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- PDB-6x1q: 1.8 Angstrom resolution structure of b-galactosidase with a 200 k... -

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Basic information

Entry
Database: PDB / ID: 6x1q
Title1.8 Angstrom resolution structure of b-galactosidase with a 200 kV cryoARM electron microscope
ComponentsBeta-galactosidase
KeywordsHYDROLASE / enzyme
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. / 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 speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.8 Å
AuthorsMerk, A. / Fukumura, T. / Zhu, X. / Darling, J. / Grisshammer, R. / Ognjenovic, J. / Subramaniam, S.
Funding support1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)
CitationJournal: IUCrJ / Year: 2020
Title: 1.8 Å resolution structure of β-galactosidase with a 200 kV CRYO ARM electron microscope.
Authors: Alan Merk / Takuma Fukumura / Xing Zhu / Joseph E Darling / Reinhard Grisshammer / Jana Ognjenovic / Sriram Subramaniam /
Abstract: We report the determination of the structure of β-galactosidase at a resolution of ∼1.8 Å using data collected on a 200 kV CRYO ARM microscope equipped with a K3 direct electron detector. ...We report the determination of the structure of β-galactosidase at a resolution of ∼1.8 Å using data collected on a 200 kV CRYO ARM microscope equipped with a K3 direct electron detector. The data were collected in a single 24 h session by recording images from an array of 7 × 7 holes at each stage position using the automated data collection program . In addition to the expected features such as holes in the densities of aromatic residues, the map also shows density bumps corresponding to the locations of hydrogen atoms. The hydrogen densities are useful in assigning absolute orientations for residues such as glutamine or asparagine by removing the uncertainty in the fitting of the amide groups, and are likely to be especially relevant in the context of structure-guided drug design. These findings validate the use of electron microscopes operating at 200 kV for imaging protein complexes at atomic resolution using cryo-EM.
History
DepositionMay 19, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Derived calculations / Category: citation / pdbx_struct_conn_angle / struct_conn
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _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
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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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)465,10220
Polymers464,7244
Non-polymers37816
Water65,1783618
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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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: Chemical
ChemComp-MG / MAGNESIUM ION


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


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


Mass: 18.015 Da / Num. of mol.: 3618 / 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: beta-galactosidase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: .465 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 25mM Tris pH 8.0, 50mM NaCl, 0.5mM TCEP, 2mM MgCl
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Purified by size exclusion chromatography
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 291 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -950 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingAverage exposure time: 1 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4949
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 30 eV

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
5RELION3.1CTF correction
8UCSF Chimeramodel fitting
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 998945
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 1.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 257202 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5A1A

5a1a


Accession code: 5A1A / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00732936
ELECTRON MICROSCOPYf_angle_d0.70745012
ELECTRON MICROSCOPYf_dihedral_angle_d22.36911632
ELECTRON MICROSCOPYf_chiral_restr0.0464772
ELECTRON MICROSCOPYf_plane_restr0.0045916

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