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50 years of fiber diffraction
Journal of Structural Biology 170 (2010) 184–191
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50 years of fiber diffraction Kenneth C. Holmes * Max Planck Institute for Medical Research, 69129 Heidelberg, Germany
a r t i c l e
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Article history: Received 11 January 2010 Accepted 11 January 2010 Available online 15 January 2010 Keywords: Tobacco mosaic virus Coiled-coils Myosin cross-bridge Actin Tropomyosin Synchrotron radiation
a b s t r a c t In 1955 Ken Holmes started working on tobacco mosaic virus (TMV) as a research student with Rosalind Franklin at Birkbeck College, London. Afterward he spent 18 months as a post doc with Don Caspar and Carolyn Cohen at the Children’s Hospital, Boston where he continued the work on TMV and also showed that the core of the thick filament of byssus retractor muscle from mussels is made of two-stranded ahelical coiled-coils. Returning to England he joined Aaron Klug’s group at the newly founded Laboratory of Molecular Biology in Cambridge. Besides continuing the TMV studies, which were aimed at calculating the three-dimensional density map of the virus, he collaborated with Pringle’s group in Oxford to show that two conformation of the myosin cross-bridge could be identified in insect flight muscle. In 1968 he opened the biophysics department at the Max Planck Institute for Medical Research in Heidelberg, Germany. With Gerd Rosenbaum he initiated the use of synchrotron radiation as a source for X-ray diffraction. In his lab the TMV structure was pushed to 4 Å resolution and showed how the RNA binds to the protein. With his co-workers he solved the structure of g-actin as a crystalline complex and then solved the structure of the f-actin filament by orientating the g-actin structure so as to give the f-actin fiber diffraction pattern. He was also able to solve the structure of the complex of actin with tropomyosin from fiber diffraction. Ó 2010 Elsevier Inc. All rights reserved.
1. Birkbeck College In 1955, at the end of my undergraduate time at Cambridge, I thought that I would like to do something crystallographic for a Ph.D. I wrote round to a number of labs and was delighted to get an offer from Rosalind Franklin, at Birkbeck College London, to work on the structure of the rod-shaped tobacco mosaic virus (TMV). It turned out this was not quite crystallography as I had learnt it. Rosalind had found out how to orientate TMV by moving a 10–15% gel (really a sol) of the virus up and down glass capillary tubes. This initially led to flow-induced orientation. However, after some weeks about 10% of the specimens spontaneously developed perfect orientation that showed complete extinction between crossed polaroids. The orientated specimens were photographed with X-ray CuKa radiation in an evacuated camera that Rosalind had designed. Using bent-quartz monochromators and fine-focus X-ray generators such specimens could be induced to yield splendid fiber diffraction patterns, concentrated in 30–40 layer lines extending out to high (2.5 Å) resolution (a rather fine example using a different X-ray camera is shown in Fig. 1). For my thesis I concentrated on the zero layer line. Because of the cylindrical geometry and cylindrical averaging, fiber diffraction patterns, which can be derived from the Fourier * Fax: +49 6221 486437. E-mail address: [email protected]. 1047-8477/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2010.01.004
transform of the fibrous molecule, are much more economically expressed in cylinder functions rather than in sines and cosines. Moreover, because most objects repeat periodically along their length the scattering is confined to layer lines. The radial functionality (that gives rise to the intensity distribution along the layer lines) is expressed as Bessel functions of integral order. The order reflects the local azimuthal symmetry. In principle Bessel functions of all orders are allowed to contribute to every layer line. However, in practice things are not as bad as that. Crick and collaborators had shown that helical symmetry led to a sparse distribution of Bessel functions so that on any one layer line often only one or two Bessel functions would contribute (Cochran et al., 1952). TMV was known to be a helical structure that repeated every three turns (Watson, 1954). Moreover, on account of the high symmetry of TMV the low angle part of the zero layer line consists entirely of contributions from the cylindrically symmetrical part of the diffraction pattern, which is made of Bessel functions of zero order (J0). The amplitude of scattering in the inner part of the zero layer line is actually the sum over all atoms of J0 Bessel functions, each depending on the weight f and radius r of the j0 th atom in the virus
FðRÞ ¼
X
fj J 0 ð2pr j RÞ
ð1Þ
j
where F is the scattering amplitude and R is the reciprocal space radius. By inverting this process one this one can calculate the radial density distribution q of the virus
K.C. Holmes / Journal of Structural Biology 170 (2010) 184–191
J n ð2pRr Hg Þ
Fig. 1. A fiber diffraction pattern from an orientated gel of tobacco mosaic virus made by K.C. Holmes in D.L.D. Caspar’s laboratory in 1960 and photographed in Caspar’s laboratory in 1980 with a flat film camera, CuKa radiation and two focussing mirrors (courtesy of D.L.D. Caspar).
qðrÞ ¼
Z
1 0
FðRÞJ 0 ð2prRÞdR
ð2Þ
where r is the radius in real space. The observed intensities yielded only F2, thus one had to find a way of determining the signs of F. This was done by adding one methyl-mercury group to each subunit of the virus. Since the subunits are quite small (MW 17,500) the differences produced by adding one mercury atom were easily measurable. Once the radius of the mercury was determined its scattering J 0 ð2pRr Hg Þ could be calculated. The signs of the TMV data with and without mercury had to be chosen so that the difference agreed with the calculated mercury scattering. The radial density distribution calculated from the signed scattering amplitudes of the zero layer line showed, among other things, that the nucleic acid (a single RNA strand) was embedded in the virus at a radius of 40 Å and that the virus is hollow (see Franklin and Holmes, 1958). Eq. (2) (a zero order Fourier-Bessel transform) was evaluated as a 50 50 matrix multiplication (Rosalind had produced cyclostyled sheets comprising a square matrix of the values of J0(2prR) in intervals of 2 Å along the top and 0.002 Å1 down the side). I did many such operations. Each time the 2500 multiplications took me three days with a Marchant calculator. The noise was grim, especially for my room mates. Thus, when a little later Bill Longley joined us bringing some experience with the IBM 650, I gladly took to programming. The IBM 650 computer, located somewhere on the Strand, was a drum machine with 2048 locations total that used machine code written in SOAP (symbolic optimum assembly program). It had a strange word structure – 2 5 bits. Nevertheless, Bill had located subroutines for zero order Bessel functions. Thus in 1959 I became an early computer geek. At this time, although it was clear that TMV had a high symmetry and repeated in three turns, the actual symmetry had not been determined. Fortunately, the mercury scattering allowed us to determine the symmetry. The zero layer line arises from the projection of the structure onto an equatorial plane. If there are n subunits in the repeat of the virus there will be n mercury atoms forming a circle in the equatorial projection. At higher angles of scattering on the zero layer line (outside the J0 region) this ring of points will give rise to a characteristic scattering peak
185
ð3Þ
Since we had determined rHg and the position of the first peak of the Bessel function was measurable it was possible to calculate that n was 49 (Franklin and Holmes, 1956). During this time I met Mary Scruby, who worked in Birkbeck College library. We married in September 1957. She was a classics major. Later she became the founding librarian of the European Molecular Biology Laboratory in Heidelberg. In the Spring of 1958 Rosalind tragically died. The leadership of the group passed to Aaron Klug, who was then working on crystalline spherical viruses with John Finch. Aaron had collaborated with Rosalind on many theoretical aspects of the TMV work. He was a great teacher and I learnt much from him. Among his many achievements, he produced the formalism that allows a threedimensional density map to be calculated from fiber diffraction data (Klug et al., 1958). Only two systems (TMV and bacterial flagella) have yielded fiber diagrams of high enough quality to allow this to be carried out (most fiber applications rely on calculating the fiber diffraction from an atomic model and then somehow improving the fit) but later the formalism proved very useful for reconstructions of electron microscope images from helical biological fibers. The calculation of the three-dimensional electron density map of TMV at high resolution became the long-term aim of the group. To do this we had to find many heavy atom derivatives: not only did we need to phase the scattering amplitudes but also to separate out the contributions from overlapping Bessel function terms. Measuring the data also became a major job. Here we were very ably assisted for two years by Susan Fenn (later Lady Fenn, her husband Nicholas became British High Commissioner for India). 2. Children’s Hospital, Boston Don Caspar at Yale had calculated the radial density distribution of TMV for his thesis a couple of years ahead of me. In 1958 he moved to the Children’s Cancer Research Foundation (Jimmy Fund) in Children’s Hospital, Boston. He shared the lab with Carolyn Cohen. Don kindly offered me a post doc position to continue with the TMV work. Thus in February 1960 we took a Cunarder from Liverpool and 6 days later we were in New York. Then on to Boston, where we stayed for 18 months (see Fig. 2). Susan Fenn continued to measure and send on data on TMV that had to be evaluated. This led me to Project Mac in the cellars of MIT. An IBM 709 (later an IBM 7090) with a Fortran compiler was available for batch users. The job-control-cards were horrendous: one tiny mistake and the job was thrown out. Furthermore, the turn-round was 2–3 days – a good training for accuracy. I made lots of TMV specimens, one of which was photographed and showed wonderful orientation (a bit like good vintage wine) some 20 years later (Fig. 1). Don was always fascinated by things that did not quite fit. Thus he was delighted to discover that the Dahlemense strain of TMV showed satellite reflections not arising from the helical symmetry of the virus. After a hint from Carolyn we drew on radio side-band theory to interpret these as a modulation phenomenon: the outer parts of the virus subunits like to group in pairs thereby breaking the helical symmetry. Our paper was quickly accepted by J. Mol. Biol. (Caspar and Holmes, 1969) but spent another 7 years going backwards and forwards between the two nit-picking authors! Carolyn was not averse to finding jobs for other peoples’ post docs. Thus I was led to discover that the anterior byssus retractor muscle from mytilus edulis could be pulled into a quartz capillary tube to yield an orientated gel that showed very good extinction. Such samples gave excellent X-ray fiber diagrams. Most of the diffraction arises from the paramyosin in the middle of the myosin thick filaments, which is pure alpha helix. Using Francis Crick’s theory of diffraction for coiled-coils (Crick, 1953), Carolyn and I were
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Fig. 2. Ken and Mary Holmes, Christmas 1960 at 45 Carlton St. Brookline, Boston Mass.
able to show that our ABRM fiber diffraction pattern was exactly what one would expect from a two-chain coiled–coiled alpha helix (Cohen and Holmes, 1963). This was my first foray into muscle.
3. Laboratory of Molecular Biology, Cambridge We returned to England in the summer of 1961 in time for the birth of out first daughter and I rejoined Aaron’s group. The plan
was to move the group to the newly founded MRC Laboratory of Molecular Biology in Cambridge. However, the building was not ready, so we continued at Birkbeck for another year. Finally, in the summer of 1962 we all moved to Cambridge. Here the search for heavy atom derivatives of TMV and data collection continued. Angela Mott replaced Susan Fenn. We were joined by Judy Gregory who made excellent orientated samples (Gregory and Holmes, 1965). Reuben Leberman helped with the chemistry. For the TMV work we used fine-focus X-ray generators and bent-quartz monochromators. Soon Bill Longley and I were involved in making fine focus rotating anode X-ray generators, ably assisted by Tony Woollard. Our efforts turned into the Elliott GX6 X-ray generator (Fig. 3). Hugh Huxley started using our apparatus to register the low-angle X-ray fiber diffraction from contracting frog muscle. It was clear that Hugh really needed a thousand times more intensity than we could give, so in 1964 I started wondering about synchrotron radiation. Julius Schwinger’s theory (Schwinger, 1949) allowed the expected intensity to be calculated. This was full of Bessel functions, but I had Bessel function subroutines to hand and could estimate the strength of synchrotron X-ray radiation. It looked good! However, at this time neither the German ring DESY at Hamburg nor the English ring at Daresbury were actually working, so the idea was put on hold. About this time a collaboration with John Pringle’s group at Oxford led to an interesting experiment on flight muscle from the giant water bug Lethocerus. Insect flight muscle shows the phenomenon of stretch activation: when turned on by Ca2+ the muscle contracts only if you stretch it. Heart muscle also shows this phenomenon to an extent. Pringle’s co-worker Richard Tregear had set up strips of insect flight muscle in a machine that oscillated the muscle, which allowed it to generate work cyclically when fed with ATP. The muscle indeed generated Lissajous figures showing that it was doing work on the machine. Richard came to Cambridge with a sample. We took a low-angle X-ray diffraction picture in rigor (in a Ringer’s solution) and then added ATP. Each exposure took ten minutes because that’s all the time we had before we dashed off for the train to London to take part in a meeting of the London muscle club. We examined the photos in the train. They showed that there was a strong 145 Å meridional reflection in the ATP-containing solution but none in rigor (Fig. 4). The experiment proved to be both reversible and reproducible. Mike Reedy subsequently used Hugh Huxley’s wonderful technology for cutting ultra-thin sections from plastic embedded muscle to show by electron microscopy that the myosin cross-bridges were giving the signal and that they were taking on two different orientations: at 90° to the axis with ATP and 45° to the axis without ATP (Reedy et al., 1965). This observation became the basis of the swinging crossbridge hypothesis of muscle contraction, which even now appears to be correct.
4. Max Planck Institute for Medical Research, Heidelberg
Fig. 3. A fine focus rotation anode X-ray generator (W. Longley, T. Woollard and K.C. Holmes) based on the Taylor rotating anode (Taylor, 1948) and the cathode from the Beaudouin X-ray Generator. This was the prototype of the Elliott GX6 X-ray generator.
This simple experiment turned out to have far reaching ramifications, not only for science but also for me personally, because it led to a ‘‘call” from the Max Planck Institute of Medical Research in Heidelberg to open a department of biophysics. The retiring director of the Department of Physiology, Hans Hermann Weber, was a famous muscle physiologist who wanted someone to continue the long tradition of muscle research in the Max Planck Institute in Heidelberg. He would have preferred Hugh Huxley but decided to settle for Ken Holmes. Hence in September 1968 Mary and I with our children moved to Heidelberg, where we are still. The department was set up as two sub-departments, for biophysics and for biochemistry. Since I have an indifferent reputation as a chemist, I entrusted setting up the biochemistry to Reuben Leberman,
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Fig. 4. X-ray fiber diagram from a strip of insect flight muscle (left) in rigors solution and (right) in rigors solution containing 10 mM Mg ATP (20 min exposure). After Reedy et al. (1965).
who complete with Pat Leberman and their children came with us from Cambridge. The department survived until 2003 when it was closed on account of my retirement. In Heidelberg we continued the TMV work. John Barrington Leigh, who had been a student in Cambridge, came and worked together with Eckhard Mandelkow, Tony Barrett, and Ute Gallwitz to make and determine the positions of five different heavy atoms at-
Fig. 5. The Rosenbaum-Barrington Leigh X-ray beam line in Bunker 2 at DESY Hamburg with the author making adjustments. The focussing system was: bent mirrors in the vertical plane and a bent-quartz monochromator in the horizontal plane. When the synchrotron beam was on all adjustments had to be made by remote control. The optical bench had over 100 motorized adjustments. The mirror box (front) was helium filled.
tached to the virus protein (Barrett et al., 1971; Holmes et al., 1972). This was the prerequisite for calculating the structure. Later two collaborators, Gerald Stubbs and Steve Warren used these heavy atoms to calculate a density map of the virus at 4.0 Å resolution (Holmes et al., 1975; Stubbs et al., 1977). Gerald left the lab to work with Don Caspar. He finally was able to calculate a 2.8 Å resolution map of TMV from fiber diffraction data. In 1969 Gerd Rosenbaum joined me to do a doctoral thesis on insect muscle. The aim was to try to get time-resolved diffraction from contracting muscle. Hugh was doing similar things with frog muscle. Gerd soon became frustrated with conventional X-ray sources and took up the idea that we go to DESY Hamburg to try to use synchrotron radiation. He had worked at DESY on synchrotron light for his diploma and knew the physical set up. In the summer of 1970 we conducted the first proof of principle experiments at DESY to show that indeed the ring was an intense source of Xrays: at least 10 times stronger than any laboratory source (Rosenbaum et al., 1971). In addition it had laser-like optics. Synchrotrons have their limitations since they dump the beam every 20 ms. Storage rings, with a continuous high intensity electron beam, are far better. Nevertheless, with the enthusiastic support of the DESY directorate, in particular of Martin Teucher, Bunker 2 was built
Fig. 6. The G-actin monomer consists of two sub-domains with ADP or ATP sandwiched between the sub-domains.
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for pro tem use of the synchrotron ring. In the next two years in Bunker 2 Gerd Rosenbaum and John Barrington Leigh built the first ever X-ray beam line (Barrington Leigh and Rosenbaum, 1974). The beam was 10–20 times more intense than a laboratory source and very much better collimated, which made it particularly attractive for low angle scattering experiments on muscle (Fig. 5). We mostly used the beam for experiments on insect flight muscle for trying out the effects of ATP analogs, made for the purpose by Fritz Eckstein and Roger Goody, to see if we could get evidence for other states in the muscle (Goody et al., 1975). Richard Tregear was an enthusiastic collaborator. The fact that we were parasitic users of the beam made our endeavor fairly cheap but sometimes led to extreme frustration since the beam (or lack of it) depended entirely on the needs of the high energy physicists. With a promise of 50–500 times more intensity clearly the future lay with storage rings. We designed another lab, Bunker 4, lo-
cated at the perimeter of the new storage ring DORIS. However, the whole enterprise was getting too big to be an adjunct of a Max Planck department. Hence we were grateful when in 1975 it was taken over by the newly formed European Molecular Biology Laboratory as the EMBL Outstation. Here in 1980 Hugh Huxley was at last able to conduct time-resolved measurements frog muscle that showed that the 145 Å meridional dropped right down in intensity as the muscle recovered tension after a quick release (Huxley et al., 1980), a result that was fully consistent with the swinging crossbridge theory of muscle contraction. 5. Actin In the late 70’s I gave up the TMV work to concentrate on actin. Valerie Lednev, while a postdoc at Kings London in the 70’s, had managed to orientate F-actin gels much as we had orientated
Fig. 7. Fitting the g-actin monomer into the f-actin helix. F-actin has thirteen subunits every six turns.
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TMV. Lednev then returned to Pushchino and after some efforts by Gillis and O’Brien the know-how went into abeyance. However, at the end of 1985 Lednev visited Germany and, having stopped at the Russian Embassy in Bonn long enough to fill his copious overcoat pockets with bottles of duty-free whisky, visited us for an extended Christmas party. During the course of this well lubricated exchange of views David Popp, then a diploma student in the lab, was instructed by Valerie on how to make orientated gels of filamentous actin (f-actin). David was successful and soon we had high quality fiber diagrams from f-actin gels (Popp et al., 1987). However, the fiber diagrams were nothing like so perfect as from TMV: the resolution was limited (about 5 Å); moreover, the layer lines overlapped. There was no hope of determining the structure from first principles as we had achieved for TMV. The alternative approach is model building: if you have a structure for the subunit (g-actin) then you can try it in all orientations in the fiber to see if you can compute the correct fiber diagram. Hence we were obliged to do the X-ray crystallography of g-actin. G-actin is difficult because its response to adding salt is not to crystallise but to make f-actin. However, in 1974 Uno Lindberg had published the observation that g-actin binds tightly to the pancreatic enzyme DNAse 1 thereby inhibiting f-actin formation. Reuben Leberman managed to make small crystals of the complex that were improved by Hans-Jörg Mannherz (Mannherz et al., 1977, 1975). However, solving the structure proved very difficult. First Wolfgang Kabsch and Dietrich Suck managed to solve the structure of DNase 1 (Suck et al., 1984). Data collection from the actinDNase1 complex was a nightmare since the crystals changed their cell size in response to a host of unknown environmental parameters. The answer was to collect all of a three-dimensional data set from one crystal. This became possible through the advent of 2D electronic detectors. Wolfgang developed his famous XDS software (Kabsch, 1988a,b) just for the purpose of data collection from actin-DNase 1 crystals using 2D detectors . After some struggles the structure of g-actin became available (Kabsch et al., 1990). It was a rather flat molecule consisting of two structurally similar sub-domains (see Fig. 6). Now we could try to fit it to the fiber diffraction pattern. In a computer search only one orientation came close to fitting the fiber
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diffraction pattern. Thus we had a model of f-actin (Holmes et al., 1990) (see Fig. 7). However, the fit to the fiber diffraction pattern was not perfect: apparently g-actin undergoes some (small) conformational change between g- and f-actin. It took another twenty years to work out what the conformational change was. I tried sub-domain refinement (Holmes et al., 2004), Monique Tirion tried normal modes (Tirion et al., 1995). Michael Lorenz ingeniously achieved a perfect fit to the fiber diagram using a genetic algorithm but somewhat at the expense of the stereochemistry of the actin monomer (Lorenz et al., 1993). He was clearly close to the correct structure but unfortunately the secondary structure had suffered. Recently Toshiro Oda, working at Spring 8 in Yuichiro Maeda’s group managed to improve the orientation of f-actin by placing the gels in an 18 tesla magnetic field. This gave more resolution. Toshiro was then able to show that between g- and f-actin the two sub-domains rotate by about 20°, and nothing much else happens (Oda et al., 2009). We should have got this result! (see Fig. 8). By observations of the second and fourth actin layer lines from intact muscle (Huxley, 1972; Haselgrove, 1972; Parry and Squire, 1973), it was shown that muscle activation causes the movement of tropomyosin on the surface of actin. The second layer line is responsive to two-fold symmetry and the fourth layer line to four-fold symmetry. The fourth actin-based layer line becomes stronger on activation. On activation of muscle, which releases Ca2+ into the sarcoplasm to bind to troponin, tropomyosin is induced to move so as to increase the four-foldedness of the filament and free up the myosin binding sites on actin. David Popp was a skillful actin orientor and a competent biochemist so that he was able to make an orientated gel from a synthetic actin-tropomyosin complex and then collect X-ray fiber data. We fitted this data with a model of tropomyosin on actin (Lorenz et al., 1995) – Fig. 9. It showed that the tropomyosin lies along the surface of actin in a way that allows each of the tropomyosin pseudo-repeats to have the same geometrical view of an actin monomer, but that there is no explicit contact between tropomyosin and actin (there is a layer of solvent between them). Tropomyosin is attracted electrostatically to actin but it only binds to actin because it binds end-to-end with itself. This simple model allows
Fig. 8. To show how a 20° twist of the sub-domains 1 and 2 against 3 and 4 (shown left) of g-actin gives an excellent fit the f-actin fiber diffraction pattern. In the fiber diffraction pattern on the right the upper right and lower left quadrants are data, the other two quadrants are the diffraction calculated from the model. I am grateful to Yuichiro Maeda for the sketch on the left.
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Fig. 9. The arrangement of tropomyosin on actin. (left cartoon, right space filling model).Tropomyosin is a coiled–coiled coil with the same shape as actin. Each tropomyosin molecule spans 7 actin monomers . Tropomyosin has a strong pseudo repeat of 39.3 residues. Each pseudo repeat of tropomyosin matches up with an actin monomer. Electrostatic interactions are made between the two molecules. (From Holmes and Lehman, 2008 with permission.)
tropomyosin to be pushed around on the actin surface by troponin in response to Ca2+ binding as had been foreseen by Parry and Squire. However, in spite of offering an easy explanation of tropomyosin movement our model was not enthusiastically embraced by workers in the field since they were looking for specific interactions of tropomyosin with actin. Recently, our simpler view that there are none has found more acceptance. Ours was a troponin-free system with no ability to study the effects of Ca2+ binding to troponin. Kate Poole worked up a system using demembranated muscle fibers pulled out so that there is no overlap between the actin (thin) filaments and the myosin (thick) filaments. This approximates to an orientated gel of native thin filaments with an intact troponin-tropomyosin system. Using this preparation she could add and subtract Ca2+ many times to watch (by fiber diffraction as in Parry and Squire) the movement of tropomyosin orchestrated by troponin. Moreover, she could get accurate data on the tropomyosin movement by taking the differences between X-ray fiber diagram with and without Ca2+. This
allowed the magnitude of the movement to be calculated and to be compared with electron microscope observations of the filaments with and without Ca2+ that had been made by Bill Lehman’s group and Roger Craig’s group (Poole et al., 2006). The agreement was satisfying and moreover, has led to some interesting collaborations with Bill Lehman (Holmes and Lehman, 2008). This was really my last experiment with fibers. Now I am reduced to interpreting other people’s data and producing models of how muscle might work.
References Barrett, A.N., Leigh, J.B., Holmes, K.C., Leberman, R., Mandelkow, E., von Sengbusch, P., 1971. An electron-density map of tobacco mosaic virus at 10 Angstrom resolution. Cold Spring Harb. Symp. Quant. Biol. 36, 433–448. Barrington Leigh, J., Rosenbaum, G., 1974. A report on the application of synchrotron radiation to low-angle scattering. J. Appl. Cryst. 7, 117–121. Caspar, D.L., Holmes, K.C., 1969. Structure of dahlemense strain of tobacco mosaic virus: a periodically deformed helix. J. Mol. Biol. 46, 99–133.
K.C. Holmes / Journal of Structural Biology 170 (2010) 184–191 Cochran, W., Crick, F.H.C., Vand, V., 1952. The structure of synthetic polypeptides. I. The transform of atoms on a helix. Acta Crystallogr. 5, 581–586. Cohen, C., Holmes, K.C., 1963. X-ray diffraction evidence for alpha-helical coiledcoils in native muscle. J. Mol. Biol. 6, 423–432. Crick, F.H.C., 1953. The Fourier Transform of a coiled-coil. Acta Crystallogr. 6, 685– 689. Franklin, R.E., Holmes, K.C., 1956. The helical arrangement of the protein sub-units in tobacco mosaic virus. Biochim. Biophys. Acta 21, 406–407. Franklin, R.E., Holmes, K.C., 1958. Tobacco mosaic virus: application of the method of isomorphous replacement to the determination of the helical parameters and the radial density distribution. Acta Crystallogr. 11, 213–230. Goody, R.S., Holmes, K.C., Mannherz, H.G., Leigh, J.B., Rosenbaum, G., 1975. Crossbridge conformation as revealed by X-ray diffraction studies on insect flight muscles with ATP analogues. Biophys. J. 15, 687–705. Gregory, J., Holmes, K.C., 1965. Methods of preparing oriented tobacco mosaic virus sols for X-ray diffraction. J. Mol. Biol. 13, 796–801. Haselgrove, J.C., 1972. X-ray evidence for a conformational change in the actincontaining filaments of vertebrate striated muscle. Cold Spring Harb. Symp. Quant. Biol. 37, 341–352. Holmes, K.C., Lehman, W., 2008. Gestalt-binding of tropomyosin to actin filaments. J. Muscle Res. Cell Motil. 29, 213–219. Holmes, K.C., Mandelkow, E., Leigh, J.B., 1972. The determination of the heavy atom positions in tobacco mosaic virus from double heavy atom derivatives. Naturwissenschaften 59, 247–254. Holmes, K.C., Stubbs, G.J., Mandelkow, E., Gallwitz, U., 1975. Structure of tobacco mosaic virus at 6.7 Å resolution. Nature 254, 192–196. Holmes, K.C., Popp, D., Gebhard, W., Kabsch, W., 1990. Atomic model of the actin filament. Nature 347, 44–49. Holmes, K.C., Schroder, R.R., Sweeney, H.L., Houdusse, A., 2004. The structure of the rigor complex and its implications for the power stroke. Philos. Trans. R. Soc. Lond. B Biol. Sci. 359, 1819–1828. Huxley, H.E., 1972. Structural changes in actin and myosin-containing filaments during contraction. Cold Spring Harb. Symp. Quant. Biol. 37, 361– 376. Huxley, H.E., Faruqi, A.R., Bordas, J., Koch, M.H., Milch, J.R., 1980. The use of synchrotron radiation in time-resolved X-ray diffraction studies of myosin layer-line reflections during muscle contraction. Nature 284, 140–143. Kabsch, W., 1988a. Evaluation of single crystal X-ray diffraction dat from a positionsensitive detector. J. Appl. Crystallogr. 21, 916–924. Kabsch, W., 1988b. Automatic indexing of rotation diffraction patterns. J. Appl. Crystallogr. 21, 67–71. Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., Holmes, K.C., 1990. Atomic structure of the actin:DNase I complex. Nature 347, 37–44.
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Klug, A., Crick, F.H.C., Wyckoff, H.W., 1958. Diffraction by helical structures. Acta Crystallogr. 11, 199–213. Lorenz, M., Popp, D., Holmes, K.C., 1993. Refinement of the F-actin model against Xray fiber diffraction data by the use of a directed mutation algorithm. J. Mol. Biol. 234, 826–836. Lorenz, M., Poole, K.J., Popp, D., Rosenbaum, G., Holmes, K.C., 1995. An atomic model of the unregulated thin filament obtained by X-ray fiber diffraction on oriented actin-tropomyosin gels. J. Mol. Biol. 246, 108–119. Mannherz, H.G., Kabsch, W., Leberman, R., 1977. Crystals of skeletal muscle actin: pancreatic DNAase I complex. FEBS Lett. 73, 141–143. Mannherz, H.G., Leigh, J.B., Leberman, R., Pfrang, H., 1975. A specific 1:1 Gactin:DNAase i complex formed by the action of DNAase I on F-actin. FEBS Lett. 60, 34–38. Oda, T., Iwasa, M., Aihara, T., Maeda, Y., Narita, A., 2009. The nature of the globularto fibrous-actin transition. Nature 457, 441–445. Parry, D.A.D., Squire, J.M., 1973. Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles. J. Mol. Biol. 75, 33–55. Poole, K.J., Lorenz, M., Evans, G., Rosenbaum, G., Pirani, A., Craig, R., Tobacman, L.S., Lehman, W., Holmes, K.C., 2006. A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fiber diagrams from non-overlap muscle. J. Struct. Biol. 155, 273–284. Popp, D., Lednev, V.V., Jahn, W., 1987. Methods of preparing well-orientated sols of f-actin containing filaments suitable for X-ray diffraction. J. Mol. Biol. 197, 679– 684. Reedy, M.K., Holmes, K.C., Tregear, R.T., 1965. Induced changes in orientation of the cross-bridges of glycerinated insect flight muscle. Nature 207, 1276–1280. Rosenbaum, G., Holmes, K.C., Witz, J., 1971. Synchrotron radiation as a source for Xray diffraction. Nature 230, 434–437. Schwinger, J., 1949. On the classical radiation of accelerated electrons. Phys. Rev. 12, 1912–1925. Stubbs, G., Warren, S., Holmes, K., 1977. Structure of RNA and RNA binding site in tobacco mosaic virus from 4-A map calculated from X-ray fiber diagrams. Nature 267, 216–221. Suck, D., Oefner, C., Kabsch, W., 1984. Three-dimensional structure of bovine pancreatic DNase I at 2.5 Å resolution. EMBO J. 3, 2423–2430. Taylor, A., 1948. On an new type of rotating anode X-ray tube. Proc. Phys. Soc. 61, 86–94. Tirion, M.M., ben-Avraham, D., Lorenz, M., Holmes, K.C., 1995. Normal modes as refinement parameters for the F-actin model. Biophys. J. 68, 5–12. Watson, J.D., 1954. The structure of tobacco mosaic virus. I. X-ray evidence of a helical arrangement of subunits around the longitudinal axis. Biochem. Biophys. Acta 13, 10.
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Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi
50 years of fiber diffraction Kenneth C. Holmes * Max Planck Institute for Medical Research, 69129 Heidelberg, Germany
a r t i c l e
i n f o
Article history: Received 11 January 2010 Accepted 11 January 2010 Available online 15 January 2010 Keywords: Tobacco mosaic virus Coiled-coils Myosin cross-bridge Actin Tropomyosin Synchrotron radiation
a b s t r a c t In 1955 Ken Holmes started working on tobacco mosaic virus (TMV) as a research student with Rosalind Franklin at Birkbeck College, London. Afterward he spent 18 months as a post doc with Don Caspar and Carolyn Cohen at the Children’s Hospital, Boston where he continued the work on TMV and also showed that the core of the thick filament of byssus retractor muscle from mussels is made of two-stranded ahelical coiled-coils. Returning to England he joined Aaron Klug’s group at the newly founded Laboratory of Molecular Biology in Cambridge. Besides continuing the TMV studies, which were aimed at calculating the three-dimensional density map of the virus, he collaborated with Pringle’s group in Oxford to show that two conformation of the myosin cross-bridge could be identified in insect flight muscle. In 1968 he opened the biophysics department at the Max Planck Institute for Medical Research in Heidelberg, Germany. With Gerd Rosenbaum he initiated the use of synchrotron radiation as a source for X-ray diffraction. In his lab the TMV structure was pushed to 4 Å resolution and showed how the RNA binds to the protein. With his co-workers he solved the structure of g-actin as a crystalline complex and then solved the structure of the f-actin filament by orientating the g-actin structure so as to give the f-actin fiber diffraction pattern. He was also able to solve the structure of the complex of actin with tropomyosin from fiber diffraction. Ó 2010 Elsevier Inc. All rights reserved.
1. Birkbeck College In 1955, at the end of my undergraduate time at Cambridge, I thought that I would like to do something crystallographic for a Ph.D. I wrote round to a number of labs and was delighted to get an offer from Rosalind Franklin, at Birkbeck College London, to work on the structure of the rod-shaped tobacco mosaic virus (TMV). It turned out this was not quite crystallography as I had learnt it. Rosalind had found out how to orientate TMV by moving a 10–15% gel (really a sol) of the virus up and down glass capillary tubes. This initially led to flow-induced orientation. However, after some weeks about 10% of the specimens spontaneously developed perfect orientation that showed complete extinction between crossed polaroids. The orientated specimens were photographed with X-ray CuKa radiation in an evacuated camera that Rosalind had designed. Using bent-quartz monochromators and fine-focus X-ray generators such specimens could be induced to yield splendid fiber diffraction patterns, concentrated in 30–40 layer lines extending out to high (2.5 Å) resolution (a rather fine example using a different X-ray camera is shown in Fig. 1). For my thesis I concentrated on the zero layer line. Because of the cylindrical geometry and cylindrical averaging, fiber diffraction patterns, which can be derived from the Fourier * Fax: +49 6221 486437. E-mail address: [email protected]. 1047-8477/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2010.01.004
transform of the fibrous molecule, are much more economically expressed in cylinder functions rather than in sines and cosines. Moreover, because most objects repeat periodically along their length the scattering is confined to layer lines. The radial functionality (that gives rise to the intensity distribution along the layer lines) is expressed as Bessel functions of integral order. The order reflects the local azimuthal symmetry. In principle Bessel functions of all orders are allowed to contribute to every layer line. However, in practice things are not as bad as that. Crick and collaborators had shown that helical symmetry led to a sparse distribution of Bessel functions so that on any one layer line often only one or two Bessel functions would contribute (Cochran et al., 1952). TMV was known to be a helical structure that repeated every three turns (Watson, 1954). Moreover, on account of the high symmetry of TMV the low angle part of the zero layer line consists entirely of contributions from the cylindrically symmetrical part of the diffraction pattern, which is made of Bessel functions of zero order (J0). The amplitude of scattering in the inner part of the zero layer line is actually the sum over all atoms of J0 Bessel functions, each depending on the weight f and radius r of the j0 th atom in the virus
FðRÞ ¼
X
fj J 0 ð2pr j RÞ
ð1Þ
j
where F is the scattering amplitude and R is the reciprocal space radius. By inverting this process one this one can calculate the radial density distribution q of the virus
K.C. Holmes / Journal of Structural Biology 170 (2010) 184–191
J n ð2pRr Hg Þ
Fig. 1. A fiber diffraction pattern from an orientated gel of tobacco mosaic virus made by K.C. Holmes in D.L.D. Caspar’s laboratory in 1960 and photographed in Caspar’s laboratory in 1980 with a flat film camera, CuKa radiation and two focussing mirrors (courtesy of D.L.D. Caspar).
qðrÞ ¼
Z
1 0
FðRÞJ 0 ð2prRÞdR
ð2Þ
where r is the radius in real space. The observed intensities yielded only F2, thus one had to find a way of determining the signs of F. This was done by adding one methyl-mercury group to each subunit of the virus. Since the subunits are quite small (MW 17,500) the differences produced by adding one mercury atom were easily measurable. Once the radius of the mercury was determined its scattering J 0 ð2pRr Hg Þ could be calculated. The signs of the TMV data with and without mercury had to be chosen so that the difference agreed with the calculated mercury scattering. The radial density distribution calculated from the signed scattering amplitudes of the zero layer line showed, among other things, that the nucleic acid (a single RNA strand) was embedded in the virus at a radius of 40 Å and that the virus is hollow (see Franklin and Holmes, 1958). Eq. (2) (a zero order Fourier-Bessel transform) was evaluated as a 50 50 matrix multiplication (Rosalind had produced cyclostyled sheets comprising a square matrix of the values of J0(2prR) in intervals of 2 Å along the top and 0.002 Å1 down the side). I did many such operations. Each time the 2500 multiplications took me three days with a Marchant calculator. The noise was grim, especially for my room mates. Thus, when a little later Bill Longley joined us bringing some experience with the IBM 650, I gladly took to programming. The IBM 650 computer, located somewhere on the Strand, was a drum machine with 2048 locations total that used machine code written in SOAP (symbolic optimum assembly program). It had a strange word structure – 2 5 bits. Nevertheless, Bill had located subroutines for zero order Bessel functions. Thus in 1959 I became an early computer geek. At this time, although it was clear that TMV had a high symmetry and repeated in three turns, the actual symmetry had not been determined. Fortunately, the mercury scattering allowed us to determine the symmetry. The zero layer line arises from the projection of the structure onto an equatorial plane. If there are n subunits in the repeat of the virus there will be n mercury atoms forming a circle in the equatorial projection. At higher angles of scattering on the zero layer line (outside the J0 region) this ring of points will give rise to a characteristic scattering peak
185
ð3Þ
Since we had determined rHg and the position of the first peak of the Bessel function was measurable it was possible to calculate that n was 49 (Franklin and Holmes, 1956). During this time I met Mary Scruby, who worked in Birkbeck College library. We married in September 1957. She was a classics major. Later she became the founding librarian of the European Molecular Biology Laboratory in Heidelberg. In the Spring of 1958 Rosalind tragically died. The leadership of the group passed to Aaron Klug, who was then working on crystalline spherical viruses with John Finch. Aaron had collaborated with Rosalind on many theoretical aspects of the TMV work. He was a great teacher and I learnt much from him. Among his many achievements, he produced the formalism that allows a threedimensional density map to be calculated from fiber diffraction data (Klug et al., 1958). Only two systems (TMV and bacterial flagella) have yielded fiber diagrams of high enough quality to allow this to be carried out (most fiber applications rely on calculating the fiber diffraction from an atomic model and then somehow improving the fit) but later the formalism proved very useful for reconstructions of electron microscope images from helical biological fibers. The calculation of the three-dimensional electron density map of TMV at high resolution became the long-term aim of the group. To do this we had to find many heavy atom derivatives: not only did we need to phase the scattering amplitudes but also to separate out the contributions from overlapping Bessel function terms. Measuring the data also became a major job. Here we were very ably assisted for two years by Susan Fenn (later Lady Fenn, her husband Nicholas became British High Commissioner for India). 2. Children’s Hospital, Boston Don Caspar at Yale had calculated the radial density distribution of TMV for his thesis a couple of years ahead of me. In 1958 he moved to the Children’s Cancer Research Foundation (Jimmy Fund) in Children’s Hospital, Boston. He shared the lab with Carolyn Cohen. Don kindly offered me a post doc position to continue with the TMV work. Thus in February 1960 we took a Cunarder from Liverpool and 6 days later we were in New York. Then on to Boston, where we stayed for 18 months (see Fig. 2). Susan Fenn continued to measure and send on data on TMV that had to be evaluated. This led me to Project Mac in the cellars of MIT. An IBM 709 (later an IBM 7090) with a Fortran compiler was available for batch users. The job-control-cards were horrendous: one tiny mistake and the job was thrown out. Furthermore, the turn-round was 2–3 days – a good training for accuracy. I made lots of TMV specimens, one of which was photographed and showed wonderful orientation (a bit like good vintage wine) some 20 years later (Fig. 1). Don was always fascinated by things that did not quite fit. Thus he was delighted to discover that the Dahlemense strain of TMV showed satellite reflections not arising from the helical symmetry of the virus. After a hint from Carolyn we drew on radio side-band theory to interpret these as a modulation phenomenon: the outer parts of the virus subunits like to group in pairs thereby breaking the helical symmetry. Our paper was quickly accepted by J. Mol. Biol. (Caspar and Holmes, 1969) but spent another 7 years going backwards and forwards between the two nit-picking authors! Carolyn was not averse to finding jobs for other peoples’ post docs. Thus I was led to discover that the anterior byssus retractor muscle from mytilus edulis could be pulled into a quartz capillary tube to yield an orientated gel that showed very good extinction. Such samples gave excellent X-ray fiber diagrams. Most of the diffraction arises from the paramyosin in the middle of the myosin thick filaments, which is pure alpha helix. Using Francis Crick’s theory of diffraction for coiled-coils (Crick, 1953), Carolyn and I were
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K.C. Holmes / Journal of Structural Biology 170 (2010) 184–191
Fig. 2. Ken and Mary Holmes, Christmas 1960 at 45 Carlton St. Brookline, Boston Mass.
able to show that our ABRM fiber diffraction pattern was exactly what one would expect from a two-chain coiled–coiled alpha helix (Cohen and Holmes, 1963). This was my first foray into muscle.
3. Laboratory of Molecular Biology, Cambridge We returned to England in the summer of 1961 in time for the birth of out first daughter and I rejoined Aaron’s group. The plan
was to move the group to the newly founded MRC Laboratory of Molecular Biology in Cambridge. However, the building was not ready, so we continued at Birkbeck for another year. Finally, in the summer of 1962 we all moved to Cambridge. Here the search for heavy atom derivatives of TMV and data collection continued. Angela Mott replaced Susan Fenn. We were joined by Judy Gregory who made excellent orientated samples (Gregory and Holmes, 1965). Reuben Leberman helped with the chemistry. For the TMV work we used fine-focus X-ray generators and bent-quartz monochromators. Soon Bill Longley and I were involved in making fine focus rotating anode X-ray generators, ably assisted by Tony Woollard. Our efforts turned into the Elliott GX6 X-ray generator (Fig. 3). Hugh Huxley started using our apparatus to register the low-angle X-ray fiber diffraction from contracting frog muscle. It was clear that Hugh really needed a thousand times more intensity than we could give, so in 1964 I started wondering about synchrotron radiation. Julius Schwinger’s theory (Schwinger, 1949) allowed the expected intensity to be calculated. This was full of Bessel functions, but I had Bessel function subroutines to hand and could estimate the strength of synchrotron X-ray radiation. It looked good! However, at this time neither the German ring DESY at Hamburg nor the English ring at Daresbury were actually working, so the idea was put on hold. About this time a collaboration with John Pringle’s group at Oxford led to an interesting experiment on flight muscle from the giant water bug Lethocerus. Insect flight muscle shows the phenomenon of stretch activation: when turned on by Ca2+ the muscle contracts only if you stretch it. Heart muscle also shows this phenomenon to an extent. Pringle’s co-worker Richard Tregear had set up strips of insect flight muscle in a machine that oscillated the muscle, which allowed it to generate work cyclically when fed with ATP. The muscle indeed generated Lissajous figures showing that it was doing work on the machine. Richard came to Cambridge with a sample. We took a low-angle X-ray diffraction picture in rigor (in a Ringer’s solution) and then added ATP. Each exposure took ten minutes because that’s all the time we had before we dashed off for the train to London to take part in a meeting of the London muscle club. We examined the photos in the train. They showed that there was a strong 145 Å meridional reflection in the ATP-containing solution but none in rigor (Fig. 4). The experiment proved to be both reversible and reproducible. Mike Reedy subsequently used Hugh Huxley’s wonderful technology for cutting ultra-thin sections from plastic embedded muscle to show by electron microscopy that the myosin cross-bridges were giving the signal and that they were taking on two different orientations: at 90° to the axis with ATP and 45° to the axis without ATP (Reedy et al., 1965). This observation became the basis of the swinging crossbridge hypothesis of muscle contraction, which even now appears to be correct.
4. Max Planck Institute for Medical Research, Heidelberg
Fig. 3. A fine focus rotation anode X-ray generator (W. Longley, T. Woollard and K.C. Holmes) based on the Taylor rotating anode (Taylor, 1948) and the cathode from the Beaudouin X-ray Generator. This was the prototype of the Elliott GX6 X-ray generator.
This simple experiment turned out to have far reaching ramifications, not only for science but also for me personally, because it led to a ‘‘call” from the Max Planck Institute of Medical Research in Heidelberg to open a department of biophysics. The retiring director of the Department of Physiology, Hans Hermann Weber, was a famous muscle physiologist who wanted someone to continue the long tradition of muscle research in the Max Planck Institute in Heidelberg. He would have preferred Hugh Huxley but decided to settle for Ken Holmes. Hence in September 1968 Mary and I with our children moved to Heidelberg, where we are still. The department was set up as two sub-departments, for biophysics and for biochemistry. Since I have an indifferent reputation as a chemist, I entrusted setting up the biochemistry to Reuben Leberman,
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Fig. 4. X-ray fiber diagram from a strip of insect flight muscle (left) in rigors solution and (right) in rigors solution containing 10 mM Mg ATP (20 min exposure). After Reedy et al. (1965).
who complete with Pat Leberman and their children came with us from Cambridge. The department survived until 2003 when it was closed on account of my retirement. In Heidelberg we continued the TMV work. John Barrington Leigh, who had been a student in Cambridge, came and worked together with Eckhard Mandelkow, Tony Barrett, and Ute Gallwitz to make and determine the positions of five different heavy atoms at-
Fig. 5. The Rosenbaum-Barrington Leigh X-ray beam line in Bunker 2 at DESY Hamburg with the author making adjustments. The focussing system was: bent mirrors in the vertical plane and a bent-quartz monochromator in the horizontal plane. When the synchrotron beam was on all adjustments had to be made by remote control. The optical bench had over 100 motorized adjustments. The mirror box (front) was helium filled.
tached to the virus protein (Barrett et al., 1971; Holmes et al., 1972). This was the prerequisite for calculating the structure. Later two collaborators, Gerald Stubbs and Steve Warren used these heavy atoms to calculate a density map of the virus at 4.0 Å resolution (Holmes et al., 1975; Stubbs et al., 1977). Gerald left the lab to work with Don Caspar. He finally was able to calculate a 2.8 Å resolution map of TMV from fiber diffraction data. In 1969 Gerd Rosenbaum joined me to do a doctoral thesis on insect muscle. The aim was to try to get time-resolved diffraction from contracting muscle. Hugh was doing similar things with frog muscle. Gerd soon became frustrated with conventional X-ray sources and took up the idea that we go to DESY Hamburg to try to use synchrotron radiation. He had worked at DESY on synchrotron light for his diploma and knew the physical set up. In the summer of 1970 we conducted the first proof of principle experiments at DESY to show that indeed the ring was an intense source of Xrays: at least 10 times stronger than any laboratory source (Rosenbaum et al., 1971). In addition it had laser-like optics. Synchrotrons have their limitations since they dump the beam every 20 ms. Storage rings, with a continuous high intensity electron beam, are far better. Nevertheless, with the enthusiastic support of the DESY directorate, in particular of Martin Teucher, Bunker 2 was built
Fig. 6. The G-actin monomer consists of two sub-domains with ADP or ATP sandwiched between the sub-domains.
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for pro tem use of the synchrotron ring. In the next two years in Bunker 2 Gerd Rosenbaum and John Barrington Leigh built the first ever X-ray beam line (Barrington Leigh and Rosenbaum, 1974). The beam was 10–20 times more intense than a laboratory source and very much better collimated, which made it particularly attractive for low angle scattering experiments on muscle (Fig. 5). We mostly used the beam for experiments on insect flight muscle for trying out the effects of ATP analogs, made for the purpose by Fritz Eckstein and Roger Goody, to see if we could get evidence for other states in the muscle (Goody et al., 1975). Richard Tregear was an enthusiastic collaborator. The fact that we were parasitic users of the beam made our endeavor fairly cheap but sometimes led to extreme frustration since the beam (or lack of it) depended entirely on the needs of the high energy physicists. With a promise of 50–500 times more intensity clearly the future lay with storage rings. We designed another lab, Bunker 4, lo-
cated at the perimeter of the new storage ring DORIS. However, the whole enterprise was getting too big to be an adjunct of a Max Planck department. Hence we were grateful when in 1975 it was taken over by the newly formed European Molecular Biology Laboratory as the EMBL Outstation. Here in 1980 Hugh Huxley was at last able to conduct time-resolved measurements frog muscle that showed that the 145 Å meridional dropped right down in intensity as the muscle recovered tension after a quick release (Huxley et al., 1980), a result that was fully consistent with the swinging crossbridge theory of muscle contraction. 5. Actin In the late 70’s I gave up the TMV work to concentrate on actin. Valerie Lednev, while a postdoc at Kings London in the 70’s, had managed to orientate F-actin gels much as we had orientated
Fig. 7. Fitting the g-actin monomer into the f-actin helix. F-actin has thirteen subunits every six turns.
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TMV. Lednev then returned to Pushchino and after some efforts by Gillis and O’Brien the know-how went into abeyance. However, at the end of 1985 Lednev visited Germany and, having stopped at the Russian Embassy in Bonn long enough to fill his copious overcoat pockets with bottles of duty-free whisky, visited us for an extended Christmas party. During the course of this well lubricated exchange of views David Popp, then a diploma student in the lab, was instructed by Valerie on how to make orientated gels of filamentous actin (f-actin). David was successful and soon we had high quality fiber diagrams from f-actin gels (Popp et al., 1987). However, the fiber diagrams were nothing like so perfect as from TMV: the resolution was limited (about 5 Å); moreover, the layer lines overlapped. There was no hope of determining the structure from first principles as we had achieved for TMV. The alternative approach is model building: if you have a structure for the subunit (g-actin) then you can try it in all orientations in the fiber to see if you can compute the correct fiber diagram. Hence we were obliged to do the X-ray crystallography of g-actin. G-actin is difficult because its response to adding salt is not to crystallise but to make f-actin. However, in 1974 Uno Lindberg had published the observation that g-actin binds tightly to the pancreatic enzyme DNAse 1 thereby inhibiting f-actin formation. Reuben Leberman managed to make small crystals of the complex that were improved by Hans-Jörg Mannherz (Mannherz et al., 1977, 1975). However, solving the structure proved very difficult. First Wolfgang Kabsch and Dietrich Suck managed to solve the structure of DNase 1 (Suck et al., 1984). Data collection from the actinDNase1 complex was a nightmare since the crystals changed their cell size in response to a host of unknown environmental parameters. The answer was to collect all of a three-dimensional data set from one crystal. This became possible through the advent of 2D electronic detectors. Wolfgang developed his famous XDS software (Kabsch, 1988a,b) just for the purpose of data collection from actin-DNase 1 crystals using 2D detectors . After some struggles the structure of g-actin became available (Kabsch et al., 1990). It was a rather flat molecule consisting of two structurally similar sub-domains (see Fig. 6). Now we could try to fit it to the fiber diffraction pattern. In a computer search only one orientation came close to fitting the fiber
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diffraction pattern. Thus we had a model of f-actin (Holmes et al., 1990) (see Fig. 7). However, the fit to the fiber diffraction pattern was not perfect: apparently g-actin undergoes some (small) conformational change between g- and f-actin. It took another twenty years to work out what the conformational change was. I tried sub-domain refinement (Holmes et al., 2004), Monique Tirion tried normal modes (Tirion et al., 1995). Michael Lorenz ingeniously achieved a perfect fit to the fiber diagram using a genetic algorithm but somewhat at the expense of the stereochemistry of the actin monomer (Lorenz et al., 1993). He was clearly close to the correct structure but unfortunately the secondary structure had suffered. Recently Toshiro Oda, working at Spring 8 in Yuichiro Maeda’s group managed to improve the orientation of f-actin by placing the gels in an 18 tesla magnetic field. This gave more resolution. Toshiro was then able to show that between g- and f-actin the two sub-domains rotate by about 20°, and nothing much else happens (Oda et al., 2009). We should have got this result! (see Fig. 8). By observations of the second and fourth actin layer lines from intact muscle (Huxley, 1972; Haselgrove, 1972; Parry and Squire, 1973), it was shown that muscle activation causes the movement of tropomyosin on the surface of actin. The second layer line is responsive to two-fold symmetry and the fourth layer line to four-fold symmetry. The fourth actin-based layer line becomes stronger on activation. On activation of muscle, which releases Ca2+ into the sarcoplasm to bind to troponin, tropomyosin is induced to move so as to increase the four-foldedness of the filament and free up the myosin binding sites on actin. David Popp was a skillful actin orientor and a competent biochemist so that he was able to make an orientated gel from a synthetic actin-tropomyosin complex and then collect X-ray fiber data. We fitted this data with a model of tropomyosin on actin (Lorenz et al., 1995) – Fig. 9. It showed that the tropomyosin lies along the surface of actin in a way that allows each of the tropomyosin pseudo-repeats to have the same geometrical view of an actin monomer, but that there is no explicit contact between tropomyosin and actin (there is a layer of solvent between them). Tropomyosin is attracted electrostatically to actin but it only binds to actin because it binds end-to-end with itself. This simple model allows
Fig. 8. To show how a 20° twist of the sub-domains 1 and 2 against 3 and 4 (shown left) of g-actin gives an excellent fit the f-actin fiber diffraction pattern. In the fiber diffraction pattern on the right the upper right and lower left quadrants are data, the other two quadrants are the diffraction calculated from the model. I am grateful to Yuichiro Maeda for the sketch on the left.
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Fig. 9. The arrangement of tropomyosin on actin. (left cartoon, right space filling model).Tropomyosin is a coiled–coiled coil with the same shape as actin. Each tropomyosin molecule spans 7 actin monomers . Tropomyosin has a strong pseudo repeat of 39.3 residues. Each pseudo repeat of tropomyosin matches up with an actin monomer. Electrostatic interactions are made between the two molecules. (From Holmes and Lehman, 2008 with permission.)
tropomyosin to be pushed around on the actin surface by troponin in response to Ca2+ binding as had been foreseen by Parry and Squire. However, in spite of offering an easy explanation of tropomyosin movement our model was not enthusiastically embraced by workers in the field since they were looking for specific interactions of tropomyosin with actin. Recently, our simpler view that there are none has found more acceptance. Ours was a troponin-free system with no ability to study the effects of Ca2+ binding to troponin. Kate Poole worked up a system using demembranated muscle fibers pulled out so that there is no overlap between the actin (thin) filaments and the myosin (thick) filaments. This approximates to an orientated gel of native thin filaments with an intact troponin-tropomyosin system. Using this preparation she could add and subtract Ca2+ many times to watch (by fiber diffraction as in Parry and Squire) the movement of tropomyosin orchestrated by troponin. Moreover, she could get accurate data on the tropomyosin movement by taking the differences between X-ray fiber diagram with and without Ca2+. This
allowed the magnitude of the movement to be calculated and to be compared with electron microscope observations of the filaments with and without Ca2+ that had been made by Bill Lehman’s group and Roger Craig’s group (Poole et al., 2006). The agreement was satisfying and moreover, has led to some interesting collaborations with Bill Lehman (Holmes and Lehman, 2008). This was really my last experiment with fibers. Now I am reduced to interpreting other people’s data and producing models of how muscle might work.
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