+Open data
-Basic information
Entry | Database: PDB / ID: 6tsh | ||||||
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Title | Beta-galactosidase in complex with deoxygalacto-nojirimycin | ||||||
Components | Beta-galactosidase | ||||||
Keywords | SUGAR BINDING PROTEIN / Bgal / nojirimycin | ||||||
Function / homology | Function and homology information alkali metal ion binding / organic substance catabolic process / lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding / carbohydrate metabolic process / magnesium ion binding / identical protein binding Similarity search - Function | ||||||
Biological species | Escherichia coli (E. coli) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å | ||||||
Authors | Saur, M. / Hartshorn, M.J. / Dong, J. / Reeks, J. / Bunkoczi, G. / Jhoti, H. / Williams, P.A. | ||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6tsh.cif.gz | 819.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6tsh.ent.gz | 680.3 KB | Display | PDB format |
PDBx/mmJSON format | 6tsh.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ts/6tsh ftp://data.pdbj.org/pub/pdb/validation_reports/ts/6tsh | HTTPS FTP |
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-Related structure data
Related structure data | 10563MC 6tskC 6tteC 6ttfC 6tthC 6ttiC 6ttqC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10645 (Title: Beta-galactosidase in complex with deoxygalacto-nojirimycin Data size: 765.8 Data #1: Data from EPU (movies have been converted to compressed TIF) [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments:
NCS ensembles :
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-Components
#1: Protein | 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: P00722*PLUS, beta-galactosidase #2: Chemical | ChemComp-MG / #3: Chemical | ChemComp-DGJ / ( #4: Water | ChemComp-HOH / | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Beta-galactosidase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.464 MDa / Experimental value: NO |
Source (natural) | Organism: Escherichia coli (E. coli) |
Source (recombinant) | Organism: Escherichia coli (E. coli) / Plasmid: pET-15b |
Buffer solution | pH: 6.8 |
Specimen | Conc.: 0.17 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Image recording | Average exposure time: 59.98 sec. / Electron dose: 59.69 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 476 / Num. of real images: 476 |
-Processing
Software | Name: REFMAC / Version: 5.8.0222 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software | Name: RELION / Version: 3.04 / Category: 3D reconstruction | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 146239 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: D2 (2x2 fold dihedral) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73037 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Cor.coef. Fo:Fc: 0.915 / Highest resolution: 2.3 Å / SU B: 3.362 / SU ML: 0.074 / ESU R: 0.157 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
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Solvent computation | Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 22.873 Å2
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Refinement step | Cycle: 1 / Total: 33536 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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