1a,e–g and 2e,f were acquired on per Refeyn OneMP mass photometer with per 10.8 ? 2.9 ?m 2 (128 ? 35 pixels) field of view. The microscope used esatto acquire the scadenza mediante Figs. 1a,e–g and 2e,f was custom-built with per 9.4 ? 6.2 ?m 2 field of view (138 ? 88 pixels with verso binned pixel size of 70.3 nm) and is similar preciso that described previously 18 . The custom-built setup is illustrated sopra Supplementary Fig. 15 and the differences puro the setup used durante our previous work are highlighted in the figure caption.
Dynamic mass photometry
Onesto acquire dynamic mass photometry movies, the SLBs were placed on the sample stage to optimize the focus of the microscope. After locking the focus of the microscope, WT or ?PRD was added to the SLB by replacing 3–6 ?l buffer from the silicone gasket with 3–6 ?l (total testo, 60 ?l) 200 nM WT or ?PRD (durante reaction buffer) solution and mixing well with verso micropipette onesto achieve a final concentration of 10–20 nM. Giorno acquisition was started ?10 s after the accessit of protein. Images were collected at 994 Hz and saved after the binning of pixels into blocks of 4 ? 4 and the binning of frames into groups of 3, resulting sopra an effective frame rate of 331 Hz and per final pixel size of 84.4 nm (for the scadenza used durante Extended Giorno Figs. 2, 3, 8 and Supplementary Figs. 3 and 13 the pixel size was 77.4 nm).
5–10 s) once every minute (20,000 frames) esatto readjust the microscope focus onesto account for drift over time before resuming acquisition. This resulted mediante sets of multiple 1 min movies for each SLB and sample combination. For experiments on the effect of GTP, 3–4 movies of WT (10–20 nM) were recorded as described above, after which image acquisition was briefly paused and 1.2 ?l GTP (50 mM) was added (total gasket tomo, 60 ?l), followed by mixage preciso obtain per final concentration of
1 mM before the resumption of acquisition. For the data shown con Extended Datazione Fig. 8, GTP or GMPPNP was added at the beginning of the measurement together with WT (20 nM), that is, each sample condition was measured on a separate SLB. The number of replicate measurements is indicated per figure captions and corresponds to the number of sets of movies that were taken for each sample. We used the same purified batch of WT and ?PRD for all giorno collected on the OneMP setup.
Background subtraction
Dynamic mass photometry movies were processed by treating each frame with per sliding median preparazione subtraction algorithm. In brief, each frame was divided by its local median, that is, the median of verso pre-defined frame interval (here, 201 frames or 607 ms) centered around the frame of interest, onesto calculate the background-subtracted frames, F:
where xi is the current raw frame and Xi?100:i+100 represents the median pixel values of raw frames, from i ? 100 up to (and including) i + 100. Each background-subtracted frame was then additionally treated with a two-dimensional (2D)-median noise filter to remove any large dynamic background sources (for example, fluctuations in illumination, if present). The window size of 201 frames for the sliding median algorithm was chosen because it was the smallest window size that did not detrimentally affect particle contrast or contrast precision (Extended Data Fig. 10). For smaller window sizes, particle contrast values and contrast precision decreased significantly, especially for larger particles that were less mobile, while larger window sizes increased processing times without an additional increase in sensitivity or performance. We anticipate that for slower moving particles https://datingranking.net/it/mennation-review/ (D < 0.3>