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北京时间10月4日下午5点45分，瑞典皇家科学院宣布将2017年诺贝尔化学奖授予：来自瑞士洛桑大学的Jacques Dubochet，美国哥伦比亚大学的Joachim Frank以及英国剑桥分子生物学实验室的Richard Henderson，表彰他们“开发在溶液中测定生物分子结构的高分辨率冷冻电镜”。很多人发现，这次获得化学奖的三位学者似乎又是物理学家，这再次体现出了诺贝尔奖对于交叉学科的重视。
首先，让我们看看近年中获得诺贝尔化学奖的物理学者。
1998年：DFT之父
Walter Kohn
1998年度诺贝尔化学奖的颁布，向人们展示了数学、物理和化学学科的交叉和融合取得的重大成果。美国物理学家瓦尔特·科恩（Walter Kohn）和英国数学家约翰·波普（John Pople）以物理和数学工具，发展了量子化学理论和计算方法，在化学领域取得了骄人成就。通过以科恩和波普为代表的量子化学工作者的不断努力，今天，量子化学无疑成为化学工作者最有用的工具之一。磁共振成像技术（MRI）的发明实质上是物理学与医学的结合，也是交叉学科能产生丰富成果的有力证明。这种能精确观察人体内部器官而又不造成伤害的影像技术，对于医疗诊断、治疗及其检查至关重要。
遗憾的是，瓦尔特·科恩于2016年4月19日在加利福尼亚家中因癌症逝世，享年93岁。
2014年：超高分辨率荧光显微镜
William E. Moerner
关于2014年的诺贝尔化学奖，不少人也有疑问：几位拥有物理学博士学位背景的人，发明的是在生命科学领域应用的激光物理技术，为什么会获得诺贝尔化学奖？甚至有些人还这样认为：对超高分辨显微技术看起来没有那么直接的贡献的William E. Moerner，为什么也是三位获奖者之一？
其实，超高分辨荧光显微成像技术之所以能够绕过所谓光学衍射极限的物理限制——200纳米，是建立在利用特殊荧光标记分子的光化学和光物理性质基础之上的科学发展，其重要的科学基础是单分子光谱和单分子显微技术。Moerner对凝聚相中单分子光谱技术和绿荧光蛋白特殊的光学开关性质研究贡献卓着，他获得此项诺贝尔化学奖，自然当之无愧。
2017年：冷冻电镜
何为冷冻电镜？水结冰后会阻碍原先在溶液状态下快速的物质交换与扩散，低温使得化学过程速率降低：绝大多数代谢过程变得非常非常慢以至于我们难以察觉，这个技术叫做冷冻固定术（Cryo-fixation）。应用冷冻固定术，在低温下使用透射电子显微镜观察样品的显微技术，就叫做冷冻电镜（CryoEM）。冷冻电镜是重要的结构生物学研究方法，与之地位相当的另两种技术是X射线晶体学（X-raycrystallography）和核磁共振（NMR），这些方法都是为了获得生物大分子的结构以了解其功能。冷冻电镜，就是把样品冻起来然后保持低温放进显微镜里面，高度相干的电子作为光源从上面照下来，透过样品和附近的冰层，受到散射。我们再利用探测器和透镜系统把散射信号成像记录下来，最后进行信号处理，得到样品的结构。
而今天的三位诺贝尔化学奖获得者有着怎样的背景呢？
雅克·迪波什（Jacques Dubochet），1942年生于瑞士，1973年博士毕业于日内瓦大学和瑞士巴塞尔大学，瑞士洛桑大学生物物理学荣誉教授。迪波什博士领导的小组开发出真正成熟可用的快速投入冷冻制样技术制作不形成冰晶体的玻璃态冰包埋样品，随着冷台技术的开发，冷冻电镜技术正式推广开来。
约阿基姆·弗兰克（Joachim Frank），德裔生物物理学家，现为哥伦比亚大学教授。他因发明单粒子冷冻电镜（cryo-electron microscopy）而闻名，此外他对细菌和真核生物的核糖体结构和功能研究做出重要贡献。弗兰克2006年入选为美国艺术与科学、美国国家科学院两院院士。2014年获得本杰明·富兰克林生命科学奖。
理查德·亨德森（Richard Henderson），苏格兰分子生物学家和生物物理学家，他是电子显微镜领域的开创者之一。1975年，他与Nigel Unwin通过电子显微镜研究膜蛋白、细菌视紫红质，并由此揭示出膜蛋白具有良好的机构，可以发生α-螺旋。近年来，亨德森将注意力集中在单粒子电子显微镜上，即用冷冻电镜确定蛋白质的原子分辨率模型。
简而言之，Richard Henderson开创了二维电子晶体学三维重构技术，奠定了基本理论；Joachim Frank发明了冷冻电镜单粒子三维重构技术；Jacques Dubochet则在早期实验方面做出了重要贡献。
未来科研的方向
其实，回顾百余年来的诺贝尔奖，近半数获奖者属于交叉学科。尤其在近几十年来，这一现象屡见不鲜。这对于新时代学者有着相当的借鉴意义。不同的学科彼此交叉综合，有利于科学上的重大突破，培育新的生长点，乃至新学科的产生。无疑，交叉学科研究是科学发展的主要方向，也将越来越是诺贝尔奖的眷顾对象。
以下内容源自诺贝尔奖官方网站
Over the last few years, numerous astonishing structures of life’s molecular machinery have filled the scientific literature (figure 1): Salmonella’s injection needle for attacking cells; proteins that confer resistance to chemotherapy and antibiotics; molecular complexes that govern circadian rhythms; light-capturing reaction complexes for photosynthesis and a pressure sensor of the type that allows us to hear. These are just a few examples of the hundreds of biomolecules that have now been imaged using cryo-electron microscopy (cryo-EM).
When researchers began to suspect that the Zika virus was causing the epidemic of brain-damaged newborns in Brazil, they turned to cryo-EM to visualise the virus. Over a few months, threedimensional (3D) images of the virus at atomic resolution were generated and researchers could start searching for potential targets for pharmaceuticals.
Figure 1. Over the last few years, researchers have published atomic structures of numerous complicated protein complexes. a. A protein complex that governs the circadian rhythm. b. A sensor of the type that reads pressure changes in the ear and allows us to hear. c. The Zika virus.
Jacques Dubochet, Joachim Frank and Richard Henderson have made ground-breaking discoveries that have enabled the development of cryo-EM. The method has taken biochemistry into a new era, making it easier than ever before to capture images of biomolecules.
Pictures – an important key to knowledge
In the first half of the twentieth century, biomolecules – proteins, DNA and RNA – were terra incognita on the map of biochemistry. Scientists knew they played fundamental roles in the cell, but had no idea what they looked like. It was only in the 1950s, when researchers at Cambridge began to expose protein crystals to X-ray beams, that it was first possible to visualise their wavy and spiralling structures.
In the early 1980s, the use of X-ray crystallography was supplemented with the use of nuclear magnetic resonance (NMR) spectroscopy for studying proteins in solid state and in solution. This technique not only reveals their structure, but also how they move and interact with other molecules.
Thanks to these two methods, there are now databases containing thousands of models of biomolecules that are used in everything from basic research to pharmaceutical development. However, both methods suffer from fundamental limitations. NMR in solution only works for relatively small proteins. X-ray crystallography requires that the molecules form well-organised crystals, such as when water freezes to ice. The images are like black and white portraits from early cameras – their rigid pose reveals very little about the protein’s dynamics.
Also, many molecules fail to arrange themselves in crystals, which caused Richard Henderson to abandon X-ray crystallography in the 1970s – and this is where the story of 2017’s Nobel Prize in Chemistry begins.
Problems with crystals made Henderson change track
Richard Henderson received his PhD from the bastion of X-ray crystallography, Cambridge, UK. He used the method for imaging proteins, but setbacks arose when he attempted to crystallise a protein that was naturally embedded in the membrane surrounding the cell.
Membrane proteins are difficult to manage. When they are removed from their natural environment – the membrane – they often clump up into a useless mass. The first membrane protein that Richard Henderson worked with was difficult to produce in adequate amounts; the second failed to crystallise. After years of disappointment, he turned to the only available alternative: the electron microscope.
It is open to discussion whether electron microscopy really was an option at this time. Transmission electron microscopy, as the technique is called, works more or less like ordinary microscopy, but a beam of electrons is sent through the sample instead of light. The electrons’ wavelength is much shorter than that of light, so the electron microscope can make very small structures visible – even the position of individual atoms.
In theory, the resolution of the electron microscope was thus more than adequate for Henderson to obtain the atomic structure of a membrane protein, but in practice the project was almost impossible. When the electron microscope was invented in the 1930s, scientists thought that it was only suitable for studying dead matter. The intense electron beam necessary for obtaining high resolution images incinerates biological material and, if the beam is weakened, the image loses its contrast and becomes fuzzy.
In addition, electron microscopy requires a vacuum, a condition in which biomolecules deteriorate because the surrounding water evaporates. When biomolecules dry out, they collapse and lose their natural structure, making the images useless.
Almost everything indicated that Richard Henderson would fail, but the project was saved by the special protein that he had chosen to study: bacteriorhodopsin.
The best so far was not good enough for Henderson
Bacteriorhodopsin is a purple-coloured protein that is embedded in the membrane of a photosynthesising organism, where it captures the energy from the sun’s rays. Instead of removing the sensitive protein from the membrane, as Richard Henderson had previously tried to do, he and his colleague took the complete purple membrane and put it under the electron microscope. When the protein remained surrounded by the membrane it retained its structure; they covered the sample’s surface with a glucose solution that protected it from drying out in the vacuum.
The harsh electron beam was a major problem, but the researchers made use of how the bacteriorhodopsin molecules are packed in the organism’s membrane. Instead of blasting it with a full dose of electrons, they had a weaker beam flow through the sample. The image’s contrast was poor and they could not see the individual molecules, but they used the fact that the proteins were regularly packed and oriented in the same direction. When all the proteins diffract the electron beams in an almost identical manner, they were able to calculate a more detailed image based on the diffraction pattern – they used a similar mathematical approach to that used in X-ray crystallography.
At the next stage, the researchers turned the membrane under the electron microscope, taking pictures from many different angles. This way, in 1975 it was possible to produce a rough 3D model of bacteriorhodopsin’s structure (figure 2), which showed how the protein chain wiggled through the membrane seven times.
Figure 2. The first rough model of bacteriorhodopsin, published in 1975. Image from Nature 257: 28-32
It was the best picture of a protein ever generated using an electron microscope. Many people were impressed by the resolution, which was 7 Ångström (0.0000007 millimetres), but this was not enough for Richard Henderson. His goal was to achieve the same resolution as that provided by X-ray crystallography, about 3 Ångström, and he was convinced that electron microscopy had more to give.
Henderson produces the first image at atomic resolution
Over the following years, electron microscopy gradually improved. The lenses got better and cryotechnology developed (we will return to this), in which the samples were cooled with liquid nitrogen during the measurements, protecting them from being damaged by the electron beam.
Richard Henderson gradually added more details to the model of bacteriorhodopsin. To get the sharpest images he travelled to the best electron microscopes in the world. They all had their weaknesses, but complemented each other. Finally, in 1990, 15 years after he had published the first model, Henderson achieved his goal and was able to present a structure of bacteriorhodopsin at atomic resolution (figure 3).
Figure 3. In 1990, Henderson presented a bacteriorhodopsin structure at atomic resolution.
He thereby proved that cryo-EM could provide images as detailed as those generated using X-ray crystallography, which was a crucial milestone. However, this progress was built upon an exception: how the protein naturally packed itself regularly in the membrane. Few other proteins spontaneously order themselves in this way. The question was whether the method could be generalised: would it be possible to use an electron microscope to generate high-resolution 3D images of proteins that were randomly scattered in the sample and oriented in different directions? Richard Henderson believed it would be, while others thought this was a utopia.
On the other side of the Atlantic, at the New York State Department of Health, Joachim Frank had long worked to find a solution to just that problem.
In 1975, he presented a theoretical strategy where the apparently minimal information found in the electron microscope’s two-dimensional images
could be merged to generate a high-resolution, three-dimensional whole. It took him over a decade to realise this idea.
Frank refines image analysis
Joachim Frank’s strategy (figure 4) built upon having a computer discriminate between the traces of randomly positioned proteins and their background in a fuzzy electron microscope image. He developed a mathematical method that allowed the computer to identify different recurring patterns in the image. The computer then sorted similar patterns into the same group and merged the information in these images to generate an averaged, sharper image. In this way he obtained a number of high-resolution, two-dimensional images that showed the same protein but from different angles. The algorithms for the software were complete in 1981.
Figure 4
The next step was to mathematically determine how the different two-dimensional images were related to each other and, based on this, to create a 3D image. Frank published this part of the image analysis method in the mid-1980s and used it to generate a model of the surface of a ribosome, the gigantic molecular machinery that builds proteins inside the cell.
Joachim Frank’s image processing method was fundamental to the development of cryo-EM. Now we’ll jump a few years back in time – in 1978, at the same time as Frank was perfecting his computer programs, Jacques Dubochet was recruited to the European Molecular Biology Laboratory in Heidelberg to solve another of the electron microscope’s basic problems: how biological samples dry out and are damaged when exposed to a vacuum.
Dubochet makes glass from water
In 1975, Henderson used a glucose solution to protect his membrane from dehydrating, but this method did not work for water-soluble biomolecules. Other researchers had tried freezing the samples because ice evaporates more slowly than water, but the ice crystals disrupted the electron beams so much that the images were useless.
The vaporising water was a major dilemma. How- ever, Jacques Dubochet saw a potential solution: cooling the water so rapidly that it solidified in its liquid form to form a glass instead of crystals. A glass appears to be a solid material, but is actu- ally a fluid because it has disordered molecules.
Dubochet realised that if he could get water to form glass – also known as vitrified water – the electron beam would diffract evenly and provide a uniform background.
Initially, the research group attempted to vitrify tiny drops of water in liquid nitrogen at –196°C, but were successful only when they replaced the nitrogen with ethane that had, in turn, been cooled by liquid nitrogen. Under the microscope they saw a drop that was like nothing they had seen before. They first assumed it was ethane, but when the drop warmed slightly the molecules sud- denly rearranged themselves and formed the fami- liar structure of an ice crystal. It was a triumph – particularly as some researchers had claimed it was impossible to vitrify water drops. We now believe that vitrified water is the most common form of water in the universe.
A simple technique for contrast
After the breakthrough in 1982, Dubochet’s research group rapidly developed the basis of the technique that is still used in cryo-EM (figure 5). They dissolved their biological samples – initially different forms of viruses – in water. The solution was then spread across a fine metal mesh as a thin film. Using a bow-like construction they shot the net into the liquid ethane so that the thin film of water vitrified.
Figure 5
In 1984, Jacques Dubochet published the first images of a number of different viruses, round and hexa- gonal, that are shown in sharp contrast against the background of vitrified water. Biological material could now be relatively easily prepared for electron microscopy, and researchers were soon knocking on Dubochet’s door to learn the new technique.
From blobology to revolution
The most important pieces of cryo-EM were thus in place, but the images still had poor resolution. In 1991, when Joachim Frank prepared ribosomes using Dubochet’s vitrification method and analysed
the images with his own software, he obtained a 3D structure that had a resolution of 40 Å. It was an amazing step forward for electron microscopy, but the image only showed the ribosome’s conto- urs. Frankly, it looked like a blob and the image did not even come close to the atomic resolution of X-ray crystallography.
Because cryo-EM could rarely visualise anything other than an uneven surface, the method was sometimes called “blobology”. However, every nut and bolt of the electron microscope has gradually been optimised, greatly due to Richard Henderson stubbornly maintaining his vision that electron microscopy would one day routinely provide images that show individual atoms. Resolution has improved, Ångström by Ångström, and the final technical hurdle was overcome in 2013, when a new type of electron detector came into use (figure 6).
Figure 6. The electron microscope’s resolution has radically improved in the last few years, from mostly showing shapeless blobs to now being able to visualise proteins at atomic resolution. Image: Martin Högbom.
Every hidden corner of a cell can be explored
Now the dream is reality, and we are facing an explosive development within biochemistry. There are a number of benefits that make cryo-EM so revolutionary: Dubochet’s vitrification method is relatively easy to use and requires a minimal sample size. Due to the rapid cooling process, biomolecules can be frozen mid-action and researchers can take image series that capture different parts of a process. This way, they produce ‘films’ that reveal how proteins move and interact with other molecules.
Using cryo-EM, it is also easier than ever before to depict membrane proteins, which often function as targets for pharmaceuticals, and large molecular complexes. However, small proteins cannot be studied with electron microscopy, but they can be visualised using NMR spectroscopy or X-ray crystallography.
After Joachim Frank presented the strategy for his general image processing method in 1975, a researcher wrote: “If such methods were to be perfected, then, in the words of one scientist, the sky would be the limit.”
Now we are there – the sky is the limit. Jacques Dubochet, Joachim Frank and Richard Henderson have, through their research, brought “the greatest benefit to mankind.” Each corner of the cell can be captured in atomic detail and biochemistry is all set for an exciting future.
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