Fifty years on: why macromolecular cryocrystallography has been so successful
Fifty years after publication, a scientific paper by Purdue researchers contributed to ending the 2020 COVID-19 pandemic by helping to quickly provide the three-dimensional structure of the SARS-CoV-2 spike protein for vaccine development (structure-guided drug design). This research project began in 1965, but the most important phase was performed in the Purdue University laboratory of Michael Rossmann between 1968 and 1969. The results were published in April 1970 (Haas & Rossmann, 1970), which described the invention of a means to cryocool protein crystals to near-liquid-nitrogen temperatures to reduce radiation damage. The paper also described the discovery that cryocooling of protein crystals actually reduced the radiation damage by more than ten times [today, cryocooling is now considered to reduce radiation damage by more than seventy times (Garman, 1997, 2005)]. Beginning 20 years later, around 1990, cryocooling technology grew into a key element when using synchrotron radiation for routine X-ray analysis. (The synchrotron was developed decades earlier but only perfected for protein crystallography after 1970, and produces X-ray beams millions of times greater than conventional X-ray tubes.) Today, with more than 50 synchrotrons operating worldwide, cryocooling is universally used to reduce X-ray damage during macromolecular crystallography X-ray data collection at every synchrotron. Synchrotrons and advanced cryocooling have been developed by brilliant scientists, enabling macromolecular cryocrystallography to become a routine method of structure determination. In addition, much of the credit for demonstrating the success of cryocooling goes to Michael Rossmann, who had the foresight and understanding of its potential! Thank you – once again – Michael.
Remarkably, as so often is the case, the 'primary' benefits of cryocooling macromolecular crystals were completely unforeseen by the Purdue scientists:
Cryocooling enables repeated X-ray data collection from a single crystal.
Cryocooling enables better crystal handling for synchrotron X-ray analysis and facilitates very rapid data collection with the intense X-ray beam (minutes, not weeks or months).
Cryocooling provides a host of operational and molecular/crystallographic benefits for structure interpretation not available at room temperature, such as better stability and easy transport.
Because of the remote access required by the X-ray shielded synchrotrons, robotic handling is used, and the synchrotron scientists have provided amazing technology just for this purpose. They are to be congratulated, big-time!
The personal story behind the April 1970 scientific paper
With the 1970 published scientific results, I left the field of crystallography to enter industry. In 2004, I had a brief demonstration of cryocrystallography by Elspeth Garman, but it completely overwhelmed me after 30 years – unfortunately, I did not pursue it. However, in 2015, I attended the ACA Meeting in Philadelphia and then the X-ray Macromolecular Crystallography Course at Cold Spring Harbor Laboratory in 2017, which finally opened my eyes to the subject. I was invited by Stephen Burley to present a seminar at Rutgers PDB on 24 April 2019, and this preparation led to the 'History of Science' paper published in 2020 detailing this subject (Haas, 2020). Surprisingly, I only realized recently that it is the 50th anniversary of this event. Eureka!
Being able to perform this 'proof of principle' research on cryocooling protein crystals in the Rossmann Laboratory was more than just fortuitous. I doubt that I would have been able to perform this experiment anywhere else, first because Michael Rossmann had substantial insight into the future of crystallography, which led him to agree to support the work (and me), and second, the Rossmann Laboratory and Purdue University had all the proper apparatus and personnel that were needed. Hence, I was able to complete the task, which might otherwise have been delayed by years, perhaps decades.
This project began two years earlier (1965) when I arrived for my first postdoctoral fellowship position with David C. Phillips at the Royal Institution of Great Britain. He had constructed the very first automated X-ray diffractometer and, while collecting data on myoglobin and lysozyme protein crystals a few years before, found that this radiation damage problem proved to be serious. From this work, he experienced how the X-rays used to analyze the crystals caused them to deteriorate – the same problem that all other protein crystallographers had observed (and lived with!), except with his automatic diffractometer, he could observe the problem in real time [he wrote a scientific paper on this topic: Blake & Phillips (1962)]. Therefore, when I arrived, David Phillips suggested that I investigate this issue and see if I could reduce the radiation damage. That was the fortuitous beginning of the 'Radiation Damage Project' in September 1965.
I worked on it by myself at the Royal Institution for more than a year without any success. Then during the summer of 1966, David Phillips told me that the crystallographic group was leaving the Royal Institution and that I would need to transfer my work to another laboratory. He had already inquired as to available locations, and the one he suggested was the Weizmann Institute of Science in Rehovot, Israel. My wife, Sandra, and I agreed, and a few months later, we drove to Israel in late December 1966 with my research supplies in our car, so I could just continue the project there. Little did I know that this was to provide a major breakthrough for the invention of macromolecular cryocrystallography.
The Weizmann Institute Crystallography Laboratory was excellent, and the Director, Wolfie Traub, provided everything I needed. However, after several more months, I realized that I had to change the direction of the project, so Wolfie and I made a list of possible alternatives. Only one seemed realistic. If I froze (cryocooled) the protein crystals with liquid nitrogen, then this could possibly prevent the X-rays from damaging the protein crystals. In addition, to my surprise, Wolfie had constructed the exact apparatus I needed several years earlier – a liquid nitrogen 'gas-jet' cooling apparatus just sitting in the rear of the laboratory – unused. I immediately began the cryocooling experiments.
Within a few weeks I was able to cryocool the lysozyme crystals that I had brought with me. And then when I continued the X-ray exposure for several days to create the radiation damage I had always observed on dozens of crystals, the X-ray diffraction photographs showed little or no changes, indicating possible radiation damage protection. This was the first time I had observed this, and it proved to be a Eureka moment in my research! I spent the next few weeks performing additional experiments to better understand the phenomena, but this was already May, and the Six-Day War hysteria had already begun in Israel. On 5 May 1967, the American Embassy announced that all Americans should leave Israel because, if a war begins, there would be no transportation out. So, that same day I drove my wife with our six-month-old son to the airport so they could fly to London to stay with our English friends. At the airport, we booked the next flight, and they were safely away within hours. Without my family and most of the staff and personnel at the Weizmann Institute, I was kept from performing all the experiments I had planned.
However, I continued with some of the work even though I had to collect the liquid-nitrogen supply from the Weizmann Institute supply building myself. I was the only person in the entire chemistry building, and there was basically no one on campus. I just continued on my own, preparing all my meals and telephoning Sandy every day in London. With the war imminent, the Weizmann Institute was essentially closed, so any chance of completing the research at the Weizmann Institute was dashed.
The war began on the morning of 5 June 1967, but we only saw a few military aircraft overhead that day. Sometime in the morning of this first day, a group of Weizmann security personnel came around and instructed the group that they had assembled as to what we were to do during this time of war. I was told I would be in charge of fire protection for the Stone Administration Building. They showed me where the fire hoses were within the building. In a 15-minute training session, I was 'fully' trained, more or less, in how I was to fight any fire that should develop and whatever I needed to do if the building was damaged. With my portable radio and a bunch of books, I camped out on the porch of the building, sleeping there for the next three nights. Some of the security staff came by occasionally, but on the third day, they said the war had progressed sufficiently that there was no danger of aircraft, so I could return to my apartment.
The war was over in just six days, but in conversations with the few Weizmann staff that remained, they indicated that the Institute would not be reopened for many months, as everyone would remain in the military forces until the peace negotiations ended. I realized that I would need to make an immediate decision on the matter.
As I had previously arranged for a position with Michael Rossmann at Purdue University (during my previous year's job search in September 1966), I only needed to check that the position was still available. I contacted Michael to confirm this. He said I could return at any time, as he was certainly aware of the Six-Day War situation. Hence, I decided to return to the United States as soon as possible. This proved a good decision, so I packed all our belongings into our car, said goodbye to several individuals I knew who were still around the Institute, and drove to the Port in Haifa. Remarkably, within minutes, I located a ship sailing directly to London (Tilbury), and so I purchased the ticket, drove to the side of the ship where they loaded our car onto the deck with a crane, and wow – I was in England within days. What luck!
If not for these fortuitous events, the proof experiments of reduced radiation damage by cryocooling crystals would probably have been delayed for decades. In addition, my final phase of work with Michael Rossmann would probably not have occurred elsewhere as Michael was only convinced by the 'perfect' lysozyme X-ray precession photographs from the Weizmann Institute. He quizzed me extensively and studied all the precession photographs as if it were my final exam. Still, I was sure that the work at Purdue would provide quantitative evidence (proof) for reduced radiation damage. And that is precisely how it happened!
The Rossmann Laboratory was a state-of-the-art crystallography laboratory and there were a host of postdocs and graduate students: Margaret Adams, Alan Wonacott, Michael Schevits and Alex McPherson. They were all working on the lactate dehydrogenase (LDH) protein project (Wonacott et al., 1968). The laboratory was very pleasant, and the group was always willing to help me learn the new methods. I had written a short paper describing my Weizmann Institute findings for reduced radiation damage at –50°C on the return trip to the United States, so this was distributed to Michael and the other members of the Rossmann group for their support of my project proposal (Haas, 1968).
Everyone was certainly skeptical of the benefits of possibly extending the useful life of the crystals, but I was adamant that it was worthwhile even though the 'perceived' complexity and difficulty of building, installing and operating cryo-apparatus may be a barrier. I think everyone assumed that the radiation-damage reduction would be only a few hours, certainly not the hundreds of hours that resulted. (No one could even imagine the importance of cryocooling crystals with synchrotron-radiation sources.) Again, the only convincing demonstrations of reduced radiation damage were the lysozyme precession photographs from the Weizmann Institute, but the general notion at the time was that cooling protein crystals would damage them to a point where the X-ray data could not be collected.
After weeks of discussion, Michael agreed to loan me one of the Picker automated X-ray diffractometers for three months (and no more!) in the spring of 1968, and he would fund the purchase and construction of the most primitive cryocooling apparatus if I put the system together myself. (It probably would only be used once for my project anyway.) The Purdue supply department arranged for liquid nitrogen to be delivered and provided me with several large Dewar flasks. The Purdue glass workshop fabricated a coaxial gas-delivery jet for directing the nitrogen gas onto the crystal with a dry-air outer barrier jet surrounding the nitrogen stream. I made it to exactly match the Weizmann Institute apparatus. Initially, the single crystals were each mounted in a Lindemann glass vial in the conventional manner along with a small amount of mother liquor exactly as had been done for more than 30 years, but soon after that, I froze the crystals positioned near the end of a single glass fiber where it remained attached by a small amount of frozen liquid. This was a major step forward. Michael wanted the work to be performed on his protein (LDH, not lysozyme), and enough of his crystals were available for me to use (this proved to be an important decision!). Sucrose was tested as a cryoprotectant to prevent ice formation in the LDH crystals as soon as the cryo-equipment was ready – it worked perfectly.
A temporary setup was rigged up for the precession camera, which successfully obtained precession photographs of cryocooled LDH crystals. Therefore, after Michael approved the project in late 1967, the cryo-apparatus was ready to perform the data collection in the spring of 1968. The Purdue glass making facility and laboratory supply group were invaluable. With the cryo-gas tube assembly ready, simple control circuits were fabricated, and we purchased a suitable air dryer and several bathroom scales for tracking the weight of the liquid-nitrogen-filled Dewars (the scale weight indicates the remaining liquid nitrogen in the Dewar). Three months of work were required for the data-collection portion of the project, and I began the work in April 1968.
We found that initially cooling the protein crystals in a cold gas jet was unsuitable because the crystal faces nearest the gas jet froze more rapidly and asymmetrically. Thus, the usual mounting procedure consisted of placing a single LDH crystal (approximately 0.5 mm of each edge) on a strip of filter paper with a dropper to remove excess liquid, scooping the crystal up on the end of a 0.25 mm diameter glass fiber, and immediately plunging it into liquid nitrogen. The crystal was instantly frozen isotropically to the glass fiber and immobile. As the glass fiber tip had previously been mounted and aligned to the center of the goniometer head, the crystal was also nearly centered for the diffractometer. Finally, the fiber with the protein crystal was transferred quickly from the Dewar full of liquid nitrogen to the diffractometer, where a streaming jet of cold nitrogen gas prevented thawing. Ice formation was prevented using a coaxial dry nitrogen jet around the cold jet and by surrounding the whole diffractometer with a polyethylene bag filled with dry-nitrogen gas. A statement in our 1970 paper says that a thermocouple placed near the end of the cold jet provided a (nearly) continuous record of the crystal temperature, approximately –75°C.
The data collection proceeded from the first day, with only a few interruptions when mechanical issues with the Picker diffractometer occurred. We also collected data from LDH crystals at room temperature to prepare a comparison graph of a radiation-damage reference reflection (showing data collected at both 273°C vs –70°C). Punched cards controlled the Picker diffractometer, and it was run 24 hours a day with only a few delays. Otherwise, I fell into a routine for several months of loading the punched cards, filling the Dewar flask with liquid nitrogen and regularly checking the intensities of the two reference reflections to ensure that the crystal was aligned correctly. The experiment continued unimpeded for three months – this was a very exciting time.
We processed the data during the late summer and fall of 1968, and Michael spent several months analyzing the electron-density maps from our collected data (Haas & Rossmann, 1970). The data produced nice results, with the most important graph from these experiments showing a comparison of the reduced X-ray spot intensity decay of the two reference reflections. This radiation-decay graph [Fig. 3 in Haas (2020)] indicated that the X-ray damage in cryocooled LDH crystals was reduced more than tenfold at –75°C compared with room-temperature data. It was the first quantitative measure of radiation-damage reduction (finally, quantitative proof!). Michael wrote the final draft of the paper and submitted it to Acta Crystallographica in July 1969. The tenfold reduction was low compared with the 70-fold number that is now accepted, probably because of the higher temperature and temperature variations due to the nitrogen gas jet on the crystals. Reduced radiation damage on cryocooling crystals was independently confirmed by Gregory Petsko in his 1975 study on protein crystals at subzero temperatures (Petsko, 1975). This certainly contributed to the recognition of the benefits of cryocooling.
In 1969, after interviewing for positions at several universities and pharmaceutical companies, a perfect industrial position became available. I left basic scientific research in 1970 and joined the electronics industry as an X-ray scientist in May 1970 (Philips Electronic Instruments in Mount Vernon, New York), ending my protein crystallography career. I regret not being more diligent with my brief encounter in 2004 at the Chicago ACA meeting and in Oxford with Elspeth Garman and Louise Johnson; but finally, in 2015 when at lunch with Fran and Alex McPherson, Alex explained to me that cryocooling of protein crystals had become a universal technology for X-ray synchrotron data collection. He said it was used at all 50 synchrotrons around the world and used regularly by all protein crystallographers. I was amazed! I have now concluded that in 1970 I had so rejected and repressed any thoughts of cryocrystallography that this explains why I was totally unaware of the subject for 30 years.
Cryocooling was slow to be implemented, even though several crystallographers experimented in the field between 1975 and 1990. After 1970, it appears that crystallographers generally knew the benefits of cryocooling. After 1995, most protein crystal structure data were collected at synchrotrons from cryocooled crystals. (This is because the synchrotrons produce X-ray beams millions of times more intense than the older conventional vacuum X-ray tubes and destroy the protein crystals within seconds.) In addition, the development of structure-guided drug design became widely used (see Goodford, 1984) and was developed rapidly during the early 1980s. Structure-guided drug design has been a motivating force for the pharmaceutical industry, and with the AIDS epidemic in the 1980s, protein crystallography, synchrotrons and cryocooling had already become a major force for attacking these issues.
In early 2017, I was fortunate to be invited to attend the Cold Spring Harbor X-ray Macromolecular Crystallography Course, which had been given for more than two decades. In addition to this being my first direct exposure to modern crystallography after more than 45 years, I was able to participate in laboratory work employing cryocrystallography. I never thought much about this until I began reading about the 'AIDS Lazarus effect' (Cold Spring Harbor Laboratory, 2016). The AIDS Lazarus effect is just one example of the successes of structural biology. Later that year, on being invited by Stephen Burley to give the weekly Rutgers Protein Data Bank Seminar on 24 April 2019, I reviewed the extensive literature regarding the history and chronology of macromolecular cryocrystallography. For more than 30 years, synchrotrons have been successfully used for the X-ray data collection on thousands of protein crystals. This is clearly demonstrated by the fact that more than 94% of all FDA-approved drugs used PDB protein structures employing cryocooling [see the Garman figure in Haas (2020)]. The cryocooling invention developed and demonstrated in the Rossmann Laboratory at Purdue University has proven itself in real-world drug design and as one of the main technologies of the structural and molecular biology fields today. Furthermore, personally, my life is being extended because of one of these pharmaceuticals – Eureka!
I cannot thank enough the many scientists and groups who have enabled me to fill in this 50-year knowledge gap, and in particular, Elspeth Garman, whom I am privileged to know.
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