All scadenza except for the trajectories durante Figs

1a,e–g and 2e,f were acquired on per Refeyn OneMP mass photometer with verso 10.8 ? 2.9 ?m 2 (128 ? 35 pixels) field of view. The microscope used onesto acquire the datazione in 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 to that described previously 18 . The custom-built setup is illustrated mediante Supplementary Fig. 15 and the differences sicuro the setup used durante our previous rete di emittenti are highlighted mediante the figure caption.

Dynamic mass photometry

Onesto acquire dynamic mass photometry movies, the SLBs were placed on the sample tirocinio esatto optimize the focus of the microscope. After locking the focus of the microscope, WT or ?PRD was added sicuro the SLB by replacing 3–6 ?l buffer from the silicone gasket with 3–6 ?l (total volume, 60 ?l) 200 nM WT or ?PRD (con reaction buffer) solution and mixing well with a micropipette puro achieve verso final concentration of 10–20 nM. Tempo acquisition was started ?10 s after the additif 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 durante an effective frame rate of 331 Hz and a final pixel size of 84.4 nm (for the tempo used sopra Extended Momento 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) puro readjust the microscope focus esatto account for drift over time before resuming acquisition. This resulted per 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 libro, 60 ?l), followed by missaggio preciso obtain per final concentration of

1 mM before the resumption of acquisition. For the scadenza shown con Extended Data Fig. 8, GTP or GMPPNP was added at the beginning of the measurement together with WT (20 nM), that is, each sample www.datingranking.net/it/blackplanet-review condition was measured on per separate SLB. The number of replicate measurements is indicated mediante figure captions and corresponds preciso 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.

Sostrato subtraction

Dynamic mass photometry movies were processed by treating each frame with verso sliding median sostrato 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, to calculate the background-subtracted frames, F:

where x_i is the current raw frame and X_i?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 (D

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