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- PDB-6tsk: Beta-galactosidase in complex with L-ribose -

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Basic information

Entry
Database: PDB / ID: 6tsk
TitleBeta-galactosidase in complex with L-ribose
ComponentsBeta-galactosidase
KeywordsSUGAR BINDING PROTEIN / Bgal / L-ribose
Function / homology
Function and homology information


organic substance catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding / carbohydrate metabolic process / magnesium ion binding
Similarity search - Function
Glycoside hydrolase, family 2, beta-galactosidase / 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 / 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. / Beta-galactosidase; Chain A, domain 5 - #10 / 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 / Beta-galactosidase; Chain A, domain 5 / Galactose mutarotase-like domain superfamily / Galactose-binding domain-like / Galactose-binding-like domain superfamily / Distorted Sandwich / Glycosidases / Glycoside hydrolase superfamily / Jelly Rolls / Immunoglobulins / TIM Barrel / Alpha-Beta Barrel / Immunoglobulin-like fold / Immunoglobulin-like / Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
beta-L-ribopyranose / Lactase / Beta-galactosidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsSaur, M. / Hartshorn, M.J. / Dong, J. / Reeks, J. / Bunkoczi, G. / Jhoti, H. / Williams, P.A.
CitationJournal: Drug Discov Today / Year: 2020
Title: Fragment-based drug discovery using cryo-EM.
Authors: Michael Saur / Michael J Hartshorn / Jing Dong / Judith Reeks / Gabor Bunkoczi / Harren Jhoti / Pamela A Williams /
Abstract: Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. ...Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. Here, we review the use of cryo-EM in fragment-based drug discovery (FBDD) based on in-house method development. We demonstrate not only that cryo-EM can reveal details of the molecular interactions between fragments and a protein, but also that the current reproducibility, quality, and throughput are compatible with FBDD. We exemplify this using the test system β-galactosidase (Bgal) and the oncology target pyruvate kinase 2 (PKM2).
History
DepositionDec 20, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 8, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 15, 2020Group: Database references / Source and taxonomy / Structure summary
Category: citation / entity ...citation / entity / entity_name_com / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _citation.title / _entity.pdbx_description ..._citation.title / _entity.pdbx_description / _entity.pdbx_ec / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _struct_ref.db_code / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq.seq_align_beg
Revision 1.2Mar 25, 2020Group: Data collection / Database references / Category: chem_comp / citation
Item: _chem_comp.type / _citation.journal_volume ..._chem_comp.type / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / pdbx_struct_conn_angle / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description ..._chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Structure viewerMolecule:
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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)474,27912
Polymers473,5814
Non-polymers6988
Water17,366964
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1111A9 - 1023
2111B9 - 1023
1121A9 - 1023
2121C9 - 1023
1131A9 - 1023
2131D9 - 1023
1141B9 - 1023
2141C9 - 1023
1151B9 - 1023
2151D9 - 1023
1161C9 - 1023
2161D9 - 1023

NCS ensembles :
ID
1
2
3
4
5
6

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-1, -4.5E-5, 6.9E-5), (4.5E-5, -1, 7.1E-5), (6.9E-5, 7.1E-5, 1)99.27766, 149.59142, -0.00742

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Components

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


Mass: 118395.336 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: lacZ / Plasmid: pET-15b / Production host: Escherichia coli (E. coli)
References: UniProt: Q8VNN2, UniProt: L0N6M2*PLUS, beta-galactosidase
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#3: Sugar
ChemComp-0MK / beta-L-ribopyranose / Ribose


Type: L-saccharide, beta linking / Mass: 150.130 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C5H10O5 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
LRibpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-L-ribopyranoseCOMMON NAMEGMML 1.0
b-L-RibpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
RibSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 964 / 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: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Beta-galactosidase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.464 MDa / Experimental value: NO
Source (natural)Organism: Human alphaherpesvirus 1 strain RH2
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET-15b
Buffer solutionpH: 6.8
SpecimenConc.: 0.17 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELDBright-field microscopy
Image recordingAverage exposure time: 59.98 sec. / Electron dose: 66.83 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 517

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Processing

SoftwareName: REFMAC / Version: 5.8.0222 / Classification: refinement
EM softwareName: RELION / Version: 3.04 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 124686
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40086 / Symmetry type: POINT
RefinementCor.coef. Fo:Fc: 0.903 / Highest resolution: 2.3 Å / SU B: 3.978 / SU ML: 0.086 / ESU R: 0.171
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.25012 --
obs0.25012 514440 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 25.347 Å2
Baniso -1Baniso -2Baniso -3
1-0.2 Å2-0 Å2-0 Å2
2---0.15 Å2-0 Å2
3----0.05 Å2
Refinement stepCycle: 1 / Total: 33632
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.01533632
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.4811.75845868
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.8954056
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.81420.8141768
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.764154684
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.01715264
ELECTRON MICROSCOPYr_chiral_restr0.10.24160
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.02126304
ELECTRON MICROSCOPYr_gen_planes_other
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_it5.0284.76416236
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it9.0546.57517396
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined15.04728.67545515
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Auth asym-ID: A / Number: 8156 / Refine-ID: ELECTRON MICROSCOPY / Type: tight thermal / Weight position: 0.87

Ens-IDDom-IDRms dev position (Å)
110.12
220.13
330.13
440.14
550.1
660.14
LS refinement shellResolution: 2.3→2.36 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.581 37988 -
obs--100 %

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