Single-molecule fluorescence technology which belongs to single molecule detection is one of the most widely used methods. This technology has higher sensitivity and spatial resolution, and the detection range can also reach to nanometer scale. A single molecule behavior can be directly observed by single-molecule fluorescence technology. FRET can be utilized to measure structure details and the process of dynamics, particularly biological macromolecules. By marking two fluorescent molecules in different sites, so-called donor and acceptor, FRET efficiency and distance information between the marker sites could be obtained by measuring fluorescence intensity of fluorescent molecules (shown as Fig. 1(a)). Over the past few decades, single-molecule Förster resonant energy transfer (smFRET) has rapid development. Ha et al. measured energy transfer between a single donor and a single acceptor.^[4] This is the first detection of single-molecule FRET. Whereafter smFRET has been successfully applied to the study of the conformational transitions of biomolecules, kinetic process of proteins, and so on. McLoughlin et al. used smFRET technology to monitor the dynamic changes of protein structure and interface behavior on the surface.^[5] Recently, Dai et al. discovered the conformational state distributions and dynamics of sensitive factor attachment protein receptors (SNARE) protein Ykt6 by the same method.^[6]

Fig. 1.

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Fig. 1. Schematic figure of several single-molecule experiments. (a) Förster resonance energy transfer can occur between the donor and the receptor within a certain range. (b) Optical tweezers. Double DNA is attached to an optically trapped bead, and the free end is attached to the surface of the trapping chamber. (c) Magnetic tweezers base on permanent magnets. A pair of small permanent magnets produces a magnetic field gradient along the axial direction, and the rotation of magnets produces rotation of the magnetic bead. (d) Atomic force microscopy. A cantilever with a sharp tip holds above a piezoelectric scanning stage.

FRET technology has a superiority that it is a ratiometric method to measure the interior distance in the molecular frame, and the technique needs the ratio of fluorescence intensity, not the intensity of fluorescence, which makes it greatly reduce noise and drifting of apparatus.^[7] The major limitation of this technology is that the absorption spectrum of organic dyes is narrow and the emission spectrum is wide. Photobleaching and low photostability have also impeded further applications.^[8] Therefore, it is urgent to find more suitable donors and acceptors, among which quantum dot had been proven as a better candidate for its unique quantum advantages and special properties of the coupling with organic molecules.^{[9, 10]} On the basis of these theories, Qiu et al. detected homogeneous and multiplexed microRNA.^[11] Over the past 70 years, FRET have been developed and perfected ceaselessly, and some new techniques and methods have emerged, such as multi-color FRET, fluorescence life detection, and single pair fluorescence resonant energy transfer (spFRET). Hohng et al. first used one donor (Cy3) and two acceptors (Cy5 and Cy5.5) to implement three-color FRET technology, and observed successfully the correlated movement of arms of the Holliday junction.^[12] Li et al. used single-molecule fluorescence technology to study the movement and transport function of molecular motors in cells. They reported a new kind of single molecule fluorescence detection technology — single-molecule surface induced fluorescence attenuation (sm-SIFA). They also studied dynamic changes of pore-forming peptide among five transmembrane positions, and discussed the key factors which caused lipid membrane permeability by using this technology.^[13]

Some of the new and exciting technical developments still are in the way. However, the fluorescence technique is passive observation, and single-molecule manipulation can control the target object, as described below.

In recent twenty years, single-molecule manipulation techniques have developed into manipulating and measuring the properties of single molecules. This kind of technology can exert a controlled external force on individual biomolecules and measure changes in real time, and can accurately measure the conformation and function of large biomolecules such as proteins and DNA. Therefore, the force spectroscopic information of the measurements is able to acquire, which associated with the characterization of the molecular conformation transition of dynamic stability and mechanics. These experimental techniques include the OT, MT, and AFM. Whereas the three techniques differ in the method used to measure, they have different suitable experimental and experimental conditions.

Over the last 60 years, with the rapid development of molecular biology as the core of biology, the structure of biological macromolecules at atomic or nearly-atomic resolution has been continually resolved especially by using an advanced technology called cryo-EM. 2017 Nobel Prize in Chemistry was awarded to three scientists, Dr. Jacques Dubochet, Dr. Joachim Frank, and Dr. Richard Henderson, for their fundamental contributions to developing cryo-EM for the high-resolution structure determination of biomolecules in solution. Cryo-EM now allows to interpret the mechanisms underneath the biological processes in terms of the disposition of atoms in space. Therefore, by using cryo-EM, structural biologists or biochemists could focus on how multiple proteins interact with other biomolecules in atomic details.^[35] What is cryo-EM? Cryo-EM begins with vitrification, in which the protein solution is cooled so rapidly that water molecules do not have time to crystallize. The sample is then screened for particle concentration, distribution and orientation. Next, a series of images are acquired, and two-dimensional classes are computationally extracted. In the final step, the data is processed by reconstruction software, yielding accurate, detailed, three-dimensional (3D) models of intricate biological structures at the sub-cellular and molecular scales. These models can reveal interactions that are impossible to visualize previously, a key to scientific results. In general, the total protocol of cryo-EM is freezing sample (an amorphous solid that does little or no damage to the sample structure); data collection (to obtain 2D projection images); 3D reconstruction (3D density map from 2D images).

Comparing with using charge-coupled device (CCD), the development of new electronic detector not only has a qualitative leap on the electron microscopy image quality, and the sharp increase in speed at the same time, but it also can obtain movie of electron microscopy images. The electron microscope technology previously had a major bottleneck on the resolution, which was the image drift and radiation damage caused by electron beam acted with biological samples. With the fast film record, we can track the image drift trajectory and can correct motion and radiation damage for the image. Thus, data quality is greatly improved. The imaging process of transmission electron microscopy (TEM) is that electron beam penetrates the sample and the 3D electric potential density distribution function of the sample is projected along the direction of propagation direction of the electron beam to the 2D plane perpendicular to the propagation direction. In 1968, Klug discovered the central section theorem and purposed the concept that was performed to resolve the three-dimensional structure of the object in computer through two-dimensional projection of a three-dimensional object from different angles. The early biological samples were prepared from negative stain. It is the biological samples that were stained with heavy metal salts, and some common negative stain include the high electron density of ammonium molybdate, uranyl acetate, uranyl formate, and phosphotungstic acid. They are able to embed in the gaps of biological samples, and perform the electron microscopy and 3D reconstruction on the basis of the contrast between heavy metal salts and samples. Since negative stain is the heavy metal salt, this can not only solve the problem of radiation damage to biological molecules, but also improve the contrast of the images.^[36]

Nowadays, cryo-EM, x-ray crystallography and NMR spectroscopy are three main means to determine the three-dimensional structure information of biological materials. As described above, cryo-EM provides a completely new idea for the study of bio-molecular structure different from x-ray crystallography. However, owing to the technical bottleneck (freezing, electron microscopic imaging, and image processing algorithms) in more than 30 years, cryo-EM could be curbed some relatively low-resolution structures and cannot be compared with x-ray crystallography in resolution. The most important revolutionary events occurred about three or four years ago: one was the invention of the direct electronic detector (DDD), the other was the improvement of the high-resolution image processing algorithm,^{[37, 38]} which makes cryo-EM indispensable to study the structure and dynamics of biomolecules complexes, included viruses, ribosomes, mitochondria, ion channels and a 4.5-Å resolution of enzyme complex as small as 170 kilo Daltons (Da).^[39–41] For instance, Yu et al. obtained the three-dimensional structure of highly symmetrical cytoplasmic polyhedrosis virus (CPV) at 3.88 Å resolution in 2008.^[42] It is the first time to get the structure of the near-atom resolution by single particle cryo-EM. In 2013, Liao et al. reconstructed 3.4-Å resolution 3D structure of membrane protein TRPV1 successfully by DDD cameras.^[43] Likewise, cryo-EM is one of most effective ways to resolve proteasome structure, because 19S regulatory particle naturally has a large number of sub-stable states and the substantial local conformation fluctuation. Moreover, the complete assembly (26S proteasome holoenzyme) consists of a 20S proteolytic core particle (CP) and two 19S regulatory particles. Through image reconstruction of cryoEM micrographs, Chen et al. resolved a near-atomic-resolution structure of the human proteasome holoenzyme,^[44] by using the independently developed software ROME (based on statistical manifold learning framework).^[45] In addition, Lu et al. revealed allosteric selection mechanism of the assembly of proteasome.^[46] In the field of cardiovascular diseases, Zhang et al. revealed the mechanism of cholesteryl ester transfer protein (CETP) interacting with high-density lipoproteins (HDL) and low-density lipoproteins (LDL) to transfer cholesterols and proposed a “tunnel model”,^[47] which provided a new insight for developing CETP inhibitors.

Addition to cryo-EM, traditional negative-staining methods could also provide insights of the dynamic structures and functions of biomolecules, especially for those who have molecular weights less than 100 kDa. For example, an optimized negative-staining (OpNS) method was proposed from a refined conventional protocol,^[49–52] through which many small and dynamic proteins including CETP,^[47] DNA,^[58] antibodies,^[53] lipoproteins,^{[54, 55]} and proteasome could be validated. At the same time, Zhang et al. revealed the morphology and structure information of lipoproteins and flexible antibodies by OpNS (see Fig. 2). Although samples will lose a lot of high resolution information by the negative staining method (in many cases the resolution only can reach ∼15 Å), such structural details of biological molecules could also provide crucial insights for understanding their functions.^{[43, 53]} Currently, our understanding of the function and of structure has led to a structure-driven approach, such as thermodynamics, dynamics, and pharmacology. Recently, this approach was utilized to determine the structural and thermodynamic factors during the biological processes, especially identifying the significant conformational changes with the ligand binding.^[56–58] For example, time-resolved cryo-EM method visualized a set of ribosome structures for following ribosome-transfer RNA (tRNA) complex movement through the ribosome during translocation.^[58] Cryo-EM snapshots combined with a manifold embedding method determined a massive rearrangement of the ribosometransfer RNA (tRNA) complexes.^[57] Free-energy landscapes derived from single-particle cryo-EM snapshots revealed multiple routes and reaction coordinates of Ryanodine receptor (RyR1), which is a highly conductive calcium ion channel and plays a key role in the muscle excitation-contraction coupling.^[56] Above descriptions clearly suggest the powerful impact of cryo-electron microscopy on the dynamic conformations of biological macromolecular machine, virus, and organelle.

Fig. 2.

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Fig. 2. (color online) The 3D reconstructions of IgG antibody particles and nascent HDLs by OpNS and IPET. (a) A tilt series of a single-instance IgG antibody by OpNS ET displayed in the left-most column. The IPET method obtains the 3D model of an individual protein via an iterative refinement processes. The final 3D reconstruction of an individual antibody particle shows as purple in the top right. (a) and (b) A good fit obtained by fitting each domain of the crystal structure (PDB) into each density model of IgG in left bottom. (b) IPET to study the 3D conformation changes of their antibodies before and after peptide conjugation. (c) and (d) For more than 40 years, nobody knew the 3D structures of nascent HDLs, by using IPET, Zhang et al. revealed the typical structures of two 17 nm HDLs. Adapted from Ref. [48].

There are several cryo-EM methods to resolve structures, including electron crystallography, single particle analysis, and cryo-electron tomography (cryo-ET). Electron crystallography uses the imaging of electron microscopy and the function of electron diffraction to obtain structural information from the 2D crystal of biomolecules, and analyzes its 3D structure. The range of suitable sample size is 10 kDa–500 kDa, with a maximum resolution of about 1.9 Å. Compared with x-ray crystallography, the similarity is that the periodic arrangement of the biological macromolecules with a highly uniformity is required, and the difference is that the electron microscopy can acquire the crystal electron diffraction and resolve the structure by obtaining the image of the crystal. However, single particle method is not required to crystallize and has the greatly extensive research field. The suitable sample molecular weight range is more than 100 kDa, and the highest resolution is about 2 Å. Single particle technology has developed rapidly in recent years, and has become the mainstream reconstruction technology, and has been continuously reported the 3D structure of macromolecular complex obtained by this technique. However, these technologies generally require the homogeneity of the sample and averaging from thousands of different proteins. For small and low-symmetry proteins, these methods have limited power in reconstruction resolution. Therefore, to study the 3D structures and functions of amorphous, asymmetrical and non-identical biological samples, other proper technologies have to be developed.^[36] Cryo-ET is a suitable technology that reconstructs and visualizes 3D structure of any individual objects by generating a series of tilted 2D projection images of the targets then backprojecting these 2D images to get their 3D density maps. Cryo-ET was mainly used in cell, subcellular organelle and the bio-molecular complexes with no fixed structure (molecular weight range > 800 kDa), and the maximum resolution was as low as several nanometers. However, with the development of hardware and reconstruction algorithms, cryo-ET is gradually becoming a mature technology with a resolution reaching sub-nanometer.^{[53, 58]} For example, Zhang and Ren developed the so-called individual particle electron tomography (IPET) method by investigating each individual macromolecule’s 3D structures with much higher resolution.^[48] By reducing the reconstructed area to only one target sample, molecules are reconstructed by using the proposed focused electron tomography reconstruction (FETR) to improve the resolution of the single large molecule. This method does not require an initial model and the average signal of a large number of molecules, as well as allows a certain angle error.^[48] By using IPET, Zhang et al. discovered the 3D dynamical structure and fluctuations of DNA-nanogold conjugates (an 84-bp double-stranded DNA and two 5-nm nanogold particles, see Fig. 3),^[59] in which its structural flexibility and dynamics cannot be studied by x-ray crystallography, NMR spectroscopy, or single particle cryo-EM methods. Such results have demonstrated the power of cryo-ET and its great potential application in small and dynamic bio-macromolecules.

Fig. 3.

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Fig. 3. (color online) 3D reconstruction of DNA-nanogold conjugates by IPET. (a) Images using ET of DNA-nanogold conjugates from a series of tilt angles. (b and (f) Tilt images of DNA-nanogold conjugates, then after CTF correction aligned to a common center for 3D reconstruction via an iterative refinement process and final 3D reconstructions at the corresponding tilt angles. (c) and (g) Final IPET 3D density maps of the targeted individual particles are obtained. (d) and (h) The final 3D density map indicated the overall conformation of the DNA-nanogold conjugates. (e) and (i) FSC analyses show that the resolution of these 3D maps are ∼14 Å. Adapted from Ref. [59].