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- PDB-6cvm: Atomic resolution cryo-EM structure of beta-galactosidase -

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Basic information

Entry
Database: PDB / ID: 6cvm
TitleAtomic resolution cryo-EM structure of beta-galactosidase
ComponentsBeta-galactosidase
KeywordsHYDROLASE / drift correction / radiation damage / drug discovery / precision medicine / computer-aided drug discovery
Function / homology
Function and homology information


alkali metal ion binding / lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding / magnesium ion binding / identical protein 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
2-phenylethyl 1-thio-beta-D-galactopyranoside / Beta-galactosidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.9 Å
AuthorsSubramaniam, S. / Bartesaghi, A. / Banerjee, S. / Zhu, X. / Milne, J.L.S.
CitationJournal: Structure / Year: 2018
Title: Atomic Resolution Cryo-EM Structure of β-Galactosidase.
Authors: Alberto Bartesaghi / Cecilia Aguerrebere / Veronica Falconieri / Soojay Banerjee / Lesley A Earl / Xing Zhu / Nikolaus Grigorieff / Jacqueline L S Milne / Guillermo Sapiro / Xiongwu Wu / Sriram Subramaniam /
Abstract: The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near- ...The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a data-driven dose compensation scheme that significantly improves the extraction of high-resolution information recorded during exposure of the specimen to the electron beam. These advances enable determination of a cryo-EM density map for β-galactosidase bound to the inhibitor phenylethyl β-D-thiogalactopyranoside where the ordered regions are resolved at a level of detail seen in X-ray maps at ∼ 1.5 Å resolution. Using this density map in conjunction with constrained molecular dynamics simulations provides a measure of the local flexibility of the non-covalently bound inhibitor and offers further opportunities for structure-guided inhibitor design.
History
DepositionMar 28, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 30, 2018Provider: repository / Type: Initial release
Revision 1.1Jun 20, 2018Group: Data collection / Database references / Category: citation / Item: _citation.page_first
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.energyfilter_name
Revision 1.3Aug 22, 2018Group: Data collection / Database references / Category: em_imaging_optics / pdbx_related_exp_data_set
Item: _em_imaging_optics.energyfilter_name / _pdbx_related_exp_data_set.data_reference
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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

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Structure visualization

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  • Deposited structure unit
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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)466,30424
Polymers464,7244
Non-polymers1,58020
Water75,5554194
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19320 Å2
ΔGint-199 kcal/mol
Surface area134860 Å2

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Components

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


Mass: 116181.031 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / Strain: K12 / References: UniProt: P00722, beta-galactosidase
#2: Sugar
ChemComp-PTQ / 2-phenylethyl 1-thio-beta-D-galactopyranoside / 2-Phenylethyl beta-D-thiogalactoside, PETG


Type: D-saccharide / Mass: 300.371 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C14H20O5S
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#4: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Na
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4194 / Source method: isolated from a natural source / Formula: H2O

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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: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.465 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 8
Details: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1.0 mM TCEP
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: plasma cleaned / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 90.15 K / Details: Blot for 2 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Parallel beam illumination
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 215000 X / Calibrated magnification: 215000 X / Nominal defocus min: 600 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79.8 K / Temperature (min): 79.6 K
Image recordingAverage exposure time: 7.6 sec. / Electron dose: 45 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 1539 / Details: Raw micrographs are available from EMPIAR-10061.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansMovie frames/image: 38 / Used frames/image: 1-38

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategoryDetails
3CTFFIND4CTF correctionLocal CTF correction
6UCSF Chimeramodel fitting
8PHENIXmodel refinement
10FREALIGN9.11final Euler assignment
12FREALIGN9.113D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 298715
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 1.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150321 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01133824
ELECTRON MICROSCOPYf_angle_d0.91946156
ELECTRON MICROSCOPYf_dihedral_angle_d16.47119768
ELECTRON MICROSCOPYf_chiral_restr0.0654844
ELECTRON MICROSCOPYf_plane_restr0.0076080

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