Lacey carbon grid

Ultrathin carbon support film, 3 nm on lacey carbon grids (Agar Scientific), was plasma-cleaned in a hydrogen environment for 30 s. Four microliters of purified sample (0.05 mg/ml) was pipetted on the grid in a Vitrobot III [Thermo Fisher Scientific/Field Electron and Ion Company (FEI)] in a 100% humidity chamber at 8°C and frozen in liquid ethane. Images were recorded in a Titan Krios G3 microscope operated at 300 kV (Thermo Fisher Scientific/FEI) with electron-optical alignments adjusted with Sherpa (Thermo Fisher Scientific) on a Gatan K3 direct electron detector in electron counting mode at a nominal magnification of ×5000, corresponding to a calibrated pixel size of 0.425 Å in superresolution mode. A total of 11,490 dose-fractionated movies were recorded using EPU software (Thermo Fisher Scientific/FEI) with an electron flux of 1.42 e⁻ pixel⁻¹ s⁻¹ over 42 fractions corresponding to a total dose of ~60 e⁻/A² in a defocus range of −1.4 to −3 μm.

For all datasets, low dose movies were collected manually using SerialEM software (Mastronarde, 2005 (link)) on a FEI Tecnai G2 Polara operating at 300 kV with a direct electron detector. Data collection details for each dataset can be found in Table 1.Table 1

Dataset and data collection details. *K2 summit direct electron detector from Gatan Inc. CA, USA. ^†DE20 direct electron detector from Direct Electron, San Diego, CA. ^‡With quantum post-column energy-filter (Gatan Inc. CA, USA), operated in zero-loss imaging mode with a 20-eV energy-selecting slit. ^§C-Flat 2/2-4C from Protochips Inc. ^‖Lacey carbon grids from Agar Scientific.

Decorator	Tubulin Ligands	Grid type	EM and energy filtration	Detector and mode	Pixel size (Å/pixel)	Defocus range	Dose (e-/Å2, weighted)	Total exposure time and frame number	EMPIAR and EMDB accession codes
CKK domain of CAMSAP1	α-tubulin: GTPβ-tubulin: GDP, Taxol	2 μm Holey Carbon§	Polara (300 kV‡)	K2 summit* Counting at 5e-/pixel/sec	1.39	0.5–3.5 μm	42 dose weighted	16 secs 64 frames	EMPIAR-465EMD-4643
Motor domain of MKLP2	α-tubulin: GTPβ-tubulin: GDP, Taxol	2 μm Holey Carbon§	Polara (300 kV)	DE20† (Direct Electron)Linear	1.53	0.5–3.0 μm	50 dose weighted	1.5 secs22 frames	EMPIAR-467EMD-10131
N-DC domain of DCX	α-tubulin: GTPβ-tubulin: GDP	Lacey Carbon‖	Polara (300 kV‡)	K2 summit*Counting at 5e-/pixel/sec	1.39	0.5–2.5 μm	24 unweighted	9 secs36 frames	EMPIAR-10300EMD-10195

VLPs were mixed with 6 nm gold fiducials. The plunge freezing was done with a Leica GP2 by blotting from the non-carbon side. The sample was applied onto lacey carbon grids, 300 mesh Cu (Agar Scientific), blotted for 3 s and plunged into liquid ethane. Grids were screened on a Talos Arctica.

The hydrodynamic diameter of the nanoparticles was assessed using nanoparticle tracking analysis (NS500 Nanosight, Malvern-Panalytical, Malvern, UK) according to protocols published previously [18 (link),19 (link)], and now an established protocol for the EUNCL (EU Nanomedicine Characterisation Laboratory) [20 ]. These results were confirmed with dynamic light scattering (DLS) measurements (Malvern Nano- ZS, Malvern-Panalytical, Malvern, UK), following the EUNCL protocol for DLS size analysis [21 ]. Zeta potential data was provided by the nanoparticle supplier (at pH 7), Chemicell. For determining the dry diameter of the nanoparticles, transmission electron microscopy was used. Here, nanoparticles were diluted 1 in 1000 from the stock (100 mg/mL) in double distilled water (ddH₂0) and adhered to Lacey carbon grids (AGAR Scientific, Stansted, UK). Images were taken using the JOEL 2100 (JOEL, Tokyo, Japan) at an acceleration of 200 kV and a beam current of 100–110 µA. The longest diameter of 200 individual nanoparticles was measured using ImageJ software to generate a size distribution (for a description of hydrodynamic size, zeta potential and dry size analysis, see Figure S1).