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

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Basic information

Entry
Database: PDB / ID: 6s6z
TitleStructure of beta-Galactosidase from Thermotoga maritima
ComponentsBeta-galactosidase
KeywordsHYDROLASE / BETA-GALACTOSIDASE / CARBOHYDRATE / GLUCOSYL HYDROLASE / THERMOTOGA MARITIMA / TRANSGLYCOSYLATION / IMMOBILIZATION / CRYOEM / GRAPHENE-OXIDE / GALACTOOLIGOSACCHARIDES
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2 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, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 18, 2020Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / refine / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _refine.ls_d_res_high / _refine.ls_d_res_low / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Beta-galactosidase
D: Beta-galactosidase
B: Beta-galactosidase
H: Beta-galactosidase
F: Beta-galactosidase
G: Beta-galactosidase
E: Beta-galactosidase
C: Beta-galactosidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,021,42316
Polymers1,021,2288
Non-polymers1948
Water24,9331384
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, cryo-EM, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area35720 Å2
ΔGint-137 kcal/mol
Surface area304590 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14A
24E
15A
25F
16A
26G
17A
27H
18B
28C
19B
29D
110B
210E
111B
211F
112B
212G
113B
213H
114C
214D
115C
215E
116C
216F
117C
217G
118C
218H
119D
219E
120D
220F
121D
221G
122D
222H
123E
223F
124E
224G
125E
225H
126F
226G
127F
227H
128G
228H

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: PRO / Beg label comp-ID: PRO / End auth comp-ID: ARG / End label comp-ID: ARG / Refine code: _ / Auth seq-ID: 2 - 1084 / Label seq-ID: 1 - 1083

Dom-IDEns-IDAuth asym-IDLabel asym-ID
11AA
21BC
12AA
22CH
13AA
23DB
14AA
24EG
15AA
25FE
16AA
26GF
17AA
27HD
18BC
28CH
19BC
29DB
110BC
210EG
111BC
211FE
112BC
212GF
113BC
213HD
114CH
214DB
115CH
215EG
116CH
216FE
117CH
217GF
118CH
218HD
119DB
219EG
120DB
220FE
121DB
221GF
122DB
222HD
123EG
223FE
124EG
224GF
125EG
225HD
126FE
226GF
127FE
227HD
128GF
228HD

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28

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Components

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


Mass: 127653.539 Da / Num. of mol.: 8
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) / Variant (production host): XL1-Blue / References: UniProt: Q56307, beta-galactosidase
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1384 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Map of beta-Galactosidase from Thermotoga maritima / Type: COMPLEX
Details: Map from Relion3: Refined 3D after postprocessing using the deposited mask and automatic filtering/sharpening. Sharpened by LocScale
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.02 MDa / Experimental value: NO
Source (natural)Organism: Thermotoga maritima MSB8 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: XL1-Blue / Plasmid: pRARE2
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium Phosphate BufferNaH2PO41
250 mMSodium ChlorydeNaCl1
32 mMDithiothreitolC4H10O2S21
SpecimenConc.: 0.03 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Calibrated magnification: 20895 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 400 nm / Calibrated defocus max: 2600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 25 sec. / Electron dose: 35 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3600
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

SoftwareName: REFMAC / Version: 5.8.0238 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION3particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7MOLREPmodel fittingMolrep was used to generate the full octamer after initial refinement of one monomer
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13Coot0.9-premodel refinement
14REFMAC5.5.0026model refinementwithin CCPEM
CTF correctionDetails: Standard CTF correction inside RELION's reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 229079
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154632 / Algorithm: FOURIER SPACE
Details: Standard CTF correction inside RELION's reconstruction
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
RefinementResolution: 2→2 Å / Cor.coef. Fo:Fc: 0.977 / SU B: 3.92 / SU ML: 0.098 / ESU R: 0.129
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.23263 --
obs0.23263 1357395 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 53.058 Å2
Baniso -1Baniso -2Baniso -3
1-0.61 Å20.15 Å20.14 Å2
2--0.55 Å20.13 Å2
3----1.17 Å2
Refinement stepCycle: 1 / Total: 73712
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.01374488
ELECTRON MICROSCOPYr_bond_other_d00.01767848
ELECTRON MICROSCOPYr_angle_refined_deg1.4731.648100928
ELECTRON MICROSCOPYr_angle_other_deg1.1241.581157584
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.71658688
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.48522.1134392
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.071512928
ELECTRON MICROSCOPYr_dihedral_angle_4_deg15.94915520
ELECTRON MICROSCOPYr_chiral_restr0.0660.28960
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.0282928
ELECTRON MICROSCOPYr_gen_planes_other0.0010.0217000
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it3.8915.04934680
ELECTRON MICROSCOPYr_mcbond_other3.895.04934679
ELECTRON MICROSCOPYr_mcangle_it5.457.60743344
ELECTRON MICROSCOPYr_mcangle_other5.457.60743345
ELECTRON MICROSCOPYr_scbond_it5.5415.95939808
ELECTRON MICROSCOPYr_scbond_other5.5415.95939808
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other9.0538.59257568
ELECTRON MICROSCOPYr_long_range_B_refined11.65155.07575616
ELECTRON MICROSCOPYr_long_range_B_other11.65155.07375605
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0.01 Å / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumber
11A71710
12B71710
21A71724
22C71724
31A71702
32D71702
41A71698
42E71698
51A71712
52F71712
61A71704
62G71704
71A71710
72H71710
81B71704
82C71704
91B71706
92D71706
101B71708
102E71708
111B71736
112F71736
121B71712
122G71712
131B71724
132H71724
141C71708
142D71708
151C71692
152E71692
161C71704
162F71704
171C71704
172G71704
181C71698
182H71698
191D71712
192E71712
201D71708
202F71708
211D71708
212G71708
221D71726
222H71726
231E71704
232F71704
241E71712
242G71712
251E71712
252H71712
261F71716
262G71716
271F71710
272H71710
281G71712
282H71712
LS refinement shellResolution: 2.1→2.155 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.636 100474 -
obs--100 %

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