How
x-ray diffraction with synchrotron radiation got started
K.C.
Holmes
Max
Planck Institut für medizinische Forschung 69120 Heidelberg
Germany
and
G.
Rosenbaum
Structural
Biology Center, Argonne National Laboratory, 9700 S. Cass Ave. Argonne
IL 60439, USA
The
need to record low angle scattering x-ray fibre diagrams from muscle
with milli-second time resolution drove the use of synchrotron
radiation as an x-ray light source. The first smudgy diffraction
patterns were obtained from a slice of insect flight muscle. Out of
this grew the EMBL Outstation at DESY.
Key
words: x-rays, diffraction, muscle, EMBL
1.Introduction
Synchrotron
radiation has proved to be of inestimable importance for extending the
scope of x-ray diffraction methodology for protein crystallography. The
laser-like optics and tremendous brilliance allow x-ray diffraction
data to be collected from ever smaller crystals of ever larger
complexes and organelles. In addition, the ability to select
wavelengths makes it possible to exploit anomalous scattering for phase
determination (MAD). Moreover, the continuous spectrum makes it
possible, in favourable cases, to obtain data in fractions of a second.
Within the next decade genome sequencing projects will provide us with
a complete menu of all DNA, RNA, and protein molecules in a
number of organisms and will thereby give us the inventory of life. To
make full use of all these data we will need to understand the
macromolecular interactions which control cell signalling, cell
locomotion and gene activation. To do this we will need a detailed
description of the structure of many of the relevant macromolecules.
At present, only x-ray protein crystallography yields a molecular
anatomy of adequate resolution and precision for this task. Thus there
would seem to be an almost limitless need for x-ray crystallography
groups working on proteins if full use is to be made of the flood of
genetic information arising from the sequencing projects.
However,
this is not how x-ray diffraction with synchrotron light got started,
it started with muscle.
2.
Muscle - the Problem
Muscle
is an isothermal engine which works by hydrolysing ATP (adenosine
triphosphate) with an efficiency near 50%. When muscle contracts, two
sets of protein filaments, the actin filaments and the myosin filaments
glide over each other. The gliding is driven by the cyclical
interactions of the myosin "cross-bridges" with actin so as
to "row" one set of filaments past the other: a cross-bridge
binds to actin in a initial position and "swings" into a
final position. This movement is driven by the binding to actin which
enables release of the products of ATP hydrolysis (ADP and phosphate).
At the end of the stroke ATP rebinds to the myosin cross bridge causing
rapid release from actin. Subequently ATP is hydrolyzed to ADP and
phosphate and the cycle repeats (Fig 1) (Lymn & Taylor, 1971). One
major aim of muscle research has been to demonstrate and understand
what actually happens when a cross bridge "swings".
The
cross bridges in muscle fibres repeat along the fibre axis with a
repeat distance of ca. 14.5nm. Thus they give rise to an x-ray
diffraction pattern with a series of strong meridional reflexions.
Alterations in shape of the cross bridges lead to changes in the
intensities of these reflexions. The sartorius muscle from frog can be
dissected out intact and made to contract by electrical stimulation.
X-ray diffraction patterns can then be recorded from an actively
contracting muscle. Pioneering work with conventional sources was
carried out in the 1960's by H.E. Huxley (Fig 2) (Huxley & Brown,
1967). However, the scattering is weak and muscles quickly become
fatigued. We in Heidelberg hoped that insect flight muscle might
provide an alternative system to frog muscle for studying the cross
bridges by x-ray diffraction. Insect flight muscle changes its
structure on adding ATP which leads to dramatic changes in the low
angle meridional reflexions (Reedy et al ., 1965). Moreover, it
is highly crystalline. However, we met all the intensity problems
encountered by H. E. Huxley for frog muscle but in a more acute form
since the specimens are much smaller. To get further we needed much
stronger x-ray sources.
3.
First diffraction experiments are carried out at DESY
In
1969 Rosenbaum started his doctorate in Heidelberg on developing x-ray
sources for diffraction studies of muscle Together with H.E. Huxley in
Cambridge a rotating anode x-ray tube of high brilliance was developed
(which became the GX18 of Elliott Bros). However, one quickly runs into
a performance barrier set by the strength of materials (Rosenbaum,
1979). Frustration with rotating anode tubes drove us to re-evaluate
the performance of synchrotrons as x-ray sources. Initial estimates of
the strength of synchrotron radiation K.C. Holmes had made in the '60s
indicated that synchrotrons at 6.0 GeV would not be much better than
existing rotating anode tubes. In the mean time the energy of the
machine had risen from 6.0GeV to 7.2GeV and the beam current was often
in excess of 10mA. Now things looked more promising. Moreover, G.
Rosenbaum had done his diploma work at DESY in the F41 VUV group so
that he could provide important know-how about the physical set up at
DESY. Therefore, with the ecouragement of Dr Heansel and the F41 group
we set about conducting trials in the VUV bunker at DESY.
Working
together with Jean Witz, who was an authority on x-ray optical systems,
we introduced a focusing x-ray quartz monochromator into the
synchrotron beam. A vacuum chamber was constructed to house a bent
quartz monochromator and slit assemblies with a berillium exit window
(Fig. 3). The quartz monochromator could be moved across the beam by
remote control. For all other adjustments the main beam shutter had to
be closed: reopening the beam shutter required retreating behind a
massive sheilding wall, setting up the interlocks, and a telephone call
to the main control room. This process made adjustment rather tedious.
However, estimates of the monochromatic beam intensity were very
encouraging. Therefore we set up a primitive x-ray diffraction camera
on the monochromatic beam and the first rather smudged x-ray
diffraction photograph using synchrotron radiation (from a strip of
insect flight muscle kindly supplied by Dr. H-G. Mannherz) was obtained
in August 1970 (Rosenbaum et al ., 1971)
4.
Bunker-2
On
the basis of these initial experiments the directors of DESY (in
particular Martin Teucher, who was responsible for buildings)
encouraged us to set up a bunker for x-ray diffraction experiments on
biological samples. The original (and final) plan was to set up an
x-ray lab on the storage ring DORIS which would have at least 100 times
more intensity than DESY. However, we were persuaded by Prof. Jenschke,
the founding director of DESY that since DORIS would not be available
as a synchrotron light source for 2-3 years it would be advantageous to
build an x-ray laboratory on the DESY synchrotron in order to gain
experience. However, we would need to act fast! The window of
opportunity was the major shut-down in 1971 engendered by the
construction of the connecting tunnels from DESY to the new strorage
ring DORIS. After this date the massive earth movements required to
create a new bunker would no longer be possible. Thus an x-ray
laboratory was built onto DESY during the shut down of 1971 and became
known as "Bunker-2". At the same time the tunnel for the
laboratory-to-be on DORIS were also built (to be completed in 1975).
Above Bunker 2 two offices and a room for biochemistry were added.
5.
EMBL
The
history of this bunker became entwined with the history of the European
Molecular Biology Laboratory, EMBL. It was clear that a sychrotron
radiation x-ray diffraction facility would become a central facility
for European research, particularly in molecular biology where the
advantages of good x-ray optics and intensity were appreciated much
earlier by the structural biologists than in the general
crystallographic community, who only became interested a decade later.
What better vehicle for such a facility could there be than EMBL. In
1969 advised by H.E. Huxley, we made a proposal for such a facility to
Sir John Kendrew, the head of the EMBL "Project" (EMBL did
not exist legally for another 5 years). John Kendrew greeted our
initiative with enthusiasm. The new EMBL laboratory was dedicated to
technological developments for molecular biology. High level contacts
ensued at which it was agreed that the developments in Hamburg should
become part of an outstation of EMBL at DESY. Thus a little later
Bunker 2 became the provisional headquarters of the EMBL outstation.
The initial financing of this project is a tribute what can be achieved
through good will. Salaries were initially paid by the Deutche
Forschungsgemeinschaft and then by EMBO (the private sister
organisation to EMBL). Building costs were covered by the
Bundesministerium für Wissenschaft und Forschung (via DESY) -
perhaps hoping this would help ensure that EMBL was established in
Germany - and the Max Planck Institute for Medical Research in
Heidelberg carried the equipment costs. With the ratification of the
EMBL agreement in 1974 the whole project was taken over by EMBL. An
official agreement between DESY and EMBL setting up the Outstation was
signed in 1975 (Fig 4).
6.
The first beam line
In
the mean time work went on. Inside Bunker 2 a massive neutron-proof
concrete wall separated the operators from the beam. Therefore, all
adjustments had to be made by remote control. In collaboration with
John Barrington Leigh, Gerd Rosenbaum set about building a fully
remotely controlled optical bench (Barrington Leigh & Rosenbaum,
1974; Barrington Leigh & Rosenbaum, 1976) (Fig. 5). A Guinier
monochromator was used to focus the fan of radiation from the
synchrotron in the horizontal plane and 2 x 20 cm. adjustable bent
mirrors were used to focus the much smaller divergence in the vertical
plane. The mirrors (fused quartz) were nearest to the synchrotron and
were housed in a helium-filled box separated from the machine vacuum by
a beryllium window. Beams were accommodated in vacuum tubes fitted with
mylar windows. It proved difficult to obtain mirrors polished to the
necessary flatness: optical mirror manufacturers had no way of
monitoring the micro-flatness necessary for x-ray mirrors. Here we were
considerably helped by the pioneering work of Franks (Franks &
Breakwell, 1974). Movements were controlled by about 100 small DC
motors with potentiometers as position sensors. DC motors were chosen
rather than stepping motors because they are light: the whole apparatus
was built on a mini budget and the apparatus could not become massive.
A SIT-vidicon camera was used to observe in line the image of
the direct beam formed on a cesium iodide crystal. Two other steerable
TV cameras fitted with zinc sulfide screens were mounted on a parallel
optical bench for visual observation. Zinc sulfide screens could be
inserted into the beam path by remote control for monitoring the beam
e.g. before and after the slits. The monochromator (quartz) was cut at
7° to the surface so as to approximate to the Guinier-condition
for the given source distance and the desired focal distance, i.e. the
desired demagnification. The deviation from the exact Guinier-geometry
resulted in a wavelength inhomogeneity across the converging beam.
However, this effect was small for the apertures being used and was not
important in small-angle diffraction. The angle of the latter part of
the optical bank to the direct beam was fixed for _=1.5Å. Since
the optical elements were about 40m from the tangent-point of the
synchrotron and focused within 2-3m a demagnification of H 15 was
achieved. The electron beam of DESY was relatively compact so that a
focused beam of dimensions 200 x 250 m could be obtained. With a flux
of H 10 9 photons/s and excellent
optical properties this was a very good beam for low angle scattering.
The flux density was two orders of magnitude better than could
be achieved with the best rotating anode tubes. Images were registered
on film or on one-dimensional single-wire (Gabriel & Dupont, 1972)
position sensitive detectors. The beam line was in operation in 1972
and for a couple of years remained a unique facility.
7.
Diffraction from insect flight muscle
Using
this beam line, the Heidelberg group (in collaboration with Richard
Tregear from Oxford) studied the diffraction from insect flight muscle.
The excellent collimation led to detailed fibre diffraction pictures
which yielded new structural information (Holmes et al ., 1980).
Time resolved experiments were set up with oscillating insect flight
muscle. The muscles were attached to a vibrator and oscillated at 5
Herz at which frequency they generate considerable work if provided
with ATP. At low amplitudes of oscillation it was expected that the
cross bridges might be partially synchronized so that one should be
able to record diffraction patterns from various parts of the
cross-bridge cycle. The diffraction was recorded, a layer line at a
time, on a position sensitive detector and the output switched into one
of 32 bins in synchrony with the oscillation. Data with usable
statistics could be obtained from the equator in about 15mins. However,
on account of the available intensity, the measurements remained
confined to the strong equatorial reflexions (Barrington Leigh &
Rosenbaum, 1976). Unfortunately, these reflexions alter little between
resting and contracting insect flight muscle and, therefore, are not
very useful for monitoring the cross-bridge swing. We were not
observing the cross bridges in flagranti . The insect flight
muscle experiments needed a storage ring!
Since
the intensity was not adequate to allow a time resolved study of the
meridional reflexions (which do alter with cross bridge
orientation) attempts were made to "freeze" the cross-bridges
in alternative conformations by the use of non-hydrolysable analogs of
ATP (Goody et al ., 1976). Quite large changes in the
diffraction pattern were induced by certain analogs. However, a large
part of the changes resulted from alterations in the pattern of binding
of the cross bridges to actin, rather than in an underlying change in
the cross-bridge orientation (Goody et al ., 1975).
The
group shared experiences with Hugh Huxley and Uli Arndt in Cambridge
who were setting up a similar beam line at NINA for experiments on frog
muscle.
8.
Protein crystallography
The
initial success at DESY sparked world wide interest. In June 1972 there
was a historical meeting in Brookhaven at which most of the subsequent
applications of x-ray synchrotron radiation were discussed (see
-BNLReport 1973). The most important application for biology later
proved to be protein crystallography. Early tests of protein
diffraction on the DESY source (Harmsen et al ., 1976) showed
improvements compared with conventional sources but the gains were
limited. The flux was about ten times better than with a conventional
source. At this stage one had failed to appreciate that the parallel
collimation of the beam was giving unusually good signal/noise. This
was the property of synchrotron radiation which ultimately made it the
source of choice for all kinds of protein crystal data collection.
About the same time studies on the Stanford storage ring SPEAR
(Phillips et al ., 1976) showed gains for crystal diffraction
even with a non-focusing monochromator which showed clearly that
storage ring sources were to be of considerable importance in protein
crystallography. These authors made use of the ability to
"tune" the wavelength across an adsorption edge to
demonstrate the potentialities of synchrotron radiation in exploiting
the effects of anomalous dispersion.
9.
Bunker 4
The
experimental facility at DORIS was housed in a small experimental hall
rather than a bunker. Nevertheless, it was known as
"Bunker-4". Duly equipped with offices, seminar room,
workshops, and a biochemistry laboratory this building became the home
of the EMBL Outstation in 1975. DORIS is a colliding beam facility with
electron and positrons circulating in opposite directions. The beams
into the EMBL bunker were from the positron ring. The first beam-line
set up (X11, designed by G. Rosenbaum and A. Harmsen, later taken over
by H. Bartunik) was a mirror-monochromator combination with 8x20cm
mirrors and a bent germanium monochromator (Rosenbaum, 1979; Rosenbaum
& Holmes, 1980). The bench carrying the specimen and detector could
be rotated around the monochromator as pivot so as to vary the
wavelength. Each of the mirrors could be individually bent. The
electron beam in DORIS was considerably larger in cross-section than
that of DESY so that fine-focused beams such as were obtained on the
DESY synchrotronwere not attainable. In fact it turned out that
although each of the mirrors was designed to be individually bent it
was not really worth bending the mirror segments at all, aligning them
appropriately without bending produced as fine a beam as one could get.
A second optical system X13 (Bordas et al ., 1980) similar in
design to X11 was soon added. The two shared a common mirror box and
mirror design. These beam lines were the workhorses of the DORIS
facility for a number of years. The EMBL Outstation in Bunker-4
expanded steadily and became one of the most widely used biological
facilities in the world.
10.
Time resolved studies on frog muscle
HE
Huxley continued with frog muscle and transferred his work to the EMBL
Outstation when this became operative (Huxley et al ., 1980).
The key experiments were carried out a little later (Huxley et al .,
1981) by a team which included a number of people who have subsequently
played an important role in the development of synchrotron radiation as
an x-ray light source. These experiments finally showed the anticipated
changes in intensity of the meridional reflexions: if a contracting
muscle is released the intensity of the 14.35nm meridional reflexion
drops within a few ms to a fraction of its initial value. If the muscle
is extended quickly, the intensity is recovered. If one waits at the
new length the intensity recovers. These experiments have recently been
repeated with very high time resolution using sophisticated mechanics
and the excellent two dimensional detectors at Daresbury (Irving et
al ., 1992). These observations are fully consistent with the
swinging cross bridge hypothesis and for many years represented the
most important time resolved experiments supporting the swinging
cross-bridge hypothesis.
Unfortunately,
the resolution of these changes is actually too low to show the
detailed molecular basis of the cross-bridge swing. Over the years it
became likely that the swinging cross bridge was actually a swinging
lever arm (Fig 6) (Cooke, 1986). Protein crystallography, which in turn
needed synchrotron light, finally yielded insight into this mechanism.
11.
Atomic structures give insight
The
structure of the actin monomer and of the actin filament have been
solved by protein crystallography (Kabsch et al ., 1990) and by
x-ray fiber diffraction (Holmes et al ., 1990) . The crystal
structure of the myosin subfragment 1 (Rayment et al ., 1993b) showed the myosin cross-bridge to have
an extended C-terminal neck which looked like the anticipated lever arm
and, moreover, a lever arm which was in the correct orientation and
position to function as a lever arm (Rayment et al ., 1993a). In
the last year a number of independent experiments provide results which
are in excellent accord with the idea that the C-terminal tail
functions as a lever arm and indeed provide evidence that it can move
(see review (Holmes, 1997)). Furthermore, new crystal structures
(Fisher et al ., 1995; Smith & Rayment, 1996a) with analogs
of ATP bound appear to show an alternative orientation of the lever of
the anticipated kind.
The
crystallographic studies cited show two distinct structural states for
the myosin cross-bridge: the "open" or "end"
conformation which is characterized by the absence of nucleotide
(rigor); and the "closed" or "beginning" state,
which is favored by binding ATP or the products complex (ADP.Pi) (Fig
7). Myosin transports actin by switching between these two states.
"Open" and "closed" refer to the status of the ATP
binding site. This in turn is coupled to the rotation of the C-terminal
lever arm. In the "closed" form the lever arm is at the
beginning of the power stroke whereas in the "open" form it
is at the end of the power stoke. The preference for "open"
or "closed" is also controlled by binding to actin. It is
likely that the closed state binds only weakly to actin. On this basis
the structural states can be correlated with the Lymn-Taylor cycle.
Starting
from an actin-myosin complex at the end of the power stroke, the
binding of ATP brings about rapid closure of the ATP binding site and
concomitant release from actin. The closed state hydrolyses ATP to ADP.
Pi without attaching to actin. Thereafter, the rebinding of myosin in
the closed or "beginning" conformation of the products
complex to actin opens the site to facilitate release of
the__-phosphate. Release of phosphate in turn induces an isomerisation
to the open "end" conformation since it is the presence of
the__-phosphate which stabilizes the closed form. The isomerisation
results in large changes of angle of the "lever arm" (at the
distal part of the myosin head). Since the S1 is strongly attached to
actin at this stage this results in a 12nm transport of actin past
myosin.
12
It would not have been possible without synchrotron light
Thus
we see that one of the most important puzzles of biology, the basis of
animal movement, which originated as a research project with the
Alexandrian school in the third century BC has yielded many of its
secrets to a structural and physico-chemical analysis. It is noteworthy
that this could not have happened without synchrotron radiation
sources. Moreover, this project opened up one of the most important
uses of synchrotron radiation yet discovered, namely its use as an
x-ray diffraction a source for protein structure determination.
Figures
Fig 1. The Lymn-Taylor
cycle (Lymn & Taylor, 1971) . A diagrammatic representation of the
crossbridge cycle: the myosin cross bridge is bound to actin in rigor
45°- position - "down" (1) ATP binds which leads to very
fast dissociation from actin (2)]. The hydrolysis of ATP to ADP and a
phosphate ion leads to a return of the myosin cross bridge to the
90° "up" position(3) whereupon it rebinds to actin (4).
This leads to release of the products and return to [1]. In the last
step actin is "rowed" past myosin.
Fig 2. X-ray fibre
diffraction pattern of a living frog muscle (Huxley & Brown, 1967).
The fibre axis is at right angles to the x-ray beam. Note the regular
layer lines (with a repeat of (1/430Å) which arise from the
helical array of myosin cross bridges. The third order (1/143.5Å)
meridional reflexion corresponds to the repeat distance between
cross-bridges. (This x-ray fibre diagram was obtained using a rotating
anode x-ray generator and mirror-monochromator optics).
(Figure still to come)
Fig 3a. Diagram of the
vacuum chamber used to house the bent quartz monochromator in the
experiments on the VUV bunker. The monochromator could be translated by
a motorised control, The exit wíndow was a disc of berillium ).
Fig3b The equatorial diffraction (A) from a piece of insect flight
muscle obtained using the synchrotron radiation beam from the apparatus
shown in Fig 3a. Also shown is (B) the equatorial diffraction from a
similar sample obtained with a conventional x-ray source. The first
strong x-ray reflexion (seen only the right side because of parasitic
scattering)is the 200 reflexion (Bragg spacing 21nm) from the hexagonal
muscle lattice.(Rosenbaum
et al., 1971).
Fig 4 In 1975 EMBL and
DESY entered into a formal agreement to set up an EMBL outstation at
DESY in Hamburg. The photograph shows H. Schopper Chairman of the
Directors of DESY, J.C. Kendrew Director General of EMBL and W.
Berghaus the Administrative Director of DESY at the signing ceremony.
(Figure still to come)
Fig 5 Diagram of the
remotely controlled low-angle diffraction camera in Bunker 2. The
mirrors were housed in a helium pespex case fitted with mylar windows.
The curved quartz monochromator was mounted in air. All other
intervening beam paths were through lead-covered vacuum tubes. Note the
primary slits (aperture 1) and the guard slits (aperture 2). The shape
and composition of the slits were important for reducing parasitic
scatter. A monotoring TV camera ran along a parallel optical bench (not
shown).
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