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Title:2.2 A resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor
Authors:Bartesaghi A, Merk A, Banerjee S, Matthies D, Wu X, Milne J, Subramaniam S
Sample:Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Method:Single particle reconstruction (2.2 angstroms resolution)
Red flagLatest update:2015-10-14
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Sample
Sample name: Escherichia coli beta-galactosidase bound to phenylethyl beta-D-thiogalactopyranoside (PETG)
Oligomeric state: tetramer
Theoretical molecular weight of the sample: 0.465
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinbeta-galactosidasetetramerfalseEscherichia coli K-12P00722GO:0004565
2ligandphenylethyl beta-D-thiogalactopyranosidemonomerfalseunidentifiedMolecular weight of PETG is 300.37 Da.
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
8.02.3 mg/mL25 mM Tris, pH 8.0, 50 mM NaCl, 2 mM MgCl2, 1.0 mM TCEP200 mesh Quantifoil R2/2 grids, plasma cleaned
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE90%90.15 KLEICA EM GPBlot for 2 seconds before plunging. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TITAN KRIOS300 kVFLOOD BEAMBRIGHT FIELD2.7 mm600 nm2000 nm215000215000FIELD EMISSION GUNGATAN K2 (4k x 4k) mmObjective lens astigmatism was corrected at 215,000 times magnification.

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Liquid nitrogen cooledFEI TITAN KRIOS AUTOGRID HOLDER°°Gatan, Inc.0-20 eV79.7 K79.6 K79.8 K mrad45 e/Å2Parallel beam illumination15-DEC-2014
Processing
Protocol:phase residual minimization
Software:FREALIGN
CTF correction:each particle
Number of particles:41123
Imposed symmetry:D2
Resolution by author:2.2 Å
Resolution method:FSC 0.143, semi-independent
Processing details:Final map was obtained from 41,123 particles. D2 symmetry was applied throughout refinement. Map was corrected using a B-factor of -75 A^2.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
1487 μm/pixelEvery image is the average of 38 frames recorded by the direct electron detector.
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails