RESUMO
Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a â¼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a â¼ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine's effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining â¼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM.
Assuntos
Apoferritinas , Microscopia Crioeletrônica , Elétrons , Microscopia Crioeletrônica/métodos , Apoferritinas/química , Apoferritinas/ultraestrutura , Imagem Individual de Molécula/métodos , Processamento de Imagem Assistida por Computador/métodosRESUMO
A high-flux sub-micrometre focusing system was constructed using multilayer focusing mirrors in Kirkpatrick-Baez geometry for 100â keV X-rays. The focusing mirror system had a wide bandwidth of 5% and a high peak reflectivity of 74%. Performance was evaluated at the undulator beamline BL05XU of SPring-8, which produced an intense 100â keV X-ray beam with a bandwidth of 1%. When the light source was focused directly in both vertical and horizontal directions, the beam size was measured to be 0.32â µm (V) × 5.3â µm (H) with a flux of 1 × 1012â photonsâ s-1. However, when a limited horizontal slit was used to form a secondary source, the focusing beam size decreased to 0.25â µm (V) × 0.26â µm (H) with a flux of 6 × 1010â photonsâ s-1. The 200â nm line and space patterns of a Siemens star chart made of tantalum were clearly resolved by the absorption contrast of the focused beam. This 100â keV focusing system is applicable to various fields of nondestructive analyses with sub-micrometre resolutions.
RESUMO
Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.