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Cryo-EM structure of heavy chain mouse apoferritinCryo-EM structure of heavy chain mouse apoferritin
Structural highlights
Function[FRIH_MOUSE] Stores iron in a soluble, non-toxic, readily available form. Important for iron homeostasis. Has ferroxidase activity. Iron is taken up in the ferrous form and deposited as ferric hydroxides after oxidation. Also plays a role in delivery of iron to cells. Mediates iron uptake in capsule cells of the developing kidney.[1] Publication Abstract from PubMedDetector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 A with a 1.346 A physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 A physical pixel size) we achieved a resolution of 1.65 A within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 A resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community. Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM.,Sun M, Azumaya CM, Tse E, Bulkley DP, Harrington MB, Gilbert G, Frost A, Southworth D, Verba KA, Cheng Y, Agard DA J Struct Biol. 2021 May 11;213(3):107745. doi: 10.1016/j.jsb.2021.107745. PMID:33984504[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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