1200 ex 2 microscope

Full-length myosin and myosin tail fragments were mixed in buffer A containing 0.5 M NaCl to give the final molar ratios indicated in the text. The ionic strength was lowered by dilution into 10 mM MOPS (pH 7.0), 0.1 mM EGTA, 2 mM MgCl₂ and the required concentration of NaCl such that the final NaCl concentration was 150 mM and the final myosin concentration (full-length plus tail fragment) was 100 nM. Samples were incubated for 30 min on ice prior to making EM grids. A 3 μl drop of sample was applied to UV-treated carbon-coated copper grids and stained with 1% Uranyl Acetate (45 min UV treatment using a type R51 UV lamp with 5 cm between the bulb and grid surface (UV Products, Pasadena, CA)). Micrographs were recorded on a JEOL 1200EX II microscope operating at room temperature. Data were recorded on an ATM XR-60 CCD camera.

0.4 mg/ml of RavA (in a 20 mM Tris-HCl,
500 mM NaCl, 10 mM MgCl₂, 1 mM
DTT, 5% glycerol, pH 6.8 buffer) was mixed with 0.3 mg/ml of either
LdcI, LdcC, LdcCI or LdcIC in the presence of 2 mM ADP and
10 mM MgCl₂ in a buffer containing
20 mM Hepes and 150 mM NaCl at pH 7.4. After 10 minutes
incubation at room temperature, 3 μl of mixture were
applied to the clear side of the carbon on a carbon-mica interface and
negatively stained with 2% uranyl acetate. Images were recorded with a JEOL 1200
EX II microscope at 100 kV at a nominal magnification of 15000 on a
CCD camera yielding a pixel size of 4.667 Å. No
complexes between RavA and LdcC or LdcIC could be observed, whereas the
LdcCI-RavA preparation manifested cage-like particles similar to the previously
published LdcI-RavA19 (link), but also unbound RavA and LdcCI, which
implies that the affinity of RavA to the LdcCI chimera is lower than its
affinity to the native LdcI. 1260 particles of
96 × 96 pixels were extracted interactively
from several micrographs. 2D centering, multivariate statistical analysis and
classification were performed using IMAGIC36 (link). Class-averages
similar to the cage-like LdcI-RavA complex were used as references for
multi-reference alignment followed by multivariate statistical analysis and
classification.

Immunoprecipitation eluents of YRS-HA-FLAG, from 17 500 cm² flasks (Nunc TripleFlasks, Thermo Fisher Scientific) using 0.4 µl anti-FLAG resin, were concentrated to 100 µl (from an original volume of 1500 µl) by ultrafiltration with a 10 kDa MWCO centrifugal unit (as described above). Samples were then diluted by 1:5 in 25 mM Tris-HCl pH 7.5, 150 mM NaCl before application of 3.5 µl onto copper electron microscope grids coated with a plasma-treated thin carbon support films (Electron Microscopy Sciences). Staining was accomplished with 2% uranyl acetate solution using the droplet method and electron micrographs were recorded by a Gatan ORIUS 2.7 k×2.7 k CCD camera at a nominal magnification of 25 000 times using a JEOL 1200 EX II microscope operating at 100 kV. Particle images (1030) were manually selected and windowed (128 pixel) from 50 micrographs (sampling of 2.7 Å/pixel) using SIGNATURE [32] (link). Rotational averages were calculated in EMAN1 [33] (link) following pre-centring and the radial profile was plotted from the resulting total average in ImageJ [34] . Iterative reference-free averaging in EMAN was used to generate the representative average of a manually selected sub-set of images (137 out of 1030) and iterative reference-free classification produced the class averages for heterogeneity assessment.