Structure: a new imaging method of frozen electron microscopy to significantly improve the resolution

Recent technological breakthroughs, such as the invention of direct electron detectors and the realization of new image processing algorithms, have enabled cryo em to determine macromolecular structures with atomic or near atomic resolution As the core instrument of this technology, modern transmission electron microscope (TEM) has quite complex electron optics, and there is still much room for further improvement Material science has witnessed the application of some new electronic optical devices and revealed the structure of subatomic resolution If low temperature electromagnetic technology can benefit from new optical tuning equipment including phase plate and CS corrector, the prospect will be very attractive According to the contrast transfer theory, the traditional TEM (CTEM) asymmetric data collection strategy has been used for decades The contrast transfer theory predicts the sinusoidal contrast transfer function (CTF) of weak phase biological specimens The CTF can be modulated into cosine function by adding a phase plate on the back focal plane of the objective lens The main advantage of using phase plate is the strong enhancement of image contrast In 2015, a new phase plate using a charged film (the so-called Volta phase plate, VPP) was proved to be able to give high-resolution images of biomolecules Combined with a direct electron detector, VPP allows the reconstruction of several protein structures at near atomic resolution Theoretically, there is no information loss in the microscope, and the perfect objective lens has no spherical aberration However, the decrease of spherical aberration in objective lens results in information distortion in image, especially in high frequency Such a problem can be overcome by using the CS corrector of the objective lens Compared with the results obtained by CTEM, the microscopes equipped with CS correction devices have also been shown to be able to solve the structural resolution of ribosomes higher than 3 μ Although theoretical analysis and simulation show that the combination will maintain more structural information at high spatial frequency, no attempt to use the combination of phase plate and CS corrector for high-resolution cryoelectron microscopy has been reported Recently, the research group led by Professor Wang Hongwei of the school of life sciences of Tsinghua University published a paper on the new imaging technology of frozen electron microscopy on structure In this paper, we first proposed and used the over focus imaging technology to obtain the high-resolution protein structure, which also provided a new idea for the application of CS correction technology and phase plate technology in the field of cryoelectron microscopy biological research The research team of CTF and cryo EM images (source: structure) obtained by VPP CS corrector combined with EM firstly carried out theoretical analysis, and pointed out that the combination of phase plate and CS corrector can produce high-resolution images suitable for low focus and over focus imaging conditions The electron microscope with CS corrector is equipped with VPP The frozen apo ferritin is imaged under the condition of low focus or over focus, and the resolution is about 3 μ The combination of VPP and CS corrector shows the novel imaging capability for high resolution cryo em For thin biological samples in TEM, the weak phase prediction uses the Fourier transform of TEM image as the product of the Fourier transform of TEM object projection and the CTF of TEM electron optical system The simplified form of CTF can be written as follows: by equipping CS corrector in TEM, CS value can be greatly reduced, CS value can be very small (0.001mm), and the second term of sine function in equation can be ignored Therefore, the equation becomes: the combination of CS corrector and phase plate allows the use of cryoelectron microscopy in the biological field using imaging conditions of low focus or over focus In order to test the above theory, the research team established a program (Star method) to collect frozen apo ferritin low-temperature EM images by using the Cryo em of VPP CS corrector They deliberately collected data sets of low focus and over focus samples with a phase shift range of 0.2 π to 0.8 π Therefore, three data sets are generated, and all three data sets are regarded as unfocused images, which are used to determine CTF parameters All images, regardless of their internal phase shift, are used for single particle analysis using the standard relation image processing strategy For all three data sets, they have successfully carried out three-dimensional classification and repair, and obtained data in 2.9 Å, 3.2 Å and 3.0 Å The three reconstructions seem to be almost identical Subtle resolution differences seem to be related to the quality of the dataset Statistics of 2.9 μ VPP apo ferritin reconstruction under low focus imaging (source: structure) the signal-to-noise ratio (SNR) of dose-dependent molecular image reconstructed from VPP dataset is the key factor of data processing For CTEM data acquisition, high electron dose or defocus is usually used to improve SNR, especially at low frequency However, high electron dose will lead to radiation damage, high defocusing value will lead to CTF correction error at high frequency, and suppress high frequency signal When using CTEM without VPP, they used a total dose of 40 to 50 e - / ޹2 and a defocus value in the range of 1.0 to 2.0 mm in order to clearly see the protein particles When VPP is added, the dose can be reduced to about 15E - / ޹2, and the minimum dose can be as low as 7e - / ޹2 Cryo-EM has introduced structural biology into a new era, in which the atomic or near atomic resolution of macromolecular structure is more easily obtained The research shows that the application of phase plate or CS correction frozen em can solve the problem of high resolution structure So far, it has not been reported to use the combination of phase plate and CS corrector to obtain high-resolution structural pattern, or to collect data from over focusing imaging conditions to obtain structural confirmation The atomic resolution image of apo feritin obtained by VPP CS corrector combined with frozen EM under the condition of over focus imaging not only proves the feasibility of this combined optical system, but also finds some interesting advantages of the system which are worth further investigation and study In addition to data acquisition performance, the combination of CS corrector and VPP also shows better optical performance than when used alone In future microscopes, the next generation of CS correctors will become more stable and self correcting, the phase plates will become more solid, and the combination of phase plates and CS correctors may become an efficient tool On the simulation of anti astigmatism ability of CS corrector (source: structure) paper link: http://www.cell.com/structure/fulltext/s0969-2126 (17) 30261-7