Resolution: 3.7→40 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms36112 0 4 3 36119
Biso mean--40.92 38.94 -
Num. residues----4332
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0040.01337204
X-RAY DIFFRACTIONr_bond_other_d0.0030.01733892
X-RAY DIFFRACTIONr_angle_refined_deg1.4441.64850404
X-RAY DIFFRACTIONr_angle_other_deg1.1241.58178716
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.26954336
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.05122.1172192
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.934156460
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.25115260
X-RAY DIFFRACTIONr_chiral_restr0.0570.24476
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.0241408
X-RAY DIFFRACTIONr_gen_planes_other0.0010.028476
Refine LS restraints NCS

Ens-ID: 1 / Refine-ID: X-RAY DIFFRACTION

Dom-IDAuth asym-IDNumberTypeRms dev position (Å)Weight position
1A11133LOOSE POSITIONAL0.25
2B11133LOOSE POSITIONAL0.215
3C11133LOOSE POSITIONAL0.25
4D11133LOOSE POSITIONAL0.235
1A6372TIGHT THERMAL10.610.5
2B6372TIGHT THERMAL8.020.5
3C6372TIGHT THERMAL7.50.5
4D6372TIGHT THERMAL12.070.5
1A11133LOOSE THERMAL11.5110
2B11133LOOSE THERMAL9.1610
3C11133LOOSE THERMAL8.7910
4D11133LOOSE THERMAL12.2410
LS refinement shellResolution: 3.7→3.796 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.393 276 -
Rwork0.346 5366 -
all-5642 -
obs--99.52 %

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