W. Lehman, R. Craig, P. Vilbert Nature, 1994, 386: 65-67. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. W. Lehman, R. Craig, P. Vilbert Nature, 1994, 386: 65-67.

Outline Background and purpose of paper Thin filament background Experimental design Figures from paper Conclusion

Background / Purpose At this point in time, there was some x-ray data, but not clear enough to be used for models of the thin filament structure. Only “on” state had been resolved by 3D reconstruction. To show the “off” and “on” state of thin filaments using Limulus. To provide support for steric effects in muscle activation.

Muscle Thin Filament

Steric Model of Regulated Muscle Contraction

Experimental Design Used EGTA for “off” state, Ca2+ for “on” state. Negatively stained Limulus thin filaments. Constructed density maps of EGTA samples. Constructed density maps of Ca2+ samples.

Negatively stained thin filaments Limulus thin filaments (in paper) Arrows point to “bulges”  troponin (in paper) A – in EGTA B – in Ca2+ Note – this is from a more recent Lehman and Craig paper, chicken gizzard thin filaments

Density Maps A – in EGTA, B – in Ca2+, C – both samples superimposed Picture on Fig. RE2 shows surface views of reconstructed densities of thin filaments, left in the presence of EGTA (a strong Ca2+ complexing agent, thus in the presence of EGTA there is practically no free Ca2+), right in the presence of Ca2+. The helically wound strands on the surface of actin have a diameter of about 20 Å (that is the width of TM). The inner (Ai) and outer (Ao) domains of one actin monomer are marked. The actin monomer shapes from both the EGTA- and Ca2+-treated filaments are very similar, but the elongated strands of density originating from TM are located in different positions on the actin filaments. Fig. RE3 shows helical projections formed by projecting the map densities of RE2 onto a plane perpendicular to the helix axis, a) thin filaments in EGTA, b) thin filaments in Ca2+. The TM strand, which makes contact with actin monomers at A0 and Ai in a) and b), respectively, is indicated by arrows. The difference map in c) was calculated by subtracting densities in the Ca2+ map from those in the EGTA map. The regions of the maps that are significantly different are indicated by white (positive differences) and by black (negative differences). The pairs of positive and negative peaks are located at the respective strand positions and demonstrate strand movement. A – in EGTA, B – in Ca2+

Averaged Layer-Line Data Derived from Fourier transforms A – in EGTA B – in Ca2+ Note – from a more recent Lehman and Craig paper

Conclusion Both the “on” and “off” states of troponin in native filaments with regulation of troponin can be resolved. This is direct evidence to support the steric model of regulated muscle contraction.