Cm200 microscope

Images of negative stain hGINS were taken on a Philips CM200 microscope at a nominal defocus range of 3 to 1 μm and at a magnification of 50,000×, resulting in a pixel size of 2 Å/pixel. Frames were CTF corrected, binned by a factor of 2 and band-pass filtered. After normalisation, ~20,000 particles were automatically picked, centered and classified based on multivariate statistical analysis (MSA). Good classes were used as references for further cycles of alignment and classification. Various reference-free models were obtained by angular reconstitution and among those in good agreement with each other the one with the lowest Euler error was used for refinement. As eigen-image analysis indicate the presence of two-fold symmetry, the asymmetric map was refined further by applying C2 symmetry. Details are presented in the Supplementary Information.

TEM measurements were performed in a 200 kV Philips CM-200 microscope (Philips, Amsterdam, Netherlands) with a supertwin objective lens, a LaB6 filament and side-entry specimen holder (point resolution 0.24 nm). To prepare the TEM samples, ~5 µL of solution containing the sample were removed from the bottom of the vial, followed by the removal of ~5 µL of the supernatant into the same pipette tip. Two drops of this suspension were deposited on a carbon-coated copper TEM grid (Ted Pella, Redding, CA, USA). The instrument is equipped with an energy-dispersive X-ray spectroscopy (EDX) detector (EDAX Inc., Tilburg, Netherlands). Signals from the elements Si, Cu and C are originated from carbon-coated copper TEM grid. Taking into consideration that EDX is based on the principle that each element has a unique atomic structure (with a unique set of peaks on its electromagnetic emission spectrum), we can discriminate between silver NPs and the TEM grid. The size histogram was prepared using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA), based on the measurements obtained from more the 150 nanoparticles.

Full-length CthACC was diluted to 0.01 mg ml⁻¹ in 30 mM MOPS pH 7.0, 200 mM ammonium sulfate, 5% glycerol and 10 mM dithiothreitol. Protein sample was adsorbed to a 200-μm copper grid and stained with 2% uranyl acetate. Grids of CthACC were imaged on a CM-200 microscope (Philips) equipped with a TVIPS F416 4k CMOS camera (Tietz Video and Image Processing Systems). The voltage used was 200 kV, and a magnification of × 50,000 results in a pixel size of 2.14 Å. Initial image processing and particle picking was carried out using Xmipp69 (link)70 (link). Overall, 22,309 particles were picked semi-automatically from 236 micrographs with a box size of 300 × 300 pixels. After extraction, particles with a z-score of more than three were discarded and 22,257 particles were aligned and classified into 48 2D class averages using the maximum-likelihood target function in Fourier space (MLF2D). After 72 iterations, 4,226 additional particles were discarded and the remaining 18,031 particles were re-aligned and classified into 36 classes using MLF2D with a high-resolution cutoff of 30 Å. After 44 iterations the alignment converged and class averages were extracted.

The crystal nanostructures of fabricated materials were achieved with recording X-ray diffraction (XRD) scans by filtered Co Kα radiation on a Phillips X'Pert MPD diffractometer. The Fourier transform infrared (FTIR) measurements were carried out on a Nicolet AVATAR 370 spectrometer within the spectral range of 600 and 4000 cm⁻¹. The Raman spectra were achieved using an Almega Thermo Nicolet dispersive Raman spectrometer with a 532 nm laser excitation source. The inductively coupled plasma optical emission spectroscopy (ICP-OES) was performed on a PerkinElmer Optima 8000 instrument. The field emission scanning electron microscopy (FESEM) and energy-dispersive X-ray spectroscopy (EDX) together with the corresponding elemental mapping were conducted on a TESCAN Mira 3-XMU. The transmission electron microscopy (TEM) images were collected on a Phillips model CM200 microscope.