Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.

Characterisation of the new microscopy hardware

The energy spread of the field emission gun was measured using the new energy filter. Post-slit multi-poles of the filter were set to spectroscopy mode in order to form an enlarged image of the spectrum on the camera. The intensity of the incoming beam was carefully reduced in order not to saturate or damage the camera. The energy dispersion at the camera was 8 meV/pixel. The exposure time was 31 ms. The energy spread as full width at half the maximum value (FWHM) of Δ E =0.71 for XFEG, and Δ E =0.28 for CFEG ( Figure 1b ) were used to calculate CTF envelope function, Δf , using the following equation 31 : Δ f = C c ( Δ E / e V ) 2 + ( Δ V / V ) 2 + ( 2 Δ I / I ) 2 , using C c =2.7mm; Δ V/V =0.02ppm and Δ I/I =0.1ppm. Note that the latter two contributions account for less than 1% drop in the CTF envelope. The stability of the energy filter was assessed by repeatedly measuring the position of the zero-loss peak with respect to the energy-selecting slit. To this end, we mechanically moved one slit edge to the optical axis and fine-tuned an offset on the high tension supply such that the slit edge blocked half of the beam intensity. The resulting offset of the high tension was read out as the shift in the zero loss position. Measurements, each taking approximately 40 seconds, were repeated continuously for 14 days. Dose response curves for the Falcon-3 and Falcon-4 cameras were measured using the flu-screen and fixed magnification steps. Before each experiment, the flu-screen was carefully calibrated with a Faraday cup in its linear range (0.4-5.0 nA at 300 kV). The read-out from the flu-screen was interpreted as the expected electron dose and plotted against the counted number of electrons on the camera. DQE measurements were performed as described 35 at 300 keV on a Titan Krios G2 microscope at the LMB for the Falcon-3 and on a Titan Krios G3i microscope at the Materials Science Department of the University of Cambridge for the Falcon-4. For calibration of the flu-screen on the microscope with the Falcon-4, a Faraday cage and a Keithley 6485 picoammeter (kindly provided by Gerard van Hoften, Thermo Fisher Scientific) were used. For the microscope with the Falcon-3, the drift tube of a Gatan energy filter (GIF; BioQuantum 686) was used as a Faraday cup and the current was measured using a Deben 087-001 SEM Probe current meter. Because the beam currents used in counting mode imaging are too low to be accurately measured, a defined beam with a measurable current was set at a low magnification and the desired electron flux on the camera was obtained by increasing the magnification. The relative magnification change was measured by using a higher current to fully illuminate the flu-screen at the low magnification and noting the change in current upon going to the higher magnification. To account for the non-linearity that is introduced by coincidence loss, DQEs were first measured at a low dose rate (0.19 e - /pixel/s for the Falcon-3; 0.32 e - /pixel/s for the Falcon-4) and then extrapolated to a typical dose rate (0.5 e - /pixel/s for the Falcon-3; 3.6 e - /pixel/s for the Falcon-4) using the measured response versus dose-rate curves ( Figure 1e ). Modulation transfer functions (MTFs) were measured using the knife-edge method from a straight part of a shadow of the pointer on the camera. The actual area of the images used was chosen carefully by looking at the difference between the observed image and that predicted from the inferred MTF, to ensure that the edge in the selected area was straight and free of defects. In the case of the Falcon-4, the only part of the pointer edge that was suitable was at a 15 degrees angle with the pixel array of the camera, the effect of which was accounted for when calculating the MTF. Even at the lowest dose rates used, there was still a small decrease in the power spectra at low-spatial frequency due to coincidence loss. To avoid overestimating the resulting DQE at low spatial frequency, the noise power spectra were extrapolated from a Gaussian fit of the power between 0.2 and 0.8 of Nyquist. The amplitudes and widths of fitted Gaussians to the noise power spectra were observed to be in excellent agreement with those expected from the number of counted electrons per frame and estimates from the point spread function of images of individual incident electrons. GABA A receptor production, purification and nanodisc reconstitution Human GABA A receptor β 3 -subunit with a truncated M3-M4 loop was transiently expressed in HEK293S GnTI - cells as previously described 3 . The sample used for XFEG datasets collected on Falcon4 contained a point mutation, K279T, thought to stabilise the receptor and a C-terminal 1D4 tag for purification. The sample used for the CFEG dataset, as well as the Falcon3 and K3 datasets, contained the wild-type sequence and an N-terminal streptavidin-binding peptide (SBP) tag for purification. Subsequent comparison of the resulting cryo-EM maps revealed that the K279T mutation had no structural impact, hence the Falcon4 datasets could be merged during data processing. Frozen cell pellets were resuspended in 50 mM HEPES pH 7.6, 300 mM NaCl, 1 mM histamine supplemented with 1% (v/v) mammalian protease inhibitor cocktail (Sigma-Aldrich) and solubilised with 1% (w/v) lauryl maltose neopentyl glycol (LMNG) and 0.1% (w/v) cholesteryl hemisuccinate Tris salt (CHS). No CHS was added to the sample that was used for the CFEG dataset. Receptors were purified by 1D4 and SBP affinity chromatography, respectively. Nanodisc reconstitution was performed as previously described 23 . Briefly, after the binding step, while the receptor was still bound to the affinity beads, an excess of bovine brain extract (BBE; type I, Folch fraction I, Sigma-Aldrich, 20 mg/mL stock) and phosphatidylcholine (POPC, Avanti, 10 mg/mL) mixture (15:85, v/v) was added to the samples and incubated for 30 min. Receptors were then reconstituted into membrane scaffold protein 2N2 (MSP2N2) lipid nanodiscs by incubating with an excess of MSP2N2 (0.6 mg/mL final concentration) and bio-beads for 2 h. The resin was then washed extensively and the GABA A R samples were eluted with a buffer containing 12.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.6, 75 mM NaCl, 1 mM histamine and 2mM 1D4 peptide or 2.5 mM biotin, respectively. Cryo-EM grid preparation Purified GABA A R was incubated with ~1 μM Megabody-25 21 and 3.5 μl sample was applied to glow discharged 300 mesh 1.2/1.3 and 2/2 UltraAuFoil gold grids (Quantifoil) for 30 s then blotted for 5.5 s prior to plunge-freezing with liquid ethane cooled by liquid nitrogen. Grid vitrification was performed using a Leica plunger (Leica Microsystems; XFEG datasets) or Vitrobot Mark IV (Thermo Fisher Scientific; CFEG datasets) at 100% humidity and 14 °C. A frozen aliquot of 7 mg/ml mouse apoferritin in 20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM dithiothreitol (DTT) and 5% trehalose, which we received from the Kikkawa Lab at Tokyo University, was thawed at room temperature and cleared by centrifugation at 10,000g for 10 min. The supernatant was diluted to 5 mg/ml with 20 mM HEPES pH 7.5 150 mM NaCl, and 3 μl of the diluted sample was applied onto glow-discharged R1.2/1.3 300 mesh UltrAuFoil gold grids (Quantifoil) for 30 s and then blotted for 5 s before plunge-freezing the grids into liquid ethane cooled by liquid nitrogen. Plunge-freezing was performed using a Vitrobot Mark IV (Thermo Fisher Scientific) at 100% humidity and 4 °C.

Cryo-EM data acquisition

All cryo-EM data were collected on Falcon or K3 cameras in electron counting mode using Titan Krios microscopes (Thermo Fisher Scientific) operating at 300 kV. Before data acquisition, two-fold astigmatism was corrected and beam tilt was adjusted to the coma-free axis using the autoCTF program (Thermo Fisher Scientific). All datasets were acquired automatically using EPU software (Thermo Fisher Scientific). Detailed data acquisition parameters for all data sets are given in Extended Data Table 1 . For GABA A R, two data sets were acquired on a Titan Krios at the Department of Biochemistry, University of Cambridge, UK. This microscope is equipped with an XFEG and a bottom-mounted (BM) Falcon-3 camera and a K3 camera behind the Gatan energy filter. All other data sets were acquired at the Thermo Fisher Scientific RnD division in Eindhoven, The Netherlands. Four GABA A R data sets were collected on a Titan Krios equipped with an XFEG and a prototype Falcon-4 camera that was mounted behind the new energy filter. These datasets were collected consecutively over a period of 5 days from the same grid to study the effect of varying energy slit widths. To keep the ice thickness similar between these experiments, all the exposure areas were selected at the start using a constant ice filter within EPU software. One GABA A R data set was acquired on a microscope with a CFEG and a prototype Falcon-4 camera that was mounted behind the new energy filter. The slit width of the energy filter was set to 5 eV. For this data set, 5573 movies were acquired using a 100 μm objective aperture and 3160 movies were collected without an objective aperture. The CFEG was automatically flashed every 5 hours using the EPU software. For the apoferritin data set, we used the same microscope with the CFEG, the new filter and the prototype Falcon-4 camera. No objective aperture was used and the slit width of the energy filter was set to 10 eV. Flashing of the CFEG had no noticeable effect on data quality ( Extended Data Figure 4f ). Data acquisition was not optimised in terms of speed and the prototype Falcon-4 version did not write movies at the highest possible rate. Typically, two movies were collected per hole, with physical stage movements in between each hole. The resulting data acquisition rate of ~95 movies per hour (including Dewar filling times and CFEG flashing). The 3,370 apoferritin movies were collected within 36 hours; GABA A R datasets allowing ~2 Å reconstructions in ~9 hours.

Fringe free imaging

(FFI) and aberration-free image shift (AFIS), which allow acquisition of multiple images per hole and multiple holes per stage position, would allow considerably higher throughputs. The EER movie format Electron event representation (EER) is a movie format that is introduced with the Falcon-4 camera 18 . Whereas in the existing MRC image format groups of subsequent camera frames, or dose fractions, are summed and represented as images, EER stores the location and time of recording of each detected electron event. The EER format records the event coordinates in a four times oversampled, that is a 16k×16k, grid. The time resolution is the original frame rate of the camera, so that dose fractionation during data acquisition is no longer needed. Instead, beam-induced sample motions or stage drift can be corrected without a loss in temporal resolution. The electron event stream is stored on disk after compression with a run-length encoding algorithm. For GABA A R, data sets were acquired at a dose rate of 4.7 electrons per pixel per second, at a pixel size of 0.727 Å and a total dose of 18.1 electrons per pixel. These settings yielded EER movies consisting of 1113 frames and with an average size of 485 MB. For apoferritin, data were acquired at a dose rate of 4.5 electrons per pixel per second, at a pixel size of 0.457 Å and a total dose of 40 electrons per Å 2 , resulting in movies of 434 frames occupying on average 160 MB. To read EER movies, we modified the publicly available RELION-3.1-beta. Inside RELION, the events from the 16k×16k grid are positioned as binary pixels in a 8k×8k grid, which is then Fourier cropped into a 4k×4k grid. Using the modified version, EER movies can be used for frame alignment using RELION’s implementation of the MotionCor2 algorithm 36 and for per-particle beam-induced motion correction using its Bayesian polishing program 20 . For both programs, the user needs to specify a dose fractionation rate.

Ice-thickness measurement

The ice thickness was measured on a GABA A R grid using tomography. Tilt series were collected on three different grid-squares with a 10eV slit on a Falcon4 camera. Data was acquired covering an angular range from -45° to +45° with 3° angular increments recorded automatically using the dose-symmetric tilting scheme under low-dose conditions using TEM Tomography software (TFS). Each tilt series was collected with a nominal defocus value of 5 μm in electron counting mode using a dose of 4 e-/Å 2 per tilt and a calibrated pixel size of 1.898 Å. Tomograms were reconstructed with patch tracking and ice thickness measured with IMOD software 37 .

Apoferritin image processing

A total of 3370 movies in EER format were motion corrected with RELION’s implementation of the MotionCor2 algorithm 36 . For this purpose, original hardware movie frames were dose fractionated into groups of 14 frames, corresponding to an accumulated dose of 1.3 e - /Å 2 per fraction. CTF estimation was performed with CTFFIND-4.1.13 38 using the sums of power spectra from combined fractions corresponding to an accumulated dose of 4 e - /Å 2 . Micrographs whose estimated resolution from CTFFIND was worse than 5 Å were removed, leaving 3080 micrographs for further processing. Using RELION’s template-based algorithm, 428,590 particles were picked and subjected to 2D classification. After selection of good classes, 380,236 particles were subjected to standard 3D auto-refinement to give an initial reconstruction with an estimated resolution of 2.3 Å. Subsequently, three runs of CTF refinement 26 were performed: first refining magnification anisotropy; then refining optical aberrations (up to the 4th order); and finally refining per-particle defocus and per-micrograph astigmatism. A 3D auto-refinement with the refined values yielded an estimated resolution of 1.7 Å. Bayesian polishing to optimise per-particle beam-induced motion tracks, followed by another round of auto-refinement resulted in a 1.43 Å map. CTF refinement was then repeated for optical aberration correction, magnification anisotropy, per-particle defocus and per-micrograph astigmatism. To compensate for changes in the tilt of the electron beam during the data acquisition session, separate optics groups were created for particles belonging to consecutive groups of 125 micrographs, or whenever the CFEG was flashed. At this point, 201 micrographs with refined scale factors ( rlnGroupScaleCorrection ) with values below 0.5 were also removed, resulting in 363,126 particles in the final set ( Extended Data Figure 4f ). Following a last round of auto-refinement, these particles were subjected to a second round of Bayesian polishing with a large particle extraction box (spanning 320 Å), such that high-resolution signals that are delocalised by the CTF were still contained in the images. At this point, seven hardware frames were grouped into each dose fraction to be able to capture more rapid motions. Since Ewald sphere curvature was predicted to limit the resolution of a particle with a diameter of 120 Å to approximately 1.3 Å 39 , the particles were then used for reconstruction with Ewald sphere correction 27 . The estimated resolution of the final map, following standard post-processing procedures in RELION, was 1.22 Å. GABA A receptor image processing GABA A R data sets were processed following the same strategy as for apoferritin, with the following exceptions. All movies, except for the ones acquired on the microscope with a CFEG, were recorded in MRC format. For the CFEG data, hardware movie frames were initially dose fractionated into groups of 24 frames, corresponding to an accumulated dose of 0.86 e - /Å 2 per fraction. For the second round of Bayesian polishing, a dose fractionation of 12 hardware frames was used. Particle picking was performed using an in-house generated Linux port of the of BoxNet2D neural network in Warp 40 . Prior to the last auto-refinement, 3D classification into three classes without alignment was performed to select the particles contributing to the best reconstruction. The first part of the CFEG data was acquired with a 100 μm objective aperture. Charging of the aperture probably led to third and fourth order optical aberrations, which were estimated during CTF refinement in optics groups of 160 micrographs, or whenever the CFEG was flashed or liquid nitrogen was refilled. The second part of the CFEG data was acquired without an objective aperture. For these images, no significant higher-order optical aberrations were observed and creating multiple optics groups was deemed unnecessary. When merging the energy-filtered CFEG and XFEG data sets, refinement of per-micrograph scale factors and per-particle normalisation corrections were unstable, and these were switched off using the --no_norm and --no_scale arguments. The mask used for resolution estimation during post-processing only contained density for GABA A R; all density corresponding to the megabodies was removed. B -factor estimation For each GABA A R dataset, a random subset of 32,000 particles was selected from the final set of particles after the second round of Bayesian polishing. This subset was subjected to 3D auto-refinement and the resulting orientations were used to calculate reconstructions for each of the two random halves used in the auto-refinement. Similar half-set reconstructions were also calculated for random subsets of 16,000, 8,000, 4,000, 2,000, 1,000 and 500 particles. This procedure was repeated seven times. For each subset and repeat, resolution estimation was again performed using standard post-processing functionality in RELION. The inverse square of the resulting estimated resolutions were then plotted against the natural logarithm of the number of particles in the subset, and B -factors were calculated from the slope of a straight line fitted through all points in the plot. For apoferritin, the same procedure was used, starting from a subset of 180,000 particles and halving the subset size eight times.

Atomic modelling

The atomic models for GABA A R and apoferritin were derived from PDB-4cof and PDB-6s61, respectively. Both models were manually adjusted in COOT 41 . Difference maps were used to rebuild the model, add alternative conformations and waters. Real-space refinement was performed using PHENIX, version 1.16.3549 42 for GABA A R. Both models were refined with anisotropic atomic B-factors in reciprocal space using REFMAC, version 5.8.0258 25 . For cross-validation, the atomic models refined against the full map was perturbed by random shifts of the atoms of up to 0.3 Å and the perturbed model was refined in REFMAC against one of the two half-maps. FSC curves for the resulting model against that half-map (FSC work ) and against the other half-map (FSC free ) are shown with blue and dashed orange lines, respectively, in Extended Data Figures 2 and 4 . Molprobity score, clash score, rotamer and Ramachandran analyses were performed using MOLPROBITY 43 . For refinement and difference map calculation, maps were first scaled globally using Gaussian scaling: i.e. min k , B ∑ | | F o | − k e − B s 2 / 4 | F c | | 2 , where F o is the Fourier transform of the post-processed map and F c is the Fourier transform of a map calculated from a model without hydrogens. Next, each resolution shell was scaled by finding a scale factor D and sigma per shell with a maximum likelihood method: min D , σ ∑ ( | F o − D F c | 2 / σ ) + ∑ log ( σ ) . Finally, difference maps were calculated as F o - DF c . Since F o is FSC weighted, the resulting map will be also FSC weighted. FSC is calculated from half maps. Structural figures were prepared using PyMOL (Schrödinger) and UCSF ChimeraX 44 . Hydrogen position calculations X-ray scattering and electron scattering factors are related through the Mott-Bethe formula, which is the solution of Poisson equations in Fourier space under certain conditions: f e ( s ) = c Z − f X ( s ) s 2 Where C = 2 π m e 2 h 2 ε 0 ; m is the mass of an electron; e is the charge of an electron, h is the Planck constant; and ϵ 0 is the dielectric constant of the vacuum; Z is the nuclear charge; f X (s) is the X-ray scattering factor; f e (s) is the electron scattering factor; and s=1/d is the resolution. Adding the position-independent, isotropic atomic B -factor gives: f e , B ( s ) = c Z − f X ( s ) s 2 e − B s 2 4 This formula is valid if the centre of electron density coincides with the position of the nucleus. If this is not the case, the formula should be modified according to the vector Δ x between the centre of electron density and the nuclear position (assuming that the centre of electron density is at the origin): f e , B ( s ) = c Z e − 2 π ι s Δ x − f X ( s ) s 2 e − B s 2 4 Note that, in general, the form factor for electron scattering becomes a complex quantity. For all non-hydrogen atoms, it is safe to assume that the centre of electron density and the nuclear position coincide. For hydrogen atoms, the centre of electron density and the nuclear position do not coincide and we are interested in the peak height for the density of electrostatic potential along the bond between a hydrogen and its parent atom. The below ignores the fact that electron densities will be non-spherical, as they are elongated along the bond, thus adding additional complexity to Mott-Bethe formula. The densities for the proton and the electron of a hydrogen atom at a resolution s max and with a given B value is given by: ψ p ( x ) = c ∫ | s | < s max ⁡ Z s 2 e − 2 π l s x d s = 4 π Z c ∫ 0 s max ⁡ e − B s 2 4 sin ⁡ ( 2 π | s | | x | ) 2 π | s | | x | d | s | , , ψ e ( x ) = 4 π c ∫ ​ 0 s max f X ( s ) e − B s 2 4 sin ( 2 π | s | | x | ) 2 π | s | | x | d | s | . To calculate the density at a position x we need to calculate: ψ p + e ( x ) = ψ p ( x − Δ x ) − ψ e ( x ) Even if both densities corresponding to the proton and the electron are spherically symmetric, the combined density will not be spherical. Extended Data Figure 5 shows density profiles and their maxima for a hydrogen bonded to a carbon atom for different resolutions and B values. These profiles were calculated with the assumption that the electron-carbon distance is 0.98 Å and the proton-carbon distance is 1.09 Å.

Supplementary Material Ext data movie 1 Ext data movie 2 Ext data movie 3 Ext data movie 4