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- PDB-3j7h: Structure of beta-galactosidase at 3.2-A resolution obtained by c... -

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Basic information

Entry
Database: PDB / ID: 3j7h
TitleStructure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy
ComponentsBeta-galactosidase
KeywordsHYDROLASE / hydrolase enzyme / homo-tetramer / protein complex / atomic resolution cryo-electron microscopy / direct electron detectors / single-particle cryo-EM / 3D reconstruction
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
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBartesaghi, A. / Matthies, D. / Banerjee, S. / Merk, A. / Subramaniam, S.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy.
Authors: Alberto Bartesaghi / Doreen Matthies / Soojay Banerjee / Alan Merk / Sriram Subramaniam /
Abstract: We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side ...We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg(2+) ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of ∼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e(-)/Å(2). In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.
History
DepositionJun 30, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 30, 2014Provider: repository / Type: Initial release
Revision 1.1Aug 13, 2014Group: Database references
Revision 1.2Aug 27, 2014Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_imaging_optics / em_software
Item: _em_imaging_optics.energyfilter_name / _em_software.image_processing_id / _em_software.name
Revision 1.4Feb 21, 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 / struct_site
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_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-5995
  • Imaged by UCSF Chimera
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  • Superimposition on EM map
  • EMDB-5995
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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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)466,5078
Polymers466,4104
Non-polymers974
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 116602.484 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P00722, beta-galactosidase
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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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 / Type: COMPLEX / Details: tetramer
Molecular weightValue: 0.465 MDa / Experimental value: NO
Buffer solutionName: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP
pH: 8
Details: 25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 200 mesh Quantifoil R2/2 grids, plasma cleaned
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Temp: 90.15 K / Humidity: 90 %
Details: Blot for 2 seconds before plunging into liquid ethane (LEICA EM GP).
Method: 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 / Date: Oct 31, 2013 / Details: Parallel beam illumination
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM / Electron beam tilt params: 5
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Astigmatism: Objective lens astigmatism was corrected at 105,000 times magnification.
Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen holder type: Liquid nitrogen cooled / Temperature: 79.7 K / Temperature (max): 79.8 K / Temperature (min): 79.6 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 45 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
EM imaging opticsEnergyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansNum. digital images: 509
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Cootmodel fitting
2UCSF Chimeramodel fitting
3EMAN23D reconstruction
4FREALIGN3D reconstruction
CTF correctionDetails: Individual frames of each movie were aligned by cross-correlation using the cumulative average of previously aligned frames as a reference to align the remaining frames. Parameters of the ...Details: Individual frames of each movie were aligned by cross-correlation using the cumulative average of previously aligned frames as a reference to align the remaining frames. Parameters of the contrast transfer function for each micrograph were estimated from power spectra obtained using periodogram averaging with tiles of size 512x512 pixels extracted from all frames of each movie. These power spectra were then radially averaged and used to estimate the defocus for each image using frequencies in the 15.0-3.0 Angstrom range. CTF correction was done for each particle as implemented in FREALIGN's reconstruction protocol.
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionMethod: phase residual minimization / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11726 / Nominal pixel size: 0.6375 Å / Actual pixel size: 0.6375 Å
Details: Reconstruction details: 24,750 particles were picked automatically from the best 509 micrographs by detecting the local maxima of correlation of each image with a Gaussian disk of 100 ...Details: Reconstruction details: 24,750 particles were picked automatically from the best 509 micrographs by detecting the local maxima of correlation of each image with a Gaussian disk of 100 Angstrom in radius and extracted using a binning factor of 2 and a box size of 384x384 pixels. Particles were then subjected to reference-free 2D classification in EMAN2. 160 classes out of a total of 250 were used for de novo initial model determination using e2initialmodel.py and imposing D2 symmetry. A subset of 23,452 particles (corresponding to the 160 classes used for the initial model building) was then used for 3D refinement using a gold-standard approach. Two stacks of 11,726 particles each were independently subjected to eight rounds of iterative refinement in FREALIGN using a high-resolution frequency limit of 8 Angstrom and using the best 50% of particles from each stack according to phase residual values (equivalent to 5,863 particles) to calculate the reconstructions at each iteration. At this point particles were re-extracted from the original unbinned micrographs using a box size of 768x768 pixels and further refined in FREALIGN starting from the most recent set of alignments obtained with the binned data. The final map was obtained by averaging the two half-reconstructions in real space and corrected by a B-factor of -85 Angstrom^2 for the purpose of visualization.
Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: METHOD--Local refinement, Flexible fitting DETAILS--Initial local fitting was done using Chimera and then COOT was used for flexible fitting and model building.
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms32824 0 4 0 32828

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