Automated image acquisition and processing using a new generation of 4K x 4K CCD cameras for cryo electron microscopic studies of macromolecular assemblies.
We have previously reported the development of AutoEM, a software package for semi-automated acquisition of data from a transmission electron microscope. In continuing efforts to improve the speed of structure determination of macromolecular assemblies by electron microscopy, we report here on the performance of a new generation of 4 K CCD cameras for use in cryo electron microscopic applications. We demonstrate that at 120 kV, and at a nominal magnification of 67000 x, power spectra and signal-to-noise ratios for the new 4 K CCD camera are comparable to values obtained for film images scanned using a Zeiss scanner to resolutions as high as approximately 1/6.5A(-1). The specimen area imaged for each exposure on the 4 K CCD is about one-third of the area that can be recorded with a similar exposure on film. The CCD camera also serves the purpose of recording images at low magnification from the center of the hole to measure the thickness of vitrified ice in the hole. The performance of the camera is satisfactory under the low-dose conditions used in cryo electron microscopy, as demonstrated here by the determination of a three-dimensional map at 15 A for the catalytic core of the 1.8 MDa Bacillus stearothermophilus icosahedral pyruvate dehydrogenase complex, and its comparison with the previously reported atomic model for this complex obtained by X-ray crystallography.
Zhang
Peijun
P
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Borgnia
Mario J
MJ
Mooney
Paul
P
Shi
Dan
D
Pan
Ming
M
O'Herron
Philip
P
Mao
Albert
A
Brogan
David
D
Milne
Jacqueline L S
JL
Subramaniam
Sriram
S
D001331:Automation; D020134:Catalytic Domain; D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000737:chemistry*; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex