從細菌到人類，所有的生物都由細胞組成。細胞由四種大型生物分子構(gòu)成：碳水化合物、脂肪、核酸（即DNA和RNA）和蛋白質(zhì)。這些生命的重要組成部分小到肉眼無法觀測，甚至用光學顯微鏡也難以成像。因此，盡管19世紀的科學家們知曉這些"隱形"分子的存在，也能夠通過實驗找出它們的化學成分，但科學家們卻看不到它們：這些分子結(jié)構(gòu)的任何細節(jié)始終是個謎題。這就是今天的主題：這些"隱形"分子是如何在20世紀被人們成功觀測到的。?

Every living thing, from bacteria to?our own bodies, is made up of cells. And those cells are built from four kinds of large biological molecules: carbohydrates, fats, nucleic acids (that’s DNA and RNA) and proteins. These vital components of life are too small to be seen by the naked eye, or even by a light microscope. So even though 19th century scientists knew these “invisible” molecules were there — and they could do experiments to work out their chemical constituents — they couldn’t see them; they couldn’t make out their shapes in any detail. This is the story of how the invisible became visible in the 20th century.?

"許多基礎的生物問題是非常容易解決的：只要能看到它們就行！"?—理查德·費曼

“It is very easy to answer many fundamental biological questions: you just look at the thing!”?—Richard Feynman

這是一個漫長而艱辛的故事：關于開發(fā)能夠解析生物分子結(jié)構(gòu)的工具和技術，以及對這些分子結(jié)構(gòu)的解析如何使我們能夠理解它們的功能，并設計出阻止或加強其作用的藥物。

It’s the story of a long, laborious slog to develop the tools and the techniques that would reveal the structure of biological molecules — and how seeing the structure of these molecules enabled us to understand how they work and to design drugs that block or enhance their actions.

為了講述這個故事，我們將重點放在蛋白質(zhì)上：這些大分子參與了我們身體中幾乎所有的化學過程：它們解讀遺傳密碼、催化化學反應、并充當我們細胞的守門員。蛋白質(zhì)由名為氨基酸的小分子鏈構(gòu)成。了解這些鏈如何折疊成三維結(jié)構(gòu)至關重要，因為正是蛋白質(zhì)的三維形態(tài)決定了它們的功能。

To tell this story, we’re focusing on proteins.?These large molecules facilitate just about every chemical process in our bodies: They “read” the?genetic code, they catalyze reactions, they act as the gatekeepers to our cells. Proteins are made up of chains of small molecules called amino acids.?Knowing how these chains fold up to create a three-dimensional structure is crucial, because it’s the 3D shape of proteins that determines how they work.

若要創(chuàng)建一個準確的蛋白質(zhì)三維模型，我們需要知道組成該蛋白質(zhì)的所有氨基酸中的所有原子在空間中的排列。?我們無法看到原子，因為它們比可見光的波長還要小。?為了探測這些原子，我們需要一種波長更短且穿透性極佳的波：這種波使我們能夠同時對蛋白質(zhì)內(nèi)部和外部的原子進行觀測。

To create an accurate 3D model of a protein, we need to know the arrangement in space of all of the atoms in all of the amino acids that make up that protein.?We can’t see atoms because they’re smaller than the wavelength of visible light.?To detect them, we need a different kind of wave — a wave with a shorter wavelength and one that can penetrate surfaces to show us not just the atoms on the outside but also the atoms within the protein.

因此，今天的故事開始于德國的維爾茨堡大學城。在那里，倫琴發(fā)現(xiàn)了X射線。

And so our story begins with the discovery of X-rays in a university town called Würzburg, in Germany.

本文轉(zhuǎn)載自Knowable Magazine

"Structural biology: How proteins got their close-up"

X射線的發(fā)現(xiàn)

Discovery of X-ray

那是1895年，威廉·倫琴正在實驗室里工作。像他那一代的許多物理學家一樣，他正在做陰極射線的實驗：在一個叫做克魯克司管的設備中產(chǎn)生的電子流。但與他同時代的人不同的是，倫琴注意到了一些意想不到的事情：離克魯克司管相當遠的一個屏幕在發(fā)光。倫琴認為，那個屏幕太遠了，發(fā)光絕不可能是由陰極射線引起的。在接下來的幾周里，他研究了這種發(fā)光的熒光，并意識到他發(fā)現(xiàn)了一種能夠穿透固體物體的新型射線。?就在圣誕節(jié)前，他把他的妻子帶到實驗室，給她的手拍了一張照片。?在照片中，她的血肉消失了，但骨頭和戒指都清晰可見。

It’s 1895 and Wilhelm R?ntgen is in the lab. Like many physicists of his generation, he’s experimenting with cathode rays — streams of electrons produced in a device called a Crookes tube. But unlike his contemporaries, R?ntgen notices something unexpected: a screen quite some distance from the Crookes tube is glowing — too far away to have been caused by cathode rays, he thinks. Over the next weeks he studies this glowing fluorescence and realizes that he’s found a new kind of ray that can penetrate solid objects.?Just before Christmas, he brings his wife into the lab to take a photograph of her hand.?In the photograph, her bones and ring — but not her flesh — are clearly visible.

關于他的發(fā)現(xiàn)，倫琴寫了一份的報告。1896年初，一份英文譯本發(fā)表了在《自然》雜志上。

R?ntgen writes an account of his findings and, in early 1896,?an English translation is published in the journal?Nature:

"我們看到，一些劑能夠穿透對紫外線、陽光或弧光不透明的黑色紙板。所以，研究其他物體能在多大程度上被同一個劑穿透是很有意義的。"

"It is seen, therefore, that some agent is capable of penetrating black cardboard which is quite opaque to ultra-violet light, sunlight, or arc-light. It is therefore of interest to investigate how far other bodies can be penetrated by the same agent."

該報告繼續(xù)說道：

The report continues:

"厚的木塊仍然是透明的。兩三厘米厚的松木板只吸收了很少的光線。一塊15毫米厚的鋁板仍然能夠讓X射線通過，但大大減少了發(fā)出的熒光。"

"Thick blocks of wood are still transparent. Boards of pine two or three centimetres thick absorb only very little. A piece of sheet aluminium, 15 mm. thick, still allowed the X-rays (as I will call the rays, for the sake of brevity) to pass, but greatly reduced the fluorescence."

倫琴的發(fā)現(xiàn)立即產(chǎn)生了影響。?在幾個月內(nèi)，醫(yī)生們就開始用X射線來拍攝骨折。?人們?yōu)閄射線寫詩，奇妙的X射線也成為各大展覽中的熱點。1901年，倫琴因其發(fā)現(xiàn)被授予第一個諾貝爾物理學獎：這是本故事中授予科學家們的眾多諾貝爾獎中的第一個。

R?ntgen’s discovery had an immediate impact.?Within months, doctors were using X-rays to photograph broken bones.?Poems were written about them and the “wondrous” X-rays became a popular attraction at exhibitions.?And, in 1901, R?ntgen was awarded the very first Nobel Prize in Physics for his discovery — the first of many Nobel Prizes awarded to scientists in this story.

與此同時，在實驗室里，物理學家們對X射線的性質(zhì)感到困惑。它們究竟是波還是粒子？另一位德國物理學家馬克斯·馮·勞厄推斷，如果X射線是波，那么它們的波長可能與晶體中原子之間的規(guī)則空間相似，從而提供一種破譯晶體結(jié)構(gòu)的方法。?

Meanwhile, back in labs, physicists puzzled over the nature of X-rays: Were they waves or particles??If X-rays were waves, reasoned Max von Laue, another German physicist, then their wavelength might be similar to the regular spaces between atoms in a crystal, providing a way to decipher the structure of crystals.?This was a very important insight.

這是一個非常重要的推斷，它啟蒙了X射線晶體學的發(fā)展，這種技術最終將使科學家們能夠弄清蛋白質(zhì)結(jié)晶的結(jié)構(gòu)，但走到這一步卻花了幾十年。起初，X射線晶體學被應用于更小的分子。而在這之前，弄清楚該技術的原理也花費了很長的時間。

It led to the development of X-ray crystallography, the technique that would eventually enable scientists to figure out the structure of crystallized proteins.?But it took several decades to get to that point. At first, X-ray crystallography was applied to much smaller molecules. And before that, the technique itself had to be figured out.

X射線晶體學時代

Era of X-ray Crystallography

1912年夏天，數(shù)學家和物理學家威廉·亨利·布拉格和他的兒子，另一位物理學家勞倫斯·布拉格在英國的海邊度假時聽聞了馮·勞厄的一個講座。?假期結(jié)束后，父子倆回到他們的大學，思考晶體對X射線的衍射問題。那年晚些時候，老布拉格給《自然》雜志寫信。?他首先描述了通過發(fā)射X射線獲得的顯著效果。

In the summer of 1912, mathematician and physicist William Bragg and his son Lawrence — also a physicist — were on holiday by the coast in Britain when they heard about a lecture given by von Laue.?After the holiday, father and son returned to their universities and thought about the diffraction of X-rays by crystals. Later that year, William Bragg?wrote to the journal?Nature.?He began by describing the remarkable effects obtained by passing…

"...細小的X射線流在通過晶體后并被發(fā)射到照相板時，有了顯著效果。在照相板上發(fā)現(xiàn)了一種奇怪的斑點排列，其中一些斑點與中心斑點相距甚遠，以至于它們必須被解釋為大角度的散射....."

"… a fine stream of X-rays through a crystal before incidence upon a photographic plate. A curious arrangement of spots is found upon the plate, some of them so far removed from the central spot that they must be ascribed to rays which make large angles…"

這些是被晶體中的原子散射的X射線，在膠片上形成了一個獨特的斑點圖案。

These are the X-rays that are scattered by the atoms in the crystal, causing a distinctive pattern of spots on the photographic plate.

"這些斑點的位置似乎取決于簡單的數(shù)字關系，以及晶體對入射流的呈現(xiàn)方式。我發(fā)現(xiàn)，當晶體（鋅閃石）被放置到入射光線平行于晶體中立方體的邊緣時，斑點的位置可以通過以下簡單規(guī)則預測。假設原子以矩形方式排列，相鄰原子產(chǎn)生的斑點距離為NA，其中A是相鄰原子之間的距離，而N是一個整數(shù)......"

"The positions of these spots seem to depend on simple numerical relations, and on the mode in which the crystal presents itself to the incident stream. I find that when the crystal (zincblende) is placed so that the incident rays are parallel to an edge of the cube in the crystal the positions of the spots are to be found by the following simple rule. The atoms being assumed to be arranged in rectangular fashion, any direction which joins an atom to a neighbour at a distance?na?from it, where?a?is the distance from the atom to the nearest neighbours and?n?is a whole number…"

布拉格父子找到的數(shù)學規(guī)則提供了一種解釋X射線產(chǎn)生的衍射圖案的方法，從而揭示了晶體中原子的排列。

The mathematical rule hit upon by the Braggs provided a way to interpret the diffraction patterns produced by the X-rays, thus revealing the arrangement of atoms in the crystal.

老布拉格設計了一種新的、更強大的方法來進行X射線衍射，發(fā)明了一種叫做X射線光譜儀的儀器。

William Bragg devised a new, more powerful method for carrying out X-ray diffraction, inventing an instrument called the X-ray spectrometer.

1914年，馮·勞埃因其工作獲得了諾貝爾獎。第二年，布拉格父子也得到了諾貝爾獎。當時只有25歲的小布拉格目前仍是最年輕的諾貝爾獎科學得主。

In 1914 von Laue was awarded a Nobel Prize for his work. The following year, William and Lawrence also got the gong. Lawrence, only 25 at the time, is still the youngest scientist to receive a Nobel Prize.

起初，布拉格的方法被應用于簡單物質(zhì)，如食鹽、苯和糖分子，揭示了它們結(jié)構(gòu)的秘密。許多科學家對像蛋白質(zhì)結(jié)構(gòu)這樣復雜的東西能否用這種方法解析持懷疑態(tài)度。1936年，《生物化學年度評論》中討論了X射線研究的進展。

DOI:?10.1146/annurev.bi.05.070136.000431

At first, the Bragg method was applied to?simple substances such as table salt, benzene and sugar molecules, revealing the secrets of their structures.?Many scientists were skeptical that something as complicated as a protein structure could ever be determined in this way.?In 1936, the progress of X-ray studies was discussed in the?Annual Review of Biochemistry.

"對于像糖和氨基酸這樣的晶體物質(zhì)，晶體內(nèi)分子和原子的排列是能被完全解析的；但對于像多糖和蛋白質(zhì)這樣的物質(zhì)，其中原子的排列不太規(guī)則，同時缺乏共同的晶體外觀，我們不能指望完全解析它們。"

"For such crystalline substances as the sugars and amino acids complete knowledge of the crystal structure would show the arrangement of the atoms within the molecule as well as the arrangement of the molecules within the crystal; but for substances such as the polysaccharides and the proteins, in which a less regular arrangement of the atoms is accompanied by the lack of a common crystalline appearance, such complete knowledge is not to be hoped for."

但幾年后，即1939年，有人提出了一個更樂觀的觀點：作者指出，像X射線晶體學這樣的技術，正在深刻地改變生物學。?當作者考慮到各種可能性時，他似乎相當興奮。

DOI:?10.1146/annurev.bi.08.070139.000553

But a few years later, in 1939, a more optimistic view was put forward.?Techniques like X-ray crystallography,?the author noted, were changing biology profoundly.?The author seems quite giddy as he considers the possibilities.

"生物學迅速成為了一門分子科學，站在物理學和化學的肩膀上，生物學的前景廣闊，人們迫切地想知道生物學會將人類帶向何方。生物分子的結(jié)構(gòu)成為了學界的主流追求。這些分子中最重要的是蛋白質(zhì)，而蛋白質(zhì)的結(jié)構(gòu)解析也是最激動人心的。"

"Biology is fast becoming a molecular science, a desire to tread as far as possible the friendly ground of physics and chemistry and see where it leads. It may be that the angels are right, but it is good to feel and take part in a foolishness that is the scientific hall-mark of our times. The search is now for the structure and arrangement of the molecules of living things. Chief among these molecules are the proteins, and the greatest excitement these days is about the proteins."

為了解決蛋白質(zhì)問題，需要取得一些進展：尋找更好的蛋白質(zhì)結(jié)晶方法，并用新的數(shù)學方法解析X射線的衍射圖案；以及用計算機計算數(shù)據(jù)。?英國劍橋的科學家們正致力于應對所有這些挑戰(zhàn)。

To tackle proteins, several advances were needed: better ways of coaxing proteins into crystals; new mathematical methods for interpreting diffraction patterns; and computers for crunching the data.?Scientists in Cambridge in the UK were working on all of these challenges.

1953年，X射線晶體學獲得了巨大突破：它被用于解析一個極其重要的結(jié)構(gòu)，?并不是蛋白質(zhì)，而是DNA，詹姆斯·沃森、弗朗西斯·克里克和莫里斯·威爾金斯為此獲得了諾貝爾獎。

In 1953 the field got a boost when X-ray crystallography was used to solve an extremely significant structure.?It wasn’t a protein — it was DNA, for which James Watson, Francis Crick and Maurice Wilkins later received a Nobel Prize.

約翰·肯德魯是沃森和克里克在劍橋的同事，作為一位非常積極的研究人員，他下決心解析肌紅蛋白的結(jié)構(gòu)。?肌紅蛋白是在肌肉中儲存氧的蛋白質(zhì)?？系卖斶x擇它的原因是尺寸：肌紅蛋白并不大。?他的首要任務是培育適合被X射線解析的晶體。在嘗試對馬、鼠海豚、海豹、海豚、企鵝、烏龜和鯉魚的肌紅蛋白進行結(jié)晶后，他終于成功地培育出從抹香鯨肉中提取的肌紅蛋白的美麗晶體。?

Working alongside Watson and Crick in Cambridge was John Kendrew, a highly motivated researcher who was determined to solve the structure of the protein myoglobin.?Myoglobin is the protein that holds oxygen in muscles. Kendrew chose it because it’s not too big.?His first challenge was to grow crystals suitable for X-ray analysis.?After trying to crystallize myoglobin from horse, porpoise, seal, dolphin, penguin, tortoise and carp, he finally managed to grow beautiful crystals of myoglobin extracted from sperm whale meat.?

與此同時，肯德魯?shù)耐埋R克斯·佩魯茲開發(fā)了一種向蛋白質(zhì)分子添加"重"原子的技術。這些重原子并不會改變蛋白質(zhì)的結(jié)構(gòu)，但它們?yōu)楸容^不同角度的X射線照片提供了一個參考框架。經(jīng)過多年的工作，肯德魯仍然不知道肌紅蛋白中每一個原子的精確位置，但他擁有了足夠的信息，使得他可以制作一個蛋白質(zhì)的三維模型。?這個模型并不像DNA的雙螺旋那樣漂亮；它看起來更像一根扭曲的香腸。

Meanwhile, Kendrew’s colleague, Max Perutz, developed a technique for adding “heavy” atoms to protein molecules. The heavy atoms don’t change the structure of the protein, but they provide a frame of reference for comparing X-ray photographs taken from different angles. After years of work, Kendrew still didn’t know the precise position of every single atom in myoglobin, but he finally knew enough to make a 3D model of the protein.?It wasn’t as pretty as DNA’s double helix; it looked more like a coiled sausage.

就在這個時候，理查德·亨德森加入了這個小組。?直到今天，亨德森仍然在劍橋從事蛋白質(zhì)結(jié)構(gòu)解析的工作，并以開拓新技術而聞名，我們稍后將聽到這些技術。但那時他剛剛畢業(yè)，正在尋找一個博士生職位。他還記得從愛丁堡到劍橋參觀實驗室的情景：

It was around this time that Richard Henderson joined the group.?Henderson is still working on protein structure determination in Cambridge today and is known for pioneering new techniques, which we’ll hear about later.?But back then he’d just graduated and was looking for a PhD position. He remembers traveling from Edinburgh to Cambridge to visit the lab:

理查德·亨德森：?"他們有一個開放日，也就是星期六上午，他們周末居然也在工作！而在我去過的其他實驗室，科學家都回家了，積極性也不夠高。所以我當時就想：“哦，這是個非常好的實驗室”。

Richard Henderson:?“They had an open day, which was Saturday morning, and they’re all working! Whereas everywhere else I’d been, you know, they went home or they weren’t highly motivated. So I said, ‘Oh, this is a very good lab.’”

亨德森加入了這個勤奮的劍橋團隊。這項工作雖令人激動，但進展極慢。

Henderson joined the hardworking team in Cambridge. The work was exciting but extremely slow.

理查德·亨德森：?"在1959年，他們以非常高的分辨率得到了肌紅蛋白的結(jié)構(gòu)，1960年這項研究成果發(fā)表，之后的五年沒有任何其他結(jié)構(gòu)被發(fā)表，直到倫敦的皇家研究所發(fā)表了溶菌酶。然后在那之后，又過了三年才有了第三個結(jié)構(gòu)。"

Richard Henderson:?“They got the myoglobin structure at very high resolution, 1959, really, 1960 published, and then there wasn’t another structure for five years, which was lysozyme at the Royal Institution in London. Then after that, it was another three years until the third structure.”

難以相信科學家們花了這么久的時間，為什么進展如此緩慢？一開始，X射線晶體學家研究的小分子包含不到50個原子，例如苯和糖環(huán)。相比之下，肌紅蛋白，一種相對較小的蛋白質(zhì)，包含了超過1000個原子。為了弄清這么多原子的位置，科學家不得不拍攝數(shù)百張X光照片，測量每張照片中每個光點的強度，并進行繁瑣的計算。這是一個對數(shù)據(jù)處理的巨大挑戰(zhàn)。

The researchers put in long hours, so why was progress so slow??The small molecules that X-ray crystallographers had worked on first — things like benzene and sugar rings — contained fewer than 50?atoms.?By contrast, myoglobin, a relatively small protein, contains over a thousand atoms.?To figure out the position of that many atoms, they had to take hundreds of X-ray photographs, measure the intensity of each spot in each photograph and perform tedious calculations. It was a massive data-handling challenge.

理查德·亨德森："在我的博士論文中，我拍攝了大約300張這樣的照片，一開始我必須親自測量它們：我得把膠片放在膠片掃描儀里，一束光沿著一排斑點移動，然后每隔三分鐘，就能得到一張印有痕跡的紙，上面可能有40個斑點。這時我需要用尺子在紙上測量斑點被衍射的強度，然后再把這個數(shù)字打到電腦紙上。而這僅僅是一排斑點的工作量。"

Richard Henderson:?“In my PhD, I took about 300 of these procession photographs, and initially you had to measure them by hand: So you put the film in a film scanner, and a beam of light moved along the row of spots, and then you got every, say, three minutes, you got a piece of paper with the trace on, with maybe 40 spots on it, and you measured the strength of the diffraction spot with a ruler on a piece of paper and then you typed that number onto a computer paper — and that was just one row of spots.”

這是非常耗費時間的。?研究人員逐漸渴望如何將這一過程的一部分自動化。他們發(fā)明了自動的X射線探測器和儀器，以加快斑點的測量。?約翰·肯德魯意識到，解析一個結(jié)構(gòu)所需的計算可以由計算機來完成。?幸運的是，劍橋大學數(shù)學實驗室剛剛建成了第一批具有存儲程序的電子計算機。?它們被稱為EDSAC，肯德魯便學習了如何為它們編程。?隨著更強大的計算機的出現(xiàn)，X射線晶體學家們開始使用借助計算進行結(jié)構(gòu)解析。亨德森回憶說，在20世紀60年代，他們前往倫敦，使用帝國學院的IBM 7090。?劍橋大學的團隊每天可以使用這臺計算機1個小時。

It was hugely time-consuming.?Researchers gradually figured out how to automate parts of the process, inventing automatic x-ray detectors and instruments to speed up the measurement of spots.?Kendrew realized that the calculations needed to solve a structure might be done by a computer.?Fortuitously, one of the first electronic computers with a stored memory program had just been built in the Cambridge Mathematics Lab.?It was known as EDSAC, and Kendrew learnt how to program it.?As more powerful computers became available, the X-ray crystallographers made use of them.?Henderson recalls that in the 1960s, they traveled to London to use the IBM 7090 at Imperial College.?The Cambridge team had access to this computer for 1 hour a day.

理查德·亨德森?："于是，每天下午4點，一輛出租車就來了，帶著一批研究人員和一箱箱打包好的電腦卡，送到劍橋的火車站。她們上了去倫敦的火車，上了地鐵，在南肯辛頓站和帝國學院之間的隧道里帶著所有這些沉重的盒子走上大約有一公里。然后從晚上7點到8點，劍橋大學的MRC程序在計算機上運行，操作程序的人大多數(shù)是被招募的年輕女性，在當時被我們稱為 "計算機女孩"，她們現(xiàn)在都是大師了。在當時，她們做的極其完美：數(shù)據(jù)會被打印好并帶回來。第二天早上9點，每個研究員都會檢視他們前一天的數(shù)據(jù)，并為下午4點的寄送工作做好準備"。

Richard Henderson:?“And so every afternoon at 4 o’clock a taxi came and took somebody to the train station in Cambridge with boxes of punched computer cards. They got on the train to London, got on the Underground, walked in the tunnel between South Kensington Station and Imperial College — there was about half a mile or so — carrying all these heavy boxes. And then from 7 till 8 o’clock in the evening the MRC programs from Cambridge were run on the computer and then the person taking it — and most of them were young women who’d been recruited; they were called ‘computer girls’ at the time, they’re all now computer managers, they’ve done really well — they would bring the paper output back. And the next morning at 9 a.m., everybody would examine their work from the previous day, and get ready for the 4 p.m. run.”

?難怪這是個緩慢的工作! 女士們不僅要攜帶著成箱的數(shù)據(jù)穿越倫敦，她們還要抽出時間去做X射線晶體學解析。在倫敦國王學院，羅莎琳·富蘭克林制作了DNA的X射線衍射圖案。她的照片使沃森和克里克能夠制作他們著名的模型。?在牛津，多蘿西·霍奇金解決了青霉素的結(jié)構(gòu)，后來又研究了其他重要的醫(yī)學分子，包括維生素B12和胰島素。她于1964年獲得了諾貝爾獎，該領域的另一個諾貝爾獎!

No wonder this was slow work! Women weren’t only carrying boxes of computer code across London, they were also doing X-ray crystallography. At King’s College London, Rosalind Franklin produced X-ray diffraction patterns of DNA. Her pictures enabled Watson and Crick to make their famous model.?In Oxford, Dorothy Hodgkin solved the structure of penicillin and later worked on other medically important molecules, including vitamin B12 and insulin. She was awarded a Nobel Prize in 1964.?Yet another Nobel Prize for the field!

隨著更多計算機的出現(xiàn)和計算能力的提高，更多的結(jié)構(gòu)被解決了。計算機的持續(xù)進步是另一個主題，我們將稍后回到這里。

As more computers became available and computing power increased, more structures were solved. Continuing advances in computers is another theme to which we will return.

對結(jié)構(gòu)生物學這一新領域的興奮之情日漸高昂。一些科學家認為，最終他們甚至不需要X射線晶體學便能弄清蛋白質(zhì)的結(jié)構(gòu)。

Excitement about the new field of structural biology was growing. Some scientists believed that eventually?they wouldn’t even need X-ray crystallography to figure out the structure of proteins.

"人們甚至希望有一天可以完全從氨基酸序列中推斷出構(gòu)象。"

"Hopes have even been raised that it will someday be possible to deduce conformations solely from amino acid sequence."

那是在1965年在《生物化學年鑒》上被提出的。?當時的想法是，如果你知道展開的蛋白質(zhì)鏈中的氨基酸序列，那么通過遵循原子和分子如何相互作用的簡單規(guī)則，你可以算出蛋白質(zhì)鏈將如何折疊起來。DOI:?10.1146/annurev.bi.34.070165.001335

That was written in 1965, in the?Annual Review of Biochemistry.?The idea was that if you knew the sequence of amino acids in the unfolded protein chain, then by following simple rules governing how atoms and molecules interact, you could work out how the chain would fold up.

化學家克里斯蒂安·安芬森在1972年的諾貝爾獎演講中重復了這一主張。?

Chemist Christian Anfinsen repeated this claim in his Nobel Prize lecture in 1972:?

"我們對序列和三維結(jié)構(gòu)之間相關性的大量數(shù)據(jù)積累，加上多肽鏈折疊的能量學理論的日益成熟，預測蛋白質(zhì)構(gòu)象的想法越來越現(xiàn)實了。"

"Empirical considerations of the large amount of data now available on correlations between sequence and three dimensional structure, together with an increasing sophistication in the theoretical treatment of the energetics of polypeptide chain folding are beginning to make more realistic the idea of the?a priori?prediction of protein conformation."

這是一個有吸引力的想法。?如果可以用蛋白質(zhì)折疊的規(guī)則對計算機進行編程，并輸入氨基酸序列，那么結(jié)構(gòu)可能在幾天而不是幾年內(nèi)得到解決，為昂貴和耗時的實驗方法提供一個替代方案。

It was an attractive idea.?If computers could be programmed with the rules of protein folding and amino acid sequences inputted, then structures might be solved in days rather than years, providing an alternative to expensive and time-consuming experimental methods.

但現(xiàn)在還不行。為了實現(xiàn)這樣的目標，生物學家首先必須通過使用和改進X射線晶體學來解決更多蛋白質(zhì)的結(jié)構(gòu)。并通過發(fā)明新的方法來觀察蛋白質(zhì)。而這項工作將產(chǎn)生更多的諾貝爾獎。

But not yet. For something like that to happen, biologists first had to solve the structures of?a lot?more proteins by using and improving X-ray crystallography. And by inventing new ways of seeing proteins. And this work would lead to more Nobel Prizes.

在1999年的最后幾周，生物化學家羅杰·科恩伯格終于抵達了他十多年工作的頂點：他在斯坦福同步輻射實驗室成功解析出他一直在研究的蛋白質(zhì)的結(jié)構(gòu)。

In the final weeks of 1999, biochemist Roger Kornberg was reaching the culmination of over a decade of work. He was at the Stanford Synchrotron Radiation Laboratory, getting results that would at last show him the structure of the protein he’d been working on.

羅杰·科恩伯格：?"一開始的時候，我們遠遠不清楚是否可以做到。當然，這是讓我們從也許永遠不會成功的恐懼中解脫出來的原因，也是對最終結(jié)果感到振奮的原因。"

Roger Kornberg:?“When we began, it was far from clear that it could be done. It was, of course, cause for relief from the fear we would perhaps never succeed, and exhilaration at the final result.”

科恩伯格和他的團隊已經(jīng)解決了RNA聚合酶的結(jié)構(gòu)。?這是一個巨大的成就，并且得到了另一個諾貝爾獎的認可。

Kornberg and his team had solved the structure of RNA polymerase.?It was a huge achievement and one that was recognized with, yup, another Nobel Prize.

羅杰·科恩伯格：?"在我們解析這個結(jié)構(gòu)的時候還是20年前，但迄今為止，這依然是通過X射線衍射法研究的最大和最具挑戰(zhàn)性的結(jié)構(gòu)。"

Roger Kornberg:?“So at the time when we solved that structure, which was 20 years ago, it was by far the largest and most challenging investigated by X-ray diffraction.”

RNA聚合酶可以說是生物學中最重要的蛋白質(zhì)。?這是一個挑戰(zhàn)，因為它不是一個單一的蛋白質(zhì)。該團隊研究了來自酵母的RNA聚合酶，它實際上是由12種蛋白質(zhì)組成的。更重要的是，它是一個有活動部件的分子機器。

RNA polymerase is arguably the most important protein in biology.?It was a challenge because it’s not a single protein. The team studied RNA polymerase from yeast, which is actually made up of 12 proteins. What’s more, it’s a molecular machine with moving parts.

羅杰·科恩伯格："RNA聚合酶實際上是在讀取遺傳信息。因此，它負責決定哪些信息將被儲存在基因組的DNA中，以指導每個生物的活動能力。簡單如病毒，或復雜如人類，沒有生物體不依賴RNA聚合酶而生存。"

Roger Kornberg:?“The RNA polymerase literally reads the genetic information. So it is responsible for the capacity of what information is stored in the?genome?in DNA to direct the activities of every living thing. There is no organism as simple as a virus or complicated as a human that doesn’t rely on an RNA polymerase for life.”

為了解決RNA聚合酶的結(jié)構(gòu)，科恩伯格和他的團隊花了數(shù)年時間，為他們的蛋白質(zhì)尋找合適的晶體和"重?"原子。但這還不夠。他們還需要更強烈的X射線束。

To solve the structure of?RNA polymerase, Kornberg and his team spent years working on the right kind of crystals and “heavy” atoms for their protein. But that wasn’t enough. They also needed more intense beams of X-rays.

羅杰·科恩伯格：?"X射線衍射的方法依賴于結(jié)構(gòu)中各個原子的X射線光子散射--原子數(shù)量越多，為此必須記錄的散射光子數(shù)量就越大。?如果光束強度太低，光子的數(shù)量就太少了，獲得的信息也會因此不足。使用強度較高的光束，可以檢測和記錄更多的原子"。

Roger Kornberg:?“The method of X-ray diffraction relies upon scattering of the X-ray photons from the individual atoms in the structure — and the greater the number of atoms, the larger the number of scattered photons that must be recorded for the purpose.?If the beam is of low intensity, there are not many photons and so insufficient information is obtained. With a beam of higher intensity, more atoms can be detected and recorded.”

這一難題的解決方案便是同步加速器。同步加速器是一種粒子加速器，它以極高的速度推動電子束，這些高速電子發(fā)出的X射線比傳統(tǒng)的X射線要亮幾百萬倍。它本質(zhì)上是倫琴發(fā)現(xiàn)X射線時使用的克魯克司管的一個升級版本。

The solution came from synchrotrons. Synchrotrons are particle accelerators that propel beams of electrons at high speed — and the high-speed electrons emit X-rays that are millions of times brighter than conventional X-rays. It’s essentially a more powerful and much larger version of the Crookes tube that R?ntgen was using when he discovered X-rays.

來自同步加速器的高強度X射線和不斷提高的計算機能力相結(jié)合，使得像科恩伯格這樣的科學家能夠解決更復雜的蛋白質(zhì)結(jié)構(gòu)。

The combination of high intensity X-rays from synchrotrons and increasing computer power enabled scientists like Kornberg to solve more complex protein structures.

2007年至2019年，當我在《自然》雜志工作時，我們經(jīng)常對結(jié)構(gòu)生物學論文的數(shù)量開玩笑：似乎每周都有一個新的、重要的蛋白質(zhì)結(jié)構(gòu)發(fā)表。

When I was working at the journal?Nature?from 2007 to 2019, we used to joke about the number of structural-biology papers: there seemed to be a new, important protein structure published every week.

但這是有限制的。X射線晶體學仍然很耗時，盡管不像早期那樣耗時。?而且一些類型的蛋白質(zhì)被證明很難或不可能結(jié)晶。

But there were limitations.?X-ray crystallography was still time-consuming, although not as much as in the early days.?And some types of protein proved hard or impossible to crystallize.

冷凍電鏡時代

Era of CryoEM

在世紀之交，一種新的技術進入了人們的視野?；蛘哒f，一種新的技術讓科學家們對蛋白質(zhì)有了新的認識。?該技術不使用X射線，而使用電子束。?這就是所謂的冷凍電鏡。稱之為冷凍，是因為蛋白質(zhì)樣品會被凍結(jié)。理查德·亨德森是最早使用該技術的人之一。

At the turn of the century, a new technique came into view. Or, rather, a new technique gave scientists a new view of proteins.?Instead of using X-rays, the technique uses beams of electrons.?It’s called cryo-EM.?Cryo, because the protein sample is frozen.?EM for electron microscopy.?Richard Henderson was one of the first to use it.

理查德·亨德森：?"當你照射任何東西時，無論是用X射線還是電子，除了得到一個美麗的圖像外，分子實際上在被破壞，在一定的曝光后，分子已經(jīng)失去了它的結(jié)構(gòu)，所以在不得不因照射次數(shù)太多而停止之前，能得到的信息量是有限的，因為樣品已經(jīng)失活了。而事實證明，對于同樣數(shù)量的有用信息，電子所造成的損害要比X射線小一千倍。"

Richard Henderson:?“When you irradiate anything, whether it’s with X-rays or electrons, in addition to giving you a beautiful image, you are actually damaging the molecules, and after a certain exposure the molecule has lost its structure, so you’re limited in the amount of information you can get before you have to stop, because you’ve killed your sample. And it turns out that for the same amount of information that’s useful, the electrons do about a thousand times less damage than X-rays.”

對于冷凍電鏡，蛋白質(zhì)不需要是一個晶體。相反，它被從細胞中分離出來，然后冷凍到液氮溫度或以下。?冷凍有助于保護蛋白質(zhì)免受輻射損害。

For cryo-EM, the protein doesn’t need to be a crystal. Instead, it is isolated from the cell and then frozen to liquid nitrogen temperature or below.?The freezing helps to protect the protein from radiation damage.

亨德森將該技術應用于嵌入細胞膜的蛋白質(zhì)。事實證明，這些大型蛋白質(zhì)復合物極難通過X射線晶體學進行研究。?冷凍電鏡變得非常流行。?在2000年代，科學家們談到了一場 "冷凍電鏡革命"，許多人從X射線晶體學轉(zhuǎn)向了這種新的、更快的技術。2017年，理查德-亨德森被授予諾貝爾獎。

Henderson applied the technique to proteins embedded in cell membranes. These large protein complexes had proved extremely hard to study by X-ray crystallography.?Cryo-EM became extremely popular.?In the 2000s, scientists talked about a “cryo-EM revolution” and many switched from X-ray crystallography to the new, faster technique.?In 2017, Richard Henderson was awarded a Nobel Prize.

與X射線晶體學一樣，隨著計算能力的提高，冷凍電鏡成為一個更強大的工具，使更多的數(shù)據(jù)能夠更快地被分析出來。

Like X-ray crystallography, cryo-EM became a more powerful tool as computing power increased, enabling more data to be analyzed more quickly.

羅杰·科恩伯格："我們不能低估計算能力的非凡進步所做出的貢獻。從這個角度來看，就RNA聚合酶而言，當我們在1999年底記錄RNA聚合酶的X射線衍射以解決其結(jié)構(gòu)時，需要在制造商提供給我們的特制計算機上進行一個多月的計算。今天，同樣的計算可以在幾分鐘內(nèi)在一臺筆記本電腦上完成"。

Roger Kornberg:?“One cannot underestimate the contribution made by the extraordinary advance in computing power. To put it in perspective, in respect to RNA polymerase, when we recorded the X-ray diffraction from RNA polymerase at the end of 1999 to solve the structure, it required more than a month of computation on advanced computers made available to us not commercially available, contributed by the manufacturers.?Today, that same computation could be performed in a few minutes on a laptop computer.”

計算機一直是X射線晶體學和冷凍電鏡成功的關鍵。?現(xiàn)在我們是否可以完全摒棄這些實驗技術，而僅僅使用計算能力來預測蛋白質(zhì)的結(jié)構(gòu)？還記得克里斯蒂安·安芬森在其諾貝爾演講中提出的挑戰(zhàn)嗎？

Computers have been key to the successes of both X-ray crystallography and cryo-EM.?Can we now do away with these experimental techniques all together and just use computing power to predict the structure of proteins? Remember the challenge set by Christian Anfinsen in his Nobel lecture?

"...使預測蛋白質(zhì)構(gòu)象的想法更加現(xiàn)實。"

"…?to make more realistic the idea of the a priori prediction of protein conformation."

AlphaFold的盛大登場

The grand debut of AlphaFold

為了預測一串氨基酸將如何折疊起來，科學家們使用了一個叫做"自由能"的概念。自由能使蛋白質(zhì)不穩(wěn)定。我們的想法是，氨基酸將以這樣一種方式折疊起來，以使自由能最小化。?

To predict how a string of amino acids will fold up, scientists use a concept called “free energy.”?Free energy makes a protein unstable.?The idea is that the amino acids will fold up in such a way as to minimize the amount of free energy.?

理查德·亨德森:?"你可以通過能量最小化來做結(jié)構(gòu)，最多可達60或70個氨基酸。所以美國西雅圖的大衛(wèi)·貝克小組在這方面做得特別好。但是一旦你想嘗試1000個氨基酸左右的蛋白質(zhì)，答案就會迅速變得遙不可及。"

Richard Henderson:?“You can do structures by energy minimization up to about 60 or 70 amino acids. So David Baker’s group in Seattle in the USA has been particularly strong in doing that. But once you are up to proteins of 1,000 or so, it gets rapidly out of reach.”

因此，這項技術對于弄清一個蛋白質(zhì)的一小部分，也許是一個重要的側(cè)鏈，是有效的。但是對于有數(shù)百或數(shù)千個氨基酸的整個蛋白質(zhì)，科學家們采用了不同的方法。他們并不是要求計算機從第一原理中找出結(jié)構(gòu)，而是利用已知的蛋白質(zhì)結(jié)構(gòu)數(shù)據(jù)庫訓練一種算法。?這就是谷歌的人工智能實驗室最近所做的，他們的蛋白質(zhì)預測算法AlphaFold在2020年的一次比賽中超過了所有其他的算法。

So the technique works for figuring out a small section of a protein — perhaps a significant side chain. But for whole proteins with hundreds or thousands of amino acids, scientists use a different approach. Instead of asking the computer to figure out the structure from first principles, they train an algorithm using a database of known protein structures.?This is what Google’s AI lab did recently, when their protein prediction algorithm, AlphaFold, outperformed all others at a competition in 2020.

羅杰·科恩伯格："AlphaFold的基礎確實來自于蛋白質(zhì)結(jié)晶學的悠久歷史和它的巨大成功，以及已經(jīng)解析并存入蛋白質(zhì)數(shù)據(jù)庫的巨量的結(jié)構(gòu)。AlphaFold的不同之處可能在于，其公司背景下大量的人工智能專家，這遠遠超出了任何個人學術研究者所能做到的，他們所擁有的計算能力，來自于分布在全球各地的頂級計算中心。從某種程度上說，他們除了將他們所擁有的資源用于解決一個經(jīng)過充分研究的、現(xiàn)在看來已經(jīng)解決的問題之外，也沒做太多貢獻嘛。

Roger Kornberg:?“The basis for it really comes from the long history of protein crystallography and its great success and the extraordinary number of structures that have been solved and deposited in the protein database. What is probably different about AlphaFold is the amount of AI expertise they could bring to bear in the corporate context, which goes so far beyond what any individual academic investigator can do, the power of the computation which they possess which is extraordinarily distributed over countless extraordinarily expensive computational centers around the globe.?In a way they, contributed little beyond bringing the resources that they possess to bear on what was a well-studied and in retrospect solved problem.”

科恩伯格當然認識到像AlphaFold這樣的蛋白質(zhì)預測程序在預測非常多的蛋白質(zhì)結(jié)構(gòu)方面的潛力，包括那些以前沒有被解決的蛋白質(zhì)。

Kornberg certainly recognizes the potential of protein prediction programs like AlphaFold to predict the structures of a very large number of proteins, including ones that have not been solved before.

羅杰·科恩伯格：?"而如果預測的數(shù)量足夠多，那么AlphaFold對生命科學，尤其是生物學的影響是深遠的。"

Roger Kornberg:?“And if the number is great enough, then the impact upon life science and biology in particular is profound.”

了解蛋白質(zhì)的結(jié)構(gòu)本身就很有啟發(fā)性和滿足感，但它也使我們能夠設計出更好的藥物，最近對新冠病毒的研究就表明了這一點。?稱為蛋白酶的酶幫助病毒進行復制，其中也包括冠狀病毒。所以它們一直是藥物的靶點。

Understanding the structure of proteins is enlightening and satisfying in itself, but it also enables us to design better drugs, as has been shown in the recent efforts to deal with Covid.?Enzymes called proteases help viruses, including coronaviruses, to replicate.?So they’ve been an obvious target for drugs.

羅杰·科恩伯格：?"針對蛋白酶的藥物已經(jīng)用X射線衍射法進行了改進，通過觀察藥物與其靶點的結(jié)合，然后看看如何改進藥物的結(jié)構(gòu)以獲得對靶點的更好效果。"

Roger Kornberg:?“The drugs directed against the protease have already been refined using X-ray diffraction, much improved by observing the drug associated with its target and then seeing how one might improve the structure of the drug to gain better effect upon the target.”

X射線晶體學和冷凍電鏡已經(jīng)非常成功，理查德·亨德森認為我們已經(jīng)接近解析所有蛋白質(zhì)的結(jié)構(gòu)了。

X-ray crystallography and cryo-EM have been so successful that Richard Henderson thinks we’re close to solving the structure of every protein.

理查德·亨德森：?"我們基本上已經(jīng)通過實驗確定了幾乎所有蛋白質(zhì)的結(jié)構(gòu)--可能是其中的一半，可能是其中的四分之三。而如果不是你感興趣的蛋白質(zhì)，例如一個瞄準病毒的藥物，就會有一些同源結(jié)構(gòu)。"

Richard Henderson:?“We basically have, experimentally, determined the structure of almost all the proteins — it may be half of them, it may be three-quarters of them. And if not the protein you’re interested in — for example, a drug targeting a virus — there’ll be some homologous structure.”

實驗技術和人工智能的結(jié)合是否會如此成功，以至于讓結(jié)構(gòu)生物學家失業(yè)？亨德森記得，多年前科學家們有一長串他們想解決的蛋白質(zhì)結(jié)構(gòu)清單。

Will the combination of experimental techniques and AI be so successful that it will put structural biologists out of a job? Henderson remembers that years ago scientists had long lists of proteins whose structures they wanted to solve.

理查德·亨德森："我記得我們年輕的時候，在會議上，每個人都在研究一種蛋白質(zhì)，然后他們會說?‘我們接下來應該研究什么？’?每個人都會有自己的預期名單。我還記得我的名單，有核糖體、肌動蛋白、肌球蛋白、ATP酶、氧化還原酶、細菌素，所有這些結(jié)構(gòu)都是幾十年前解決的。所以現(xiàn)在如果你問人們什么結(jié)構(gòu)，他們會告訴你他們正在研究的那個，但他們已經(jīng)沒有一個大的名單了。"

Richard Henderson:?“I remember when we were younger, in meetings, everybody would be working on one protein, then they would say ‘What shall we work on next?’ And everybody would have their favorite list. I remember mine, we had ribosomes, actin, myosin, ATPases, redoxin, bacteriocin, all of these structures solved decades ago now. And so now if you ask people what structure, they’ll tell you the one they’re working on, but they don’t have a big list left anymore.”

現(xiàn)在他們已經(jīng)把大多數(shù)蛋白質(zhì)從名單上勾掉了，那么結(jié)構(gòu)生物學家還能做什么？

Now that they’ve ticked most proteins off their lists, what will be left for structural biologists to do?

理查德·亨德森：?"一旦你知道了所有東西的結(jié)構(gòu)，并且你已經(jīng)有了一種激活劑或抑制劑的藥物，之后你總是可以發(fā)明東西，盡管這個話題存在一些爭議。有這樣一個思路，即從所謂的發(fā)現(xiàn)科學到發(fā)明科學，在那里你為一些東西申請專利并開發(fā)一種新的化合物，這可能是一種新的蛋白質(zhì)。"

Richard Henderson:?“Once you know the structure of everything and you’ve got a drug that’s an activator or inhibitor, after that you can always — this is obviously contentious discussion, but — after that you can always invent things. There is this trajectory from what you could call discovery science to invention science, where you patent something and develop a new compound, which could be a new protein.”

亨德森正在談論的是合成生物學，這是一個相對較新的領域，在這個領域中，科學家們試圖制造新型的氨基酸和蛋白質(zhì)，對遺傳密碼進行設計，或從頭開始構(gòu)建簡單的細胞。

Henderson is talking about synthetic biology, a relatively new field in which scientists try to make new kinds of amino acids and proteins, to engineer the genetic code or to build simple cells from scratch.

生物學家們似乎有很多樂觀的看法。

There seems to be plenty of optimism among biologists.

“分子生物學家孤軍奮戰(zhàn)的日子已經(jīng)一去不復返了。實驗室、研究團隊和國家正在進行前所未有的合作，以解決那些緊迫的問題，從污染到能源到大流行?！?/p>

“Gone are the days where biomolecular scientists worked in isolation. Labs, teams, and nations are collaborating as never before to address pressing problems, from pollution to energy to pandemics.”

這句振奮人心的話出自2021年的《生物物理學年度評論》

DOI: 10.1146/annurev-biophys-091720-102019

These final words come from the Annual Review of Biophysics published in 2021.

隨著基因編輯、結(jié)構(gòu)解析等令人眼花繚亂的方法改進，以及AI計算的預測可靠性不斷提高，科學家們完全有能力解決科學、健康和工業(yè)方面的許多重要問題。

With gene editing approaches, dazzling improvement in structural determination, and increasing reliability of computational predictions, scientists are well positioned to address many important problems in science, health, and industry.

水木未來·視界iss.8