Jeffrey J. Harford

Fine structure of the A-band in cryo-sections: III. Crossbridge distribution and the axial structure of the human C-zone

Abstract Optical analysis of the C-zones of negatively stained cryo-sectioned A-bands from human m. tibialis anterior muscle, combined with computer modelling of the C-zone staining pattern, has led to the following conclusions. 1. (1) At a 99.9% confidence level there are two slightly different axial repeats in the C-zone. One repeat of 434 (± 1.2) A probably corresponds to the C-protein repeat, whereas the other of 429 A (used for calibration) is probably due to myosin crossbridges. 2. (2) The myosin crossbridge array in (1) does not consist of crossbridge rows evenly spaced at 143 A. The crossbridge positions are periodically shifted from such positions to give a true 429 A crossbridge repeat. 3. (3) The myosin crossbridges are oriented almost perpendicular to the long axis of the muscle; the axial extent of the crossbridge profiles is less than 80 A. It is shown that computed model structures which explain satisfactorily the appearances of cryo-sectioned C-zones can also explain several anomalous features of published X-ray diffraction patterns from vertebrate muscle.

Actin filament organization and myosin head labelling patterns in vertebrate skeletal muscles in the rigor and weak binding states

SummaryThe structures of vertebrate skeletal muscles (particularly from frog and fish) in the rigor state are analysed in terms of the concept of target areas on actin filaments. Assuming that 100% of the heads are to be attached to actin in rigor, then satisfactory qualitative low-resolution modelling of observed X-ray diffraction data is obtained if the outer ends of these myosin heads can move axially (total range about 200Å) and azimuthally (total range less than 60°) from their original lattice sites on the myosin filament surface to attach in defined target areas on the actin filaments. On this basis, each actin target area comprises about four actin monomers along one of the two long-pitched helical strands of the actin filament (about 200 Å) or an azimuthal range of actin binding sites of about 100° around the thin filament axis. If myosin heads simply label in a non-specific way the nearest actin monomers to them, as could occur with non-specific transient attachment in a ‘weak binding’ state, then the predicted X-ray diffraction pattern would comprise layer lines at the same axial spacings (orders of 429 Å) as those seen in patterns from resting muscle.It is shown that actin target areas in vertebrate skeletal muscles are probably arranged on an approximate 62 (right-handed) helix of pitch (P) of about 720 Å, subunit translation P/6 and near repeat P/2. Troponin position need not be considered in defining the labelling pattern of cross-bridges on this 62 helix of target areas; the target areas appear to be defined solely by the azimuthal position of the actin binding sites. The distribution of actin filament labelling patterns could be regular in fish muscle which has a ‘crystalline’ A-band, but will be irregular in higher vertebrate muscles such as frog sartorius muscle.

X-ray Diffraction Studies of Striated Muscles

In this short review a number of recent X-ray diffraction results on the highly ordered striated muscles in insects and in bony fish have been briefly described. What is clear is that this technique applied to muscles which are amenable to rigorous analysis, taken together with related data from other sources (e.g. protein crystallography, biochemistry, mechanics, computer modelling) can provide not only the best descriptions yet available on the myosin head organisations on different myosin filaments in the relaxed state, but can also show the sequence of molecular events that occurs in the contractile cycle, and may also help to explain such phenomena as stretch-activation. X-ray diffraction is clearly an enormously powerful tool in studies of muscle. It has already provided a wealth of detail on muscle ultrastructure; it is providing ever more fascinating insights into molecular events in the 50-year old sliding filament mechanism, and there remains a great deal more potential that is as yet untapped.

Myosin Filament Structure and Myosin Crossbridge Dynamics in Fish and Insect Muscles

The muscle crossbridge power stroke on actin appears to involve a change in angle between the actin-attached motor domain and the neck region of the myosin heads (the ‘tilting neck hypothesis’). However, this mechanism has not been proved beyond doubt and a reasonable question to ask is how actual proof might be achieved. This is essentially a structural question. The question can be put as ‘what are the molecular shapes that the myosin head adopts during the crossbridge cycle on actin?’. A further question that also needs answering is ‘how do the biochemical stages of the actin-myosin ATPase cycle map onto the structural changes that are seen?’. The purpose of the present paper is to address how the first question might be answered and to start to make suggestions about the second.

Muscle Crossbridge Positions from Equatorial Diffraction Data: An Approach Towards Solving the Phase Problem

Following a discussion of the problems involved in the analysis of X-ray diffraction data from muscle, a description is given of a possible procedure for solving the phase problem in the case of equatorial diffraction data. The approach involves the use of the Patterson Function which can be determined unambiguously from the observed diffracted intensities. The method is tested using five different muscle-like model density distributions for which the correct phases can be calculated directly. It is then applied to the equatorial X-ray diffraction data from relaxed frog sartorius muscle where it selects a phase set which is also the most likely to be correct on the basis of other available data on frog muscle. This phase set gives rise to a Fourier synthesis map in which the crossbridges form a uniform shelf of density around the myosin filament backbones. Possible lateral movements of the crossbridges from this relaxed configuration in active and rigor muscle are discussed. The approach to solving the phase problem is now being applied to data from fish muscle, insect flight muscle and crab muscle. It should also have its application to other fibrous materials apart from muscle.

Millisecond time-resolved low-angle x-ray fibre diffraction:a powerful, high-sensitivity technique for modelling real-time movements in biological macromolecular assemblies

The modern post-Genomic era heralds the elucidation of many thousands of protein and nucleic acid structures by X-ray crystallography and other techniques. But knowledge of individual macromolecular structures is often not enough. In some cases such macromolecules function as components of much larger molecular assemblies, some of which are filamentous in nature. Striated muscle is a particularly wellordered example of an organised macromolecular assembly and, in addition, it is dynamic; it functions as a mechanical motor using molecular movements which occur in a millisecond timescale. It, therefore, provides a good test case for the development of structural methods. Here we show that time-resolved, lowangle, X-ray fibre diffraction can be used to follow the molecular movements in contracting muscle in real time and with high spatial sensitivity. More generally, the techniques discussed here can be applied to any uniaxially ordered macromolecular assembly.

Millisecond molecular events in dynamic biological systems recorded using fast area detectors: current achievements and future needs

Abstract Excellent progress is being made in the study of rapid molecular movements in intact biological systems such as muscle using low-angle X-ray diffraction, synchrotron sources and very fast area detectors. However, there is still a need for faster detectors of higher spatial resolution.

Different Myosin Head Conformations in Bony Fish Muscles Put into Rigor at Different Sarcomere Lengths

At a resting sarcomere length of approximately 2.2 µm bony fish muscles put into rigor in the presence of BDM (2,3-butanedione monoxime) to reduce rigor tension generation show the normal arrangement of myosin head interactions with actin filaments as monitored by low-angle X-ray diffraction. However, if the muscles are put into rigor using the same protocol but stretched to 2.5 µm sarcomere length, a markedly different structure is observed. The X-ray diffraction pattern is not just a weaker version of the pattern at full overlap, as might be expected, but it is quite different. It is compatible with the actin-attached myosin heads being in a different conformation on actin, with the average centre of cross-bridge mass at a higher radius than in normal rigor and the myosin lever arms conforming less to the actin filament geometry, probably pointing back to their origins on their parent myosin filaments. The possible nature of this new rigor cross-bridge conformation is discussed in terms of other well-known states such as the weak binding state and the ‘roll and lock’ mechanism; we speculate that we may have trapped most myosin heads in an early attached strong actin-binding state in the cross-bridge cycle on actin.