Crystallography of biological macromolecules at ultra-low temperature.

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Rev. Binphys. Binphys, Chern, 1990, 19: /07-26 Copyrighl © 1990 by Annual Reviews Inc, All righls reserved

Annu. Rev. Biophys. Biophys. Chem. 1990.19:107-126. Downloaded from www.annualreviews.org by NORTH CAROLINA STATE UNIVERSITY on 01/10/13. For personal use only.

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CRYSTALLOGRAPHY OF BIOLOGICAL MACROMOLECULES AT ULTRA�LOW TEMPERATURE Hakon Hope Department of Chemistry, University of California, Davis, California 95616 KEY WORDS:

biocrystallography, cryocrystallography, low-temperature data collection, x-ray diffraction methods.

CONTENTS PERSPECTIVES AND OVERVIEW,,,

108

DEVELOPMENT OF CRYOCRYSTALLOGRAPHY . ."""...... , ......"" ........"""....""".......""......"

110

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110 112

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113

Heater-Generated Gas Flow". ""...""""......"......""......"""......""......"....."""".. He at-Exchanger System Ice Prevention ,.."" .."""",..""", ....""......".....,........ ,, ..,,'......................................

114 115 116

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EQUIPMENT FOR CRYOCRYSTALLOGRAPHY

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THE METHOD

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117 118 119 120 120 121 122

Prevention of Radiation Damage "...............""" ...."......."........ " ......,,...................... Internal A lignme nt of the Crystal " ..""...... """.... "...... "............"........,,.......,,....,," Displacement (Temperature) "Factors ..."", ... ,' ..,... ,..,', ..... ,", ........"................".,,...., Absorption , ..,.....,",....."......".......,',..,............., ...........................,......." ....."',.....,',. Effect on Resolution and Structural Details .....".........""...."....."........."....,,"....,,"

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CONCLUDING NOTES

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PERSPECTIVES AND OVERVIEW

The spectacular progress in understanding life processes in recent years is intimately tied to advances in biocrystallography. Our understanding of a natural phenomenon is closely related to our ability to visualize inter relationships and processes connected to the phenomenon. In chemistry and biochemistry, a central feature of understanding is the ability to construct models of molecules. Unless we can describe the three-dimen sional structure of the molecular species or aggregates involved in a process, our understanding is severely limited. Consequently, major effort is expended in chemistry and biochemistry just determining thc structure of molecules. The advent of x-ray crystallography as a tool for structure determination in 1912 signaled a dramatic change in the way chemistry is perceived. From structure determinations of elements and simple com pounds, we have progressed, via small organic molecules with a few atoms, to large proteins, viruses, and perhaps in the not-too-distant future, to ribosomal particles. Single-crystal x-ray diffraction (with other diffraction methods) is the only method of structure elucidation that provides a mathematically direct path from primary measurements to a three-dimensional description of a chemical entity. The formula for the electron density p in a crystal at a point r from the unit cell origin: per) LHF(H) exp (-2niHr) illustrates this direct path. F(H) is closely related to the observed intensity of a diffraction maximum; the intensity is proportional to the product of F and its complex conjugate F*. The determination of an x-ray structure thus requires the measurement of a sufficient number of diffraction intensities to allow the calculation of the electron density per). For small molecules, this requirement generally does not cause serious problems. Biological macromolecules, however, often present formidable obstacles to these measurements. To begin with, the preparation of suitable crystals can be very difficult. The crystals invariably contain a large proportion of water, in the range of 30% to more than 80%. If this water escapes from the crystal, the structure will collapse, so special precautions must be taken to prevent drying. The classic solution has been to enclosc the crystal with some mother liquor in a thin-walled glass capillary. Lack of strict order, an inherent problem with biocrystals, results in a progressive weakening of diffraction intensities with increasing diffraction angle and limits the quality of the electron density derived from the data. Perhaps the most insidious problem arises because the very radiation needed to unravel a structure often severely damages the crystal. A dozen or more crystals are commonly needed to record a complete data set. The radiation damage problem was recognized during the early stages =


CRYOCRYSTALLOGRAPHY OF BIOMOLECULES

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of the development of biocrystallography. Researchers expected that low ering the temperature to near that of liquid nitrogen would reduce the rate of crystal destruction, but early attempts in this direction were not success ful. The prevailing thought was that freezing of the water in the crystal disrupted the structure. A moderate lowering of the temperature to just above the freezing point of water is helpful. Such techniques have been widely used because of their simplicity. They are not, however, a topic for this article. Later studies demonstrated that the addition of certain organic solvents prevented freezing of the crystal water, and some successful measurements were made (22, 23, 26, 3 1). These techniques are demanding, however, and did not find wide acceptance. In small-molecule crystallography, much less severe, but related prob lems were encountered. Ice formation was not a factor because of the general absence of water from the crystals, and cryogenic techniques found useful applications. Some development of apparatus and methods took place, and commercial equipment became available. The development of techniques that did not require the use of capillaries for crystal protection at any stage was particularly important (11). Investigators eventually real ized that the high water content of biocrystals need not cause the formation of ice on cooling to cryogenic temperatures, but that the mother liquor accompanying the crystals could freeze and be a source of difficulty. Cryogcnic tcchniques were then transferred from small-molecule crys tallography and adapted to biocrystallography with generally excellent results. We now believe that virtually all biocrystals can be cooled to cryotemperatures, where serious radiation damage does not occur. A full data set can be collected from a single specimen. Crystals so susceptible to radiation damage at room temperature that data collection was impos sible are now available for study. The necessity of preparing large numbers of suitable crystals has been practically eliminated. Great improvements in data quality have enabled us to uncover structural details previously obscured. The full effect of the development of cryocrystallographic methods on biocrystallography cannot be assesscd yet, but it will undoubtedly be important. Production applications of the new methods are only a couple of years old. The lack of fully satisfactory, turnkey, commercial equipment impedes the rapid spread of expertise. The near future will most likely see a substantial improvement in the design of cryocrystallographic apparatus. This article presents details of the historical development of biological cryocrystallography and its present status. We developed the first new methods at the University of California, Davis ( 12), and carried them to a state of practical, routine applications in collaboration with others at

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the Weizmann Institute of Science, in Rehovot, Israel. Other significant advances were made at the Max Planck Research Unit for Structural Molecular Biology, Hamburg, in connection with the development of ribosome crystallography (13). An excellent, full discussion of low-temperature apparatus and tech niques for x-ray diffraction is available (25). DEVELOPMENT OF CRYOCRYSTALLOGRAPHY

Biocrystallography The damaging effects of x-ray irradiation on biocrystals became apparent with the earliest x-ray studies. Researchers also realized that lowering the temperature could potentially reduce radiation damage. As early as 1966, Low et al (18) described results of cooling experiments on insulin crystals. Although the crystals did diffract after cooling, the authors did not find the resulting reflection profiles acceptable and apparently concluded that crystal decay with time is better than sudden deterioration in reflection shape. Their crystals had been sealed in capillaries, in keeping with stan dard practice.

In another notable study, from 1970, Haas & Rossman (6) cooled crystals of lactate dehydrogenase after soaking them in sucrose solution. These crystals were mounted directly on glass fibers without capillary protection. Problems, such as the inability to obtain fully reproducible cell dimensions, discouraged the authors from continuing their low-tem perature work. They attributed their loss of success to the incorporation of sucrose in the structure. Although the method of Haas & Rossman did not directly lead to further development, it did contain two very important elements: the crystals were mounted without a capillary, and the need for rapid, uniform cooling was explicitly recognized. An underlying belief that the water in biocrystals would behave like normal water and undergo its own expansion upon freezing, thereby com promising the integrity of the crystals, probably diminished interest in pursuing further developments. The way to cryotemperatures went via modification of the solvent incorporated in the crystals. Undoubtedly, the inherent difficulty in conducting cryogenic data collection was another deterrent to development. The approach by Haas & Rossmann, and later a more systematic devel opment by Petsko and coworkers (22, 23), aimed at deliberately disrupting the water structure to prevent the formation of normal ice. Petsko's method was designed to replace the water with a cryosolvent ("antifreeze"). A commonly used solvent was 2-methyl-2,4-pentanediol (MPD). Crystals were readied for low-temperature treatment by soaking them in an aqueous



II I

solution of MPD, and were mounted in capillaries. Walter et al (31) reported an interesting application of this technique. They studied tryp sinogen crystals treated with 70% aqueous methanol at 173 K and 103 K after slow, step-by-step cooling. The authors were surprised that no phase separation took place in the crystals, although at 103 K the solvent outside the crystal did freeze, as evidenced by the appearance of powder diffraction lines. They used the phrase "solvent phase in the crystal" to indicate that the solvent might be considered crystallographically separate from the protein. A full data set could be obtained from just one crystal with synchrotron radiation. They also reported superb orientational stability throughout data collection. Parak et al (21) established that separation of an ice phase could be prevented by immersing the crystals in liquid propane. This technique is thought to result in extremely rapid cooling. The value of the procedure was demonstrated in an 80-K study of myoglobin by Hartman et al (7). Nothing indicated crystal decay, and a refinement revealed dramatically decreased B factors from an average of 14 N at 300 K to 5 N at 80 K. Frauenfelder et al (5) later expanded this study into a careful analysis of the thermal expansion of the protein part of metmyoglobin crystals. Although the available equipment limited the accessible data to 2-A res olution, the decrease in B values suggests a significant improvement in potential resolution over that attainable at room temperature. Drew et al (3) used a very slow cooling process to bring a capillary mounted DNA crystal from room temperature to 16 K. The reflection shape deteriorated somewhat but clearly no ice phase developed, in spite of the slow cooling. Again, B factors substantially decreased. Other reports of cooling experiments have appeared, but they were either variations of those described here or involved modifications to the macromolecules and did not represent promising, general approaches. The results reported by Low et al (18), Parak et al (21), Walter et al (31), and Drew et al (3) contain an important message: cooling the crystal to well below the freezing point of the solvent does not necessarily result in destruction of the crystal from internal ice formation. Although Hartmann et al (7) recognized the significance of this point, no further development was reported. The technical difficulties in performing the procedures and the lack of convenient, reliable cryocrystallographic equipment most likely hampered continued research. New development was tied to improvements in equipment and crystal handling in small-molecule work. We avoid using the terms shockJreezing or jiashJreezing to denote very rapid cooling. The word cooling is used consistently for this process because the termJreezing usually implies a liquid to solid phase transition, a process we want to prevent.

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Small-Molecule Crystallography To obtain the data required for high-precision studies of electron densities in small molecules (9, 15, 20), it had become necessary to establish reliable, convenient, and economical cryocrystallographic attachments in my lab oratory. The availability of this equipment, together with acquired exper tise in its use, provided the necessary background for developing methods of wider applicability, initially to support synthetic chemistry, and later biological crystaIIography. In a set of experiments designed to increase the utility of crystallography to synthetic chemistry, Hope & Nichols (14) demonstrated that fuIIy sat isfactory structural results could be obtained in much less time than had been thought necessary. The time for a structure determination was reduced from days or weeks to hours, without l oss of essential information. This was accomplished by deliberate experiment design aimed at just the necessary counting statistics and by use of up-to-date computing methods in structure solution. The decreased intensity-damping from thermal motion effects at low temperature by itself aIIows increased data collection speed. Five- to ten-fold intensity enhancement at (sin 8)/A around 0.5 A-I (I A resolution) is commonplace when cooling from room temperature to about 100 K. Numerous small molecule data sets also revealed a dramatic reduction in radiation damage at cryotemperatures. Initially, we had no special provision for the handling of highly reactive or air-sensitive samples. This deficiency became acutely apparent when excellent crystals of what was thought to be phenyllithium became avail able, as reported by Hope & Power ( 16). These crystals could not be exposed to air without disastrous results. We did have the option of a glove box and glass-mounting capillaries but their use conflicted with our general idea of ease and speed in structure determination. Therefore, we devised an approach that would protect the crystal with a viscous, nonreactive, saturated hydrocarbon oil (l0, 1 1). We worked with the crystals under a layer of oil at room temperature, attached an oil-coated crystal to a mounting pin, and then flash cooled it, oil and all, directly on the diffractometer. We would not have been successful without the dependable, convenient cryo-attachment for the diffractometer that we had available in our laboratory. Technically, the step from reactive small molecules to sensitive bio molecules should not be large. The general consensus among crys tallographers, however, was that the high water content in biocrystals would give rise to ice formation, phase separation, and destruction of the crystal. The counter-indications described above for myoglobin, tryp sinogen, and a DNA fragment did not dispell belief in the inevitability of



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phase separation. Because of this belief, perhaps, most biocrystallogra phers did not see a need for cryogenic diffraction equipment in their labora tories, and did not pursue systematic low-temperature studies. In any event, the decisive break with tradition came from a small-molecule laboratory. Experiments on crambin revealed that crystals of this protein could be cooled in a cold gas stream on the diffractometer without loss of crystal integrity. Since the first report of these experiments by Hope (10) and Teeter & Hope (27, 28), interest in biological cryocrystallography has rapidly increased. Cryo-methods now seem destined to become part of the repertoire of practitioners of biocrystallography. EQUIPMENT FOR CRYOCRYSTALLOGRAPHY

The methods discussed in this article cannot be implemented without the appropriate expertise and well-functioning cooling equipment. The operation of a cryogenic laboratory may seem like a relatively simple addition to the complexities of biocrystallography, but all aspects of cryo crystallography must be taken seriously, and all techniques must be applied with thorough discipline. There is little leeway; procedures must be carefully established and adhered to at all times. Not appreciating the thin margin between success and failure causes many difficulties with the use of cryocrystallographic equipment. The advice of experienced users can save needless frustration. Rudman (25) has written an excellent review of crystal cooling methods and equipment. Only its essential points will be described here. Almost all researchers now cool the sample crystal by passing a cold stream of nitrogen gas over it. An effective setup should satisfy three requirements: l . Ice formation on and near the sample must be prevented. 2. Temperatures of 130 K or lower should be readily attainable. 3. Temperature fluctuations should be within ± 2 K for the duration of intensity measurement. A major advance was made by Post et al (24) in 1951. They introduced the dual-stream technique (a cold stream surrounded by a sheath of warm gas for frost prevention) that made low-temperature crystallography acces sible to many laboratories and thus made further development possible. A gas flow cooling apparatus will have these key components; l. 2. 3. 4.

a gas generator, a system for delivery of cold gas to the crystal site, a frost prevention nozzle, and a coolant (normally liquid nitrogen) supply system.

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Additional devices may be used for temperature control and measurement. Two methods for producing the cold gas stream are commonly used: boiling liquid nitrogen with an electrical heating element, and cooling dry nitrogen gas in a liquid nitrogen heat exchanger. These methods will be discussed separately.


Heater-Generated Gas Flow Figure 1 shows a schematic setup for electrically controlled gas generation. Nitrogen gas evolution can be accurately controlled with a stabilized, variable-voltage power supply. A heating element inserted in the cold stream outlet tube serves as the main means of temperature adjustment. A temperature sensor is also inserted in this tube, as close to the outlet as is practicable (typically about 20 cm from the exit nozzle) without causing turbulent flow. A level sensor activates a pumping system which replenishes the liquid nitrogen. A very simple pump can be made by inserting a heating element near the inlet of the liquid transfer tube. Gas bubbles will push 9 d

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transfer tube supplying nitrogen to evaporator vessel; (e) exit tube for transfer of cold gas to crystal; if) filling funnel with air vent; (g) refill line from transport container (not shown). The pumping device is inserted into the lower end of tube

d. A thermocouple and a stream

heater are inserted through the inlet of tube e. An electrical heater keeps the outlet nozzle

of e frost free. These devices are connected to the control box. The liquid in the evaporator provides the reference temperature bath for the thermocouple.



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liquid through the tube, similar to the action in many coffee-brewing machines. The system requires no pressure control, so the supply container can be opened and refilled, without affecting pumping action, allowing continuous operation. This pump stops and starts quickly and cannot develop mechanical problems. It has been used successfully in this lab oratory for many years. This device can maintain temperature within ± 0 .1 K over several weeks. Electronically controlled precision in boiloff rate, a near constant (± 0.3 mm) liquid level in the evaporator vessel, good temperature insulation, and a well-thermostated laboratory all contribute to stability. Measured from the supply container, typical liquid nitrogen consumption with a crystal temperature around 85 K is 0.7 liter h-I. Transfer losses from transport containers increase the total nitrogen consumption, depending on individual installations. The apparatus sketched in Figure I is a modi fied version of equipment originally manufactured by Enraf-Nonius, based on the design principles of van Bolhuis (29). The main disadvantage of this system would be electronic complexity, with a corresponding increased possibility of component failure.

Heat-Exchanger System Figure 2 shows a schematic representation of a heat-exchanger cooling system. Nitrogen gas is produced by evaporation of liquid nitrogen directly

Figure 2 Schematic representation of heat-exchanger cooling apparatus. (a) 160- L transport container; (b, e) gas shut-off valves; (c) main liquid shutoff valve; (d) solenoid valve for liquid-ni trogen supp ly; if) p ressure regulator; (g) flow me ter and regulato r ; (h) coil of copper tubing to warm gas to room temperature; (0 heat exchanger with immersed coil of copper tubing; (j, k) optional level sensor and control box for refill; (I) exit tube for transfer of cold gas to crystal. Temperature control and frost prevention are similar to that shown in Figure

I. In normal operation, cooli ng gas is generated from the liquid outlet of (a) with valve (e) open. During tank exchange, gas from the vent outlet of (a) can be routed via valve (b) with valve (e) closed.


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from a transport container. The flow rate is controlled by a pressure and flow regulator combination. Coolant for the heat exchanger is supplied from the same container that supplies the gas, through a T joint and an electrically activated valve. Temperature control and measurement are similar to that described for the electrically controlled gas flow system. The simplicity of this device produces extreme reliability in operation. Accidental introduction of moist air into the gas stream when changing supply containers, however, will eventually lead to obstruction of the gas path. In practice, such clogging has occurred only after six to eight months of uninterrupted use. The main disadvantage of the device is lack of precise temperature control; when the stream heating element is used, control becomes more difficult. This difficulty stems mainly from inherently impre cise flow rate control. Without stream heating, temperature fluctuations remain within ± 2 K. For most applications in biocrystallography, such variations are of no consequence.

lee Prevention The most difficult aspect of low-temperature diffraction experiments is the prevention of ice formation at and around the sample crystal. The situation might seem impossible. How is a crystal in the open, surrounded by normal laboratory air, exposed to temperatures of 100 K or less, to remain free of ice for the days needed to measure a full protein data set? Experience shows, however, that frosting can be prevented by very simple means. The key lies in the construction of both the exit nozzle from the transfer line and the crystal mount and in their interaction. The dual-stream apparatus introduced by Post et al (24) was the first practical solution. More recent developments in my laboratory show that one can eliminate the outer warm gas sheath, which allows simpler design of the apparatus and also lowers the liquid nitrogen consumption. Figure 3 illustrates an effective device. The cold stream exits from the transfer Dewar tube through a mouthpiece heated by an electrical heating element. It must be warm enough to prevent condensation around the outiet, but no warmer. The crystal is mounted on a thin glass fiber soldered into the tapered tip of a copper mounting pin. The gas flow should be virtually laminar, and the parts of the mounting pin in contact with the stream should be smooth and free of turbulence-causing irregularities. So long as the metal tip sticks approximately 1 mm into the cold stream and the angle between the mounting fiber and the stream is obtuse, as shown, no frosting will occur. Copper should be used because it conducts heat better than materials such as brass, which are easier to work with, and therefore might appear preferable to the instrument maker. The conducted heat keeps the metal parts just outside the stream ice free. Using the solder method of



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Figure 3 Gas outlet with crystal in position. (a) Gas delivery tube with electrically heated nozzle; (b) laminar cold stream; (c) crystal attached to glass fiber (d); (e) solder attaching glass fiber to copper mounting pin (f). Note the angle between stream and crystal mount. This orientation results in much simpler and safer operation than the head-on mounting often used with orbitting nozzle equipment on four-circle diffractometers.

attaching the glass fiber is fast, produces a mechanically stable mount, and allows for easy reuse of the metal pin. The cold stream is deflected (vide infra) to avoid ice deposits that can form if the crystal is moved through the cold stream/room-air interface; the technique also provides a means of rapid cooling. THE METHOD

The first protein selected for cooling experiments in my laboratory was crambin, a small, relatively dry protein. Dr. M. Teeter, who first reported

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the structure of crambin with Hendrickson (8), supplied a sample and thorough instructions on crystallization method. The first crystal tried was just picked up from its mother liquor with a standard glass mounting fiber and flash cooled on the diffractometer. Peak profiles, similar to those at room temperature and resolution beyond 0.8 A showed that the crystal suffered no ill effect from the cooling Obviously no separation of an ice phase had taken place, and, in that sense, the experiment had yielded the desired result. Getting the crystal out of the mother liquor was not par ticularly easy, and we feared that less robust crystals would not tolerate the handling and subsequent transport through air, albeit brief. We tried the oil protection method that had been used to advantage with small molecules (10, I I ) with immediate success. Dr. W. A. Hendrickson pro vided a sample of bovine pancreatic trypsin inhibitor (BPTJ), which we subjected to the same treatment, with corresponding success. The observed resolution for cryotreated BPTJ was approximately 1.05-l. l A. Neither crambin nor BPTI showed any sign of radiation damage during data collection. These experiments have been described elsewhere in detail


.

(12).

The results were extremely encouraging and carried the promise of relief

from the problems of radiation damage that had always plagued bio crystallographers. We planned, and carried out more extensive studies in the next couple of years. The initial promise has largely been fulfilled. About 2/3 to 3/4 of all molecules studied could be successfully treated by the simple methods used for crambin and BPTI. Using an adaptation of the cryosolvent method (22, 23), one can cool virtually all remaining crystals to near liquid nitrogen temperature without loss of crystal integ rity. Greatly reduced radiation damage has characterized all cases for which data collection has been completed. Perhaps the most conspicuous application of the new cryotechnique has been the successful handling of many different crystals of ribosomal particles. Full data sets could be collected from single specimens, and indications of only minor radiation damage were observed (13). A sum mary is given here.

Crystal-Handling Techniques In our experience, the most straightforward approach is the oil protection method (12). It comprises three main steps:

1. Following the preparation of suitable crystals, a specimen is transferred to a hydrocarbon environment.

2. The mother liquor around the crystal is removed. The crystal is picked


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up on a mounting pin with a small amount of oil that serves initially as a barrier against drying and later as an adhesive to hold the crystal rigidly. 3. The crystal is flash cooled. A summary of instructions for this technique follows: Place a small drop of mother liquor containing a crystal on a microscope slide. The drop can be from a hanging or sitting drop crystallization, or it can be pipetted from a larger volume. Then cover the solvent drop with a layer of oil. We have used Paratone-N from Exxon, either neat or mixed with mineral oil to adjust the viscosity. Move the crystal into the hydrocarbon phase with a suitable tool-a fine-tip needle, a small pusher made from clear plastic, or other implement that can move the crystal without inflicting damage to it. One can often pull the crystal into the oil without actually touching it. Dry off any mother liquor remaining on the crystal using fine-pointed strips of slightly moistened filter paper. A dry paper tends to get clogged with oil and is less effective. Many crystals can be dried simply by dragging them rapidly through the oil. Most compounds tolerate the oil environment quite well. Some crystals, however, begin to crack almost immediately. We recommend that no crystal be exposed to the oil at room temperature for very long.

Transfer to Mounting Pin Transfer the dried crystal to a mounting pin and cool. Crystals that are not thin plates or needles can be picked up with a straight, round glass fiber. This is especially easy if the oil is reasonably viscous. Use tissue paper or dry filter paper to remove excess oil. We recommend a more elaborate mounting procedure if the crystals are easily deformed. Instead of the straight glass fiber, use a microspatula fashioned from an ultrathin piece of flat glass and a mounting fiber. Blow a glass bubble from a tube until it bursts. Cut one of the flimsy sheets formed in this way to size under the microscope, and attach it to a glass fiber, much the same way one would mount an air-stable crystal. A clear silicone rubber cement is the only adhesive that works well. It is very tacky, and remains soft long enough to allow the spatula blade to be positioned in the desired orientation. A very small amount of adhesive will provide a strong bond. Figure 4 shows sketches of three types of spatula. When one uses extremely thin glass, the straight spatula's crystal support can be made virtually absorption free. One may therefore choose to use the spatula method even for crystals that are mechanically robust to avoid introducing extraneous absorbing material in the x-ray beam path. We have discussed uses and preparation of microspatulas (12, 13).


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a

b

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Figure 4 Microspatulas. (a) Two views ofa straight spatula; (b) 900 spatula; (c) 900 double ("sandwich") spatula.

The Cooling Step Before selecting and mounting a crystal, the cryo-attachment must be cooled down to operating temperature, and function smoothly. Position the mounting pin on the goniometer head so that the crystal will be well positioned in the cold stream. Make several practice runs to find convenient hand positions and movements. Once this is well practiced and a crystal has been selected, action should be as quick as possible. Deflect the cold stream before inserting the mounting pin with the crystal into the goniometer head. A small piece of stiff paper or a microscope slide placed in front of the outlet nozzle will deflect the gas. Do not expose the crystal to the deflected stream. When the crystal has been positioned, suddenly remove the deflector and the crystal will be cooled rapidly and uniformly. If it remains clear after cooling, phase separation or phase transition has probably not occurred. An opaque crystal signals trouble. Fortunately, a significant majority of biocrystals fares well with this treat ment. Individual crystals from the same preparation usually behave in a very similar manner: if one crystal has been cooled successfully, the others will be too. Conversely, if one crystal disintegrates, the remaining ones most likely will do the same (F. Frolow & H. Hope, unpublished results). After successful cooling, carry out the usual procedures-optical center ing, determination of cell dimensions and orientation matrix, and data collection. Unlike working at room temperature, one has time to carry out all operations to a desired, optimal precision. The crystal will not move on its support, and it will not decay.

Trouble Cases Some crystals will cause difficulty. Phase transformation may take place, or, in spite of all precautions, there may be separation of an ice phase. If ice forms upon cooling, our remedy has been to treat it with a cryosolvent in an adaptation of Petsko's method (22, 23). We have needed only two



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solvents, ethylene glycol or MPD (F. Frolow, personal communication). Adding 20% to 30% of one of these solvents to mother liquor with crystals usually prevents ice separation. It is usually best to increase the cryosolvent concentration in steps, e.g. 5 to 10 to 15% and so on. Test a drop of the solution by cooling it as if it were a crystal. If the drop remains clear, the crystal will probably be safe. Although we may have too little experience for a firm conclusion, crystals grown from low-salt solutions apparently fare better with MPD, whereas those from high-salt solutions do better with ethylene glycol.

Propane Cooling We used the liquid-propane cooling technique of Parak et al (21) exten sively in the ribosome work ( 13). Figure 5 shows the main elements of the process. The crystal is picked up as described above and then immediately plunged into a reservoir of liquid propane near its freezing point. A pivot mechanism is used to flip the crystal into the cold stream. This technique provides for a minimum time between crystal pickup and cooling, thereby reducing the risk of precooling damage. The rapid cooling is presumably also beneficial. The pivot mechanism can be used to remove the crystal from the diffrac tion apparatus by reversing the process used to introduce it into the cold stream. The crystal, imbedded in frozen propane, can be stored indefinitely

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Figure 5 Schematic view of the positioning mechanism used with the liquid· propane tech nique. Left, crystal in liquid propane; (a) liquid-propane bath; (b) crystal mounting pin with crystal on microspatula; (c) pivot mechanism allowing a 900 flip of the crystal holder (arrow); (d) spring-clip holding the crystal mounting pin. Right, crystal in position in the cold stream after 900 flip; (e) top end of goniometer head. The goniometer head carries an electrically heated stream deflector (not shown). The crystal positioning operation is reversible, allowing

the crystal to be returned to the liquid propane.

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in liquid nitrogen (the freezing point of propane is just above the boiling point of liquid nitrogen). A. Yonath and coworkers (personal com munication) have subjected a ribosomal crystal to several back-and-forth transfers over a period of months, and the crystal has completely retained its diffracting power. A major advantage of this development is thc ability to select and mount crystals for use in synchrotron data collection in advance. This enables one to make better use of precious beam time. RESULTS

Prevention of Radiation Damage Our work (10, 12) has focused upon simplification of the data collection process, a major element of which is the elimination of radiation damage. All data available indicate that cryogenic data collection eliminates crystal decay. Moderate lowering of the temperature had been observed to be useful. Haas & Rossmann (6) reported a tenfold decrease in decay at approximately 200 K. Based on their myoglobin work, Hartmann et al (7) suggested cryotechniques might be useful in preventing radiation damage. Singh et al (26), in their work on bovine trypsinogen and Fe fragment at 213 K, did not observe radiation damage. We saw no sign of decay in crambin or BPTI at 130 K (10, 12). Dewan & Tilton (2), studying ribo nuclease near 100 K, reported eliminating radiation damage for a crystal mounted in the open, but saw evidence of decay under similar conditions for a capillary-mounted crystal. Radiation damage likely did not cause the intensity decay. In many low-temperature data sets measured at the Weizmann Institute of Science, radiation damage has never been observed. In addition to those described elsewhere ( 17, 30), the crystals studied include: y-chymotrypsin (native and heavy-atom derivative), avidin (native and biotin complex), ferredoxin from Halohacterium marismortui, and acetylcholinesterase (F. Frolow & 1. L. Sussman, personal com munication). The most severe test appears to bc that of ribosomal crystals cxposed to intense synchrotron radiation ( 13). Around 270 K, these crystals retain only low-resolution diffraction after a few minutes of exposure, whereas at 85 K, full data sets, requiring many hours of exposure, can be recorded without loss of resolution. One crystal that had been used in data collection was allowed to warm up. While at 85 K, the crystal did not change shape or color but when warmed, it suddenly turned black and curled up like a drying leaf. Pre sumably the photochemical production of free radicals is not affected by the low-temperature, but migration and subsequent reaction with the macromolecules are hindered until the temperature has been raised.


123

These results indicate that data collection at cryotemperatures eliminates radiation damage as an experimental hindrance. In a report on human Mn superoxide dismutase, Wagner et al (30) state matter-of-factly: "At room temperature the crystals are not stable against radiation, so we cooled them to 90

K and collected a data set to 3 A".


Internal Alignment of the Crystal Singh et al

(26) have given a detailed description of the effect of cooling on

spot shape and mosaic spread for trypsinogen. They concluded that cool ing improved internal alignment, as judged from spot size and extinction effects. Alber et al (1) also reported a decrease in mosaic spread for elastase as a result of moderate cooling. These results were obtained for crystals that had been treated with cryosolvents and then cooled relatively slowly. With the shock-cooling technique, we have not seen evidence of lower mosaic spread. Ribosomal crystals show either no change or a change toward greater mosaic spread. For crystals of a DNA fragmcnt, Eisenstein et al (4) reported an increase in mosaic spread that was large enough to interfere with effective data collection at higher resolution. The gener alization that cooling always results in increased reflection width (4) has not been supported by further experiments. The differences in crystal behavior most likely reflect differences in treatment, rather than intrinsic properties of the crystals.

The effect of

encasing the crystal in a hydrocarbon shell must be considered. Frauen felder et al (5) have discussed the thermal expansion of metmyoglobin in relation to that of other substances. The linear thermal expansion coefficient of the protein is clearly much smaller than that of a hydro carbon. This condition possibly produces strong mechanical forces, which act on the crystal and lead to increased misalignment. Very thin, platelike crystals almost certainly will be damaged. Future studies will focus upon these effects.

Displacement (Temperature) Factors The effect of molecular and atomic motion, or displacement on the inten sity of a reflection, can be expressed as a function of an overall displacement coefficient

B; i.e. the intensity is proportional to exp ( - 2B sin2 OJ).2). As a

first approximation, the variation of B with temperature can be given as

BT Bo+bT, where BT is the B value at temperature T, Eo the zero-point B, and b a proportionality constant (12). For relatively small values of Bo, lowering the temperature from about 300 K to 100 K can have a substantial effect. For myoglobin, the overall B dropped from 14 A2 at 300 to 5 A2 at 80 K (7). In crambin, the B values at 130 K are generally about one half =

the values at room temperature.

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HOPE

BT suggests that small Bo values will result in pro B than will large Bo values. Experiments on structures in which B varies systematically with position in the molecule The expression for

portionately larger reduction in

bear out this prediction. Eisenstein et al (4) reported observing a near uniform decrease of 9

A2

on cooling an A-DNA fragment to 1 15

K,

although different groups, bases, sugars and phosphates, have significantly


different

B values. Drew et al (3) made a similar observation for another

DNA fragment. An unusual result was obtained for bovine trypsinogen, for which the

B value at 2 13 K was found to be less than half the value at room temperature, 6. 1 vs 13.3

A2 (26).

For this result, one would expect causes

other than lower temperature. The phenomenon warrants further study.

Absorption Crystals mounted in capillaries,with or without a drop of mother liquor, will likely give rise to strongly anisotropic absorption effects. Oil-drop mounting, especially with a microspatula, can dramatically reduce absorp tion anisotropy. The absorption coefficients for crystal and surrounding medium (oil or cryo-mixture) will often be of similar magnitude, and the rounded shape of the crystal-medium assembly will lead to more isotropic beam paths than can be attained with capillary mounting. Data sets and IjJ scans indicate that absorption correction often can be safely neglected with spatula mounts.

Effect on Resolution and Structural Details Low temperature has not been found to affect the structure of macro molecules in any major way. In studies in which comparisons have been made (3,4,5,7,28,3 1),the observed differences have been small and can be reasonably explained as a result of reduced thermal motion or improved data. In the absence of a true phase transition, we would expect that structural results obtained from eryoerystallographie data will show no unwanted bias attributable to low temperature. For crystals in which no appreciable increase in mosaic spread occurs after cooling we can expect that lower values of the displacement

A relationship has been pro B of the form '2 'I [Bo+bT )/(Bo+bTI)f/2, where 'I and ' are resolutions at 2 2 temperatures T] and T2, respectively. Unfortunately, under the parameters coefficients to result in improved resolution.

posed ( 12) between resolution, and displacement parameter =

of this equation, improvement in resolution will be relatively smaller the lower the room temperature resolution is. A crystal with room-temperature resolution of approximately 1.5 A may yield about I

A at eryotemperature,

whereas one can expect little improvement for resolutions of less than 3


or

125

4 A. However, when low resolution at room temperature results from

rapid radiation damage that prevents measurement of higher order reflec tions, this rule does not hold. A crystalline DNA duplex with looped out bases reported by Joshua-Tor et al ( 17) provides an example. Data collection beyond

4 A had been virtually impossible at room temperature

because of radiation sensitivity. Measurements at 123 K were problem free.


Usually, a protein or DNA structure is based on data obtained from a number of crystab. Each crystal will have been used until radiation damage has become severe. The data will therefore have significant errors. Also different crystals can have slightly different structures. Merging data will yield an average. The ability to obtain all data from one specimen, without decay problems, will improve data quality, concomitantally with improv ing reliability and clarity of detail in the resulting structure. The structure based on the 130-K data set for crambin illustrates this improvement well. The details of the solvent structure have been con spicuously enhanced (28). The largest solvent-containing region shows mostly diffuse electron density with the 300-K data, whereas a map of the same region, based on the 130-K data, shows a clear, detailed picture of the solvent structure. Muchmore and Watenpaugh ( 19) have reported corresponding results for chicken egg white lysozyme.

CONCLUDING NOTES Cryocrystallographic methods clearly have an important function, and interest in them is increasing. Working methods have been established in a few laboratories. One can reasonably expect widespread adoption of the best of these, but substantial effort should go into solving the engineering problems. Commercially available cryocrystallographic equipment needs to be made less demanding on the untrained user. Development of crystal handling techniques and design of new laboratory implements continues. Transferring well-tested techniques from one laboratory to others presents another problem. Workshops and laboratory visits accomplish this transfer effectively. Establishing a cryocrystallographic laboratory is a serious matter, and unless one invests in the appropriate equipment and acquires expertise, the results may not turn out well. The potential for increased productivity and richer structural information, however, is the reward for such efforts. ACKNOWLEDGMENTS

Much of the author's work in biological cryocrystallography has been supported through a grant from the US-Israel Binational Science Foun-

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HOPE

dation (J. L. Sussman and H. Hope). The cheerful help from members of

the Exxon Corporation staff in providing the necessary samples of Para tone-N is greatly appreciated.


Literature Cited I. Alber, T., Petsko, G. A., Tsernoglou, D. 1976. Na/ure 263: 297 2. Dewan, J. C., Tilton, R. F. 1987. 1. Appl.

Cryst. 20: 1 30 3. Drew, H. R., Samson, S., Dickerson, R. E. 1 982. Proc. Natl. A cad. Sci. USA 79: 4040 4. Eisenstein, M . , Hope, II., Haran, T. E., Frolow, F., Shakked, Z., Rabinovich, D. 1 988. Acta Crystallogr. B 44: 624 5. Frauenfelder, H . , Hartmann, H . , Kar plus, M., Kuntz, 1. D. Jr., Kuriyan, J., et al. 1 9 87 . Biochemistry 26: 2 54 6. Haas, D. J., Rossmann, M . G. 1970. Acta Crystallogr. B 26: 99� 7. Hartmann, H . , Parak, F., Steigemann, W., Petsko, G. A., Ringe Ponzi, D., et al. 1 9 8 2. Proc. Natl. A cad. Sci. USA 79: 4967

8. Hendrickson, W. A., Teeter, M. M. 1 98 1 . Nature 290: 1 0 7 9. H irshfeld, F. L., Hope, H . 1980. Ac ta Crystallogr. B 36: 406 10. Hope, H . 1985. Am. Crystallogr. Assoc. Meet. Abstr. 1 3 (PA3): 24 1 1 . Hope, H. 1 9 8 7 . In A CS Symposium Ser ies No. 357: Experimental Organo metallic Chemistry, ed. A. L. Wayda, M. Y. Darensbourg, pp. 257-60 1 2 . Hope, H. 1988. Acta Crystallogr. B 44: 22 1 3 . Hope, H . , Frolow, F., von Bohlen, K., Makowski, I., Kratky, c., et al. 1989. Acta Crystallogr. B 45: 1 90 1 4. Hope, H., Nichols, B. G. 1 98 1 . Acta Crystal/ogr. B 27: 1 58 15. Hope, H., Ottersen, T. 1 979. Acta Crystallogr. B 35: 370

1 6. Hope, H., Power, P. P. 1 983 . .T. Am. Chem. Soc. 105: 5320 17. Joshua-Tor, L., Rabinovich, D., Hope, H., Frolow, F., Appella, E., Sussman, J. L. 1 988. Nature 334: 82 1 8. Low, B. W., Chen, C. C. H . , Berger, J. E., Singman, L., Pletcher, J. F. 1 966. Proc. Natl. Acad. Sci. USA 56: 1 746 19. Muchmore, S. W., Watenpaugh, K. D. 1 989. Am. Crystallogr. Assoc. Meet. Abstr. 1 7 ( HB3): 43 20. Ottersen, T., Hope, H. 1 979. Acta Crystallogr. B 35: 373 2 1 . Parak, F., Mossbauer, R. L., Hoppe, W., Thomanek, U . F., Bade, D. 1 9 76. J. Phys. (Paris) Colloq. C6 37: 703 22. Petsko, G. A. 1975 . .T. Mol. Bioi. 96: 381 23. Petsko, G. A., Douzou, P., Hoa, G. H. G. 1 975. J. Mol. BioI. 96: 367 24. Post, B., Schwartz, R. S., Fankuchen, I. 1 95 1 . Rev. Sci. Instrum. 22: 2 1 8 25. Rudman, R . 1976. Low-Temperature X Ray Diffraction. New York: Plenum 26. Singh, T. P., Bode, W., Huber, R. 1 980. Acta Crystallogr. B 36: 62 1 27. Teeter, M. M., Hope, H. 1 985. Am. Crystal/ogr. Assoc. Meet. Abstr. 1 3 (PB39): 47 28. Teeter, M. M., Hope, H. 1 986. Ann. N Y A cad. Sci. 482: 1 63 29. van Bolhuis, F. 1 97 1 . J. Appl. Cryst. 4: 263 30. Wagner, U. G., Werber, M. M., Beck, Y., Hartman, J. R., Frolow, F., Suss man, J. L. 1 989. J. Mol. Biul. 206: 787 3 1 . Walter, 1 . , Steigemann, W., Singh, T. P., Bartunik, H . , Bode, W., Huber, R. 1 982. Acta Crystal/ogr. B 38: 1 462

Evolutionary adaptability of biological macromolecules.

Ion binding to biological macromolecules.

New perspectives in biological crystallography.

Assessment of resolution in biological electron crystallography.

Cathodoluminescence of biological molecules, macromolecules and cells.

Application of Raman spectroscopy to biological macromolecules.

Optimization of crystallization conditions for biological macromolecules.

Ultralow-threshold electrically injected AlGaN nanowire ultraviolet lasers on Si operating at low temperature.

CH-π hydrogen bonds in biological macromolecules.

Biological crystallography: new methods, new challenges.

Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature.

Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature.

Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons.

The place of symmetry in the study of biological macromolecules.

Temperature-amplitude coupling for stable biological rhythms at different temperatures.

Imaging biological macromolecules by STM: quantitative interpretation of topographs.

Application of high-resolution scanning electron microscopy to biological macromolecules.

Physicochemical Properties of Ion Pairs of Biological Macromolecules.

Crowding induced collective hydration of biological macromolecules over extended distances.

Biological properties of staphylococcal lipoteichoic acid and related macromolecules.

Developments in x-ray crystallographic structure determination of biological macromolecules.

Nucleation-conversion-polymerization reactions of biological macromolecules with prenucleation clusters.

Room temperature structures beyond 1.5 Å by serial femtosecond crystallography.

Room-temperature macromolecular serial crystallography using synchrotron radiation.