hca ii  (R&D Systems)

Journal: Acta Crystallographica Section D: Structural Biology

Article Title: Limiting the effects of radiation damage in MicroED through dose selection during data processing

doi: 10.1107/S205979832500912X

Figure Lengend Snippet: Effect of electron dose on the strength of integrated and unmerged mean reflection intensities 〈 I 〉. The flux density is approximately 0.10 e − Å −2 s −1 (dose rate 0.45 MGy s −1 at 200 keV and 0.38 MGy s −1 at 300 keV) and the exposure time is 1.5 s per frame. ( a ) Loss of diffraction-spot strength/intensity and resolution due to radiation damage with increasing frame number. Panels (I) and (II) show the trend of intensity decay in lysozyme:native and HCA II:AZM MicroED data, respectively, collected on a Timepix detector installed on a Jeol JEM-2100 microscope operated at 200 kV. Panel (III) shows diffraction patterns of lysozyme:GdCl 3 collected on a OneView Themis Z microscope operated at 300 kV. ( b ) Intensity of diffraction spots as a function of resolution for the four data sets. 1-10F, first ten frames; 1-20F, first 20 frames; 21-35F, frame range 21–35; All F, all data. Overall, the 1-10F data set yielded reflections with higher mean intensities compared with other data sets. Data are plotted as 〈 I 〉 = mean I hkl ± standard error of the mean versus resolution for a representative 24 crystals (lysozyme:native), 16 crystals (HCA II:AZM) and nine crystals (lysozyme:GdCl 3 ). Note that in each case the same crystals are compared for the different doses. Lysozyme:native refers to native tetragonal lysozyme crystals deposited on a holey carbon grid with nylon support, HCA II:AZM refers to HCA II bound to its inhibitor acetazolamide and lastly lysozyme:GdCl 3 refers to lysozyme soaked in GdCl 3 .

Article Snippet: MicroED data for lysozyme:native, lysozyme:Na_I 3 C, lysozyme:Gd, HCA II and HCA II:AZM were collected on a Jeol JEM-2100 transmission electron microscope with a LaB 6 filament operated at 200 kV, equipped with a hybrid electron detector (Amsterdam Scientific Instruments, Timepix).

Techniques: Microscopy

Journal: Acta Crystallographica Section D: Structural Biology

Article Title: Limiting the effects of radiation damage in MicroED through dose selection during data processing

doi: 10.1107/S205979832500912X

Figure Lengend Snippet: Site-specific damage of aspartic and glutamic acids in the HCA II:AZM protein complex. The 2 mF o − DF c maps are shown as blue meshes with a contour level of 1.5σ above the mean. The mF o − DF c difference densities are shown in green and red for positive and negative density, respectively, contoured at 3.0σ above and below the mean. A section covering 2 Å around atoms is shown for all densities. The electron doses are as specified in the figure.

Article Snippet: MicroED data for lysozyme:native, lysozyme:Na_I 3 C, lysozyme:Gd, HCA II and HCA II:AZM were collected on a Jeol JEM-2100 transmission electron microscope with a LaB 6 filament operated at 200 kV, equipped with a hybrid electron detector (Amsterdam Scientific Instruments, Timepix).

Techniques:

Journal: Acta Crystallographica Section D: Structural Biology

Article Title: Limiting the effects of radiation damage in MicroED through dose selection during data processing

doi: 10.1107/S205979832500912X

Figure Lengend Snippet: How radiation damage affects the quality of protein–ligand binding properties. ( a ) Polar contacts between HCA II and its inhibitor AZM. ( b ) The quality of the density maps around the HCA II:AZM complex and zinc atomic B factors. 2 mF o − DF c maps are shown as blue meshes with a contour level of 1.5σ above the mean. mF o − DF c difference densities are shown in green and red for positive and negative density, respectively, contoured at 3σ above and below the mean. The 1-10F (6.75 MGy) structure shows some signs of incompleteness, while the 21-35F and All F structures exhibit signs of radiation damage as judged from 2 mF o − DF c maps and increased mF o − DF c features. The 1-20F data set is more complete with low signs of radiation damage and has the lowest isotropic B factor for the active-site zinc metal. The electron doses are as specified in the figure.

Article Snippet: MicroED data for lysozyme:native, lysozyme:Na_I 3 C, lysozyme:Gd, HCA II and HCA II:AZM were collected on a Jeol JEM-2100 transmission electron microscope with a LaB 6 filament operated at 200 kV, equipped with a hybrid electron detector (Amsterdam Scientific Instruments, Timepix).

Techniques: Ligand Binding Assay