Single-cell structural biology with intracellular electron crystallography

Domain: chemistry
Published: 2026-02-27
Editorial outcome: major_revision

Abstract
Intracellular crystallization is an emerging approach in structural biology that bypasses the need for protein purification. In 2024, the InCellCryst pipeline was introduced for structural studies of intracellular crystals by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of cells containing intracellular crystals, precluding high-resolution studies on proteins that crystallize only in a few cells. Here we introduce IncelluloED, a method that combines intracellular crystallization with in situ 3D electron diffraction in cells and achieves high-resolution structures from just one crystal inside one cell. Experiments on a microcrystal of the HEX-1 protein from Magnaporthe protein from Magnaporthe grisea, grown inside an insect cell, give a structure at 1.9 Å resolution from a volume of ~1.6 µm3 as compared to 1.8 Å resolution achieved by serial X-ray crystallography from a combined volume exceeding eleven million µm3. IncelluloED uses widely available cryo-EM tools and brings high-resolution structural biology into home laboratories while also advancing a vision for a “single-cell structural laboratory”.

Introduction
Protein crystallization inside living cells occurs naturally and serves roles such as storage, protection, solid-state catalysis, and, in some cases, arises from progression of disease or infection1. Recombinant gene expression can also induce intracellular crystallization, and targeted in cellulo crystallization of proteins1,2 is an emerging approach in structural biology. In certain cases, the quasi-native cellular environment enables binding and identification of physiologically relevant ligands and cofactors2,3, which AI-based structure prediction cannot yet replicate. Moreover, fully glycosylated proteins, such as T. brucei cathepsin B, have been crystallized in living insect cells4,5 while conventional crystallization trials were not successful. Since the first structure determination of a recombinantly produced and intracellularly crystallized protein in 20076, structure elucidation of in cellulo protein crystals has evolved significantly. The development of micro- and nano-focus beamlines at advanced synchrotrons and X-ray free-electron lasers (XFELs), coupled with serial data collection strategies, made atomic-resolution7 studies of tiny intracellular crystals possible. As a result, over 30 in cellulo structures are now deposited in the Protein Data Bank (PDB)1,3,5,8,9.

Systematic strategies for testing crystallization of recombinant proteins in living insect cells have been developed10,11, including the InCellCryst pipeline2 where a recombinant baculovirus (rBV) is employed to drive target gene expression to increase local protein concentrations, triggering crystal nucleation and efficient crystal growth2,4,5,12,13. Structures are then determined via serial X-ray crystallography directly from the cells2, avoiding purification and preserving quasi-native states. The efficiency of intracellular crystallization can be very low, and frequently less than 0.1% of the cultured cells may contain a protein crystal. This is a serious problem, because serial crystallography with small intracellular crystals requires the exposure of tens of thousands of individual crystals for a full dataset, and the low number of cells containing a crystal may preclude structure elucidation even at advanced synchrotron and XFEL facilities. This constitutes a major bottleneck in the InCellCryst pipeline.

Exploiting the strong interactions of electrons with matter14, three-dimensional electron diffraction (3D ED or MicroED)15,16 allows structure determination from sub-micrometer-sized protein crystals, like a 2.1 Å lysozyme structure from a 0.14 µm3 crystal17. Developments with 3D ED have produced a number of protein and peptide structures (e.g., see refs. 15, 18–22), offering the ability to record atomic resolution diffraction data (≤1.2 Å resolution7) from protein crystals of sub-micrometer dimensions23,24. The small crystal volume and the low number of crystals required makes 3D ED a promising technique for in cellulo structure elucidation of proteins crystals.

The possibility of collecting ED datasets from multiple intracellular crystals in frozen cells was recently demonstrated for the major basic protein-1 (MBP-1)25, which naturally forms numerous nanocrystals in the cytoplasmic secretory granules of eosinophils. The study also showed that combining cryo-electron tomography26 with 3D ED enables analysis of physiologically relevant structural changes associated with the release of MBP-1 in situ and on multiple length scales, from molecular structure to nanometer-scale changes in the crystal packing during MBP-1 release.

In a typical 3D ED experiment15,27, a protein crystal is deposited on a transmission electron microscopy (TEM) grid, vitrified, and an ED dataset is recorded under cryogenic conditions as the crystal is continuously rotated under parallel illumination in a high-energy electron beam (typically 200–300 kV). Optimal sample thickness is 200–300 nm28,29. Thicker samples, like a cell containing a crystal, require thinning to reduce multiple scattering and absorption15. Thinning a crystal to a lamella of desired thickness can be performed by focused ion-beam milling under cryogenic conditions (cryo-FIB milling), using a dual-beam focused ion-beam scanning electron microscope (FIB/SEM)26,30–33. Protein crystals have been shown to retain crystalline order after thinning23,31,32, making cryo-FIB a standard and reliable method for 3D ED sample preparation. However, cryo-FIB/SEM provides only topographical information and cannot localize deeply embedded crystals. Cryo-light microscopy (cryo-LM) and correlative light-electron microscopy (CLEM), established for cryo-electron tomography26,34, offer a solution.

Here we present the IncelluloED pipeline, which combines the InCellCryst intracellular-crystallization pipeline with in situ 3D ED, enabling high-resolution structure determination from a single crystal within a single cell. A schematic overview of the IncelluloED workflow is given in Fig. 1. Using microcrystals of the HEX-1 protein from the filamentous fungus Magnaporthe grisea (MgHEX-1) grown inside High Five insect cells, we demonstrate that our approach delivers lamellae from appropriate cellular sections containing the target crystals. The lamellae are suitable for high-resolution structure determination by 3D ED and yield the previously unknown MgHEX-1 structure at 1.9 Å and 2.2 Å resolution from volumes of ~1.6 µm3 and ~0.8 µm3, respectively. For comparison, we also determine the structure of MgHEX-1 by serial synchrotron X-ray crystallography (SSX), using 62,496 crystals, and obtain a structure at 1.8 Å resolution from a total volume of about 11 million µm3. IncelluloED achieves similar structural details from a single crystal, overcoming the key limitation of InCellCryst, and opening possibilities for cellular structural sciences.

Results
Intracellular crystallization of MgHEX-1
To establish the IncelluloED approach, MgHEX-1, a structurally uncharacterized HEX-1 protein derived from the filamentous fungus M. grisea, has been selected. HEX-1 proteins are highly conserved among ascomycetes and self-assemble into a hexagonal crystalline core within peroxisome-derived organelles (Woronin bodies) and help to seal septal pores during cellular stress35–37. A Neurospora crassa HEX-1 (NcHEX-1) was found to produce regular, hexagonal crystals with average dimensions of 9.1 ± 3.2 µm (± standard deviation) in length and 3.5 ± 0.7 µm in width when overexpressed in insect cells using the rBV system8,38. This protein was quickly established as a test protein for intracellular crystal detection by various techniques (e.g., small angle X-ray scattering-X-ray powder diffraction, SAXS-XRPD38, fixed-target serial diffraction data collection at XFELs8 and synchrotron sources39) and led to the development of the InCellCryst pipeline2. The crystallization efficiency and low-micrometre size range position HEX-1 crystals as ideal candidates for IncelluloED method development. Moreover, the elucidation of a second HEX-1 structure can provide insights into biological self-assembly processes and into the structural conservation within this protein family. Thus, we selected MgHEX-1 as a pilot system in this study that shares 74% sequence identity with NcHEX-1.

Applying the InCellCryst approach, up to three hexagonal-shaped structures per cell were detected by light microscopy in approximately 40% of the rBV-infected High Five insect cells at day four post infection, exhibiting a hexagonal morphology similar to that of previously reported NcHEX-1 crystals2,37 (Supplementary Fig. 1). The mean size of MgHEX-1 crystals, which ranged from 4 to 15 µm in the longest dimension, was estimated at 8.6 ± 2.5 µm (± standard deviation) (Fig. 2a, b). Infected High Five cells also produce enhanced yellow fluorescent protein (EYFP) as a fluorescence marker encoded on the bacmid from the DH10EmBacY cells40, which enables 3D visualization of intracellular MgHEX-1 crystals in a negative imaging manner using fluorescence (FL) microscopy (Fig. 2c–e). This was used for 3D targeting of crystals in subsequent cryo-FIB experiments. The High Five cells containing the MgHEX-1 crystals were used both for 3D ED experiments and for X-ray serial crystallography measurements.

Workflow of in cellulo crystal preparation for ED experiments
Building upon the development of cryo-FIB milling procedures for crystals31,33,41 and also on the idea of using 3D negative fluorescence imaging for accurate 3D localization of target crystals inside a cryo-FIB/SEM microscope, we established the protocol for ED sample preparation. The procedure is shown schematically in Fig. 1 with key steps described below. Technical details are provided in the corresponding sections of “Methods” and in Supplementary Figs. 2–7.

In the first step (Fig. 1, panel 1), presence of MgHEX-1 crystals in cells was verified by light microscopy at room temperature (Fig. 2). Then, cell aliquots were collected, deposited onto a TEM grid and plunge-frozen. The frozen sample grids were sputtered with platinum prior to cryo-LM imaging (Supplementary Figs. 2 and 3). The ~20 nm metallic Pt sputter coating layer improved both reflected light contrast and subsequent FIB/SEM imaging, and assisted coordinate transfer between cryo-light and FIB-SEM platforms.

In the second step (Fig. 1, panel 2), regions of interest (ROIs) containing crystals were first identified using combined negative fluorescence (FL), reflected light (RL) and transmitted light imaging (Supplementary Fig. 2) of the frozen grids in a cryo-LM microscope. This step is of particular importance if only few cells contain crystals since the cryo-LM platform enables rapid screening of wide areas. Then, the negative FL imaging yielded 3D coordinates of crystal centers (Supplementary Fig. 3). These coordinates were further referenced to the top sample surface, established by 3D RL imaging. The RL data provided surface topography information analogous to subsequent FIB/SEM imaging and aided transfer of the crystal coordinates (Supplementary Fig. 4).

In the third step (Fig. 1, panel 3), the samples were moved into a Ga+ cryo-FIB/SEM microscope, and the crystal coordinates were transferred to the microscope frame by correlating the FIB/SEM imaging data with the previously acquired cryo-LM data. Specifically, the ROIs containing the crystals were located by aligning the SEM overview images with the FL/RL data (Supplementary Fig. 4a). For each ROI, the RL data coupled with a 3D crystal position (relative to the top sample surface) were correlated with the FIB images of the ROI via shared surface topographical features (Supplementary Fig. 4b). This enabled accurate localization of target crystals. Fiducial markers were generated by the Ga+ FIB (Supplementary Fig. 5) to guide the subsequent crystal lamella production using cryo-FIB milling.

Finally (Fig. 1, panel 3), lamellae containing parts of target crystals were prepared using a two-step cryo-FIB milling process with 30 kV Ga+ ions. First, ~300 nm thick protective organoplatinum coating was applied via a gas injection system (PtGIS)28,31,41, and rough high-current milling at 90° and 20° angles was run to yield ~4.5 µm thick lamellae. This step was designed to remove enough surrounding material to provide a wide tilt range for subsequent continuous rotation ED measurements in a cryo-TEM (Supplementary Fig. 6). Then, a gentle low-current FIB milling at 20° (Supplementary Fig. 7) gradually decreased the lamella thickness to final values (~250–300 nm) desired for 3D ED experiments (Fig. 1, panel 4). The MgHEX-1 crystals selected for the measurements were typically in the range of 5–8 µm (see e.g., Fig. 3), slightly below the average observed crystal size of ~8.6 µm, and only the crystals fully contained within the cell were used for processing.

Electron diffraction data collection
A lamella of an in cellulo grown MgHEX-1 crystal (~6 µm size; Fig. 3) was used to assess whether the sample preparation method yields ED data for high-resolution structure determination. The lamella was prepared with a thickness of ~250 nm (Fig. 3b, c), an optimal value reported for 200 kV ED28,29. Following localization of the crystal in imaging mode in a 200 kV cryo-TEM (Fig. 3d), ED data were recorded on a scintillator-based electron detector (see “Methods”) from a ~4.8 µm diameter zone with a crystal volume of ~4.4 µm3 (blue circle in Fig. 3d, e). This was followed by data collection from a 1 µm diameter zone (red circle in Fig. 3d, f), corresponding to a crystal volume of only ~0.2 µm3. Both 200 kV ED signals were acquired as still images with the same electron fluence of ~0.087 e−/Å2 (corresponding to an absorbed radiation dose of ~0.38 MGy42).

Further illumination of the crystal lamella with the ~4.8 µm diameter beam of 200 kV electrons was used to follow the radiation-induced decay42–45 of the MgHEX-1 diffraction pattern (Supplementary Fig. 8). The summed intensity of high-resolution ED peaks (1.9–2.5 Å range; Supplementary Fig. 8d) exponentially dropped to 50% of its initial value at a fluence of ~1.09 e−/Å2 (an absorbed dose of ~4.8 MGy42). 50% intensity loss often sets the dose limit44–46 in high-resolution crystallography investigations. All further measurements were far from this limit.

The sample preparation method yields crystal lamellae diffracting to high resolution even at low crystal volumes while using scintillator-based (indirect) electron detection. However, direct electron detection (DED) combined with energy filtration provides superior sensitivity and signal-to-noise ratios, yielding significantly improved ED data at lower electron fluences24,47. Therefore, data for the MgHEX-1 structure determination were collected using a 300 kV microscope equipped with a DED K3 electron-counting camera in a continuous rotation mode while filtering out inelastically scattered electrons with energy loss greater than 10 eV (see “Methods” for full detail).

We produced four lamellae containing crystals ranging from 5 to 8 µm size for the 3D ED structure determination experiments. The thickness of these four cryo-FIB milled lamellae was ~300 nm, optimal for 300 kV ED28,29. For two of the lamellae, 3D ED data were acquired using a 2.5 µm diameter collection zone (volume of ~1.6 µm3; denoted as “microvolume”) with a total fluence of ~0.3 e−/Å2. This corresponds to ~1.1 MGy absorbed dose, which is much below the 70% (D0.7 ~2.5 MGy) or 50% (D0.5 ~4.8 MGy) radiation dose limits marked in Supplementary Fig. 8d. These two microvolume ED datasets gave very similar data processing statistics (Supplementary Table 1). One of these datasets was selected for MgHEX-1 structure determination via molecular replacement. The previously reported NcHEX-1 structure (PDB 1KHI)37, which shares 74% sequence identity, was used as a search model. This yielded a 1.9 Å resolution crystal structure of MgHEX-1 with P622 symmetry (Table 1 and Fig. 4a), same as previously reported for NcHEX-12,8,37. The structure model was refined to 0.205/0.229 of Rwork/Rfree.

The other two lamellae were tested using a collection zone of 1.75 µm in diameter (~0.8 µm3 volume, denoted as “nanovolume”). Three-dimensional ED data were collected from one lamella at a total fluence of ~0.6 e−/Å2, which was double of the fluence used for the microvolume ED data collection to compensate for the reduced sample volume. This corresponds to an absorbed dose of ~2.2 MGy42, which is still much smaller than the value of the half-intensity decay (~4.8 MGy) shown in Supplementary Fig. 8d. Processing of the nanovolume ED data resulted in a 2.2 Å resolution structure of MgHEX-1 that was refined to 0.211/0.258 of Rwork/Rfree (Table 1 and Fig. 4b). Interestingly, the nanovolume ED signal from the second lamella reached 1.7 Å resolution. This was the highest resolution detected for all lamellae tested in this study at the same electron fluence, regardless of the crystal volume (Supplementary Fig. 9). Unfortunately, this lamella was contaminated with ice microcrystals (Supplementary Fig. 9c) during cryo-transfer into the cryo-TEM from storage, preventing reasonable 3D data collection for structure determination. Since the four crystal lamellae showed different diffraction strengths, testing more crystals could lead to improved resolution and provide better data quality in 3D ED structural studies.

During the development and optimization steps of the IncelluloED sample preparation workflow, we used 14 cells containing MgHEX-1 microcrystals, selected by RL/FL imaging. All crystals were successfully milled into lamellae, and all lamellae provided high-resolution ED signals. Although the localization and targeting process looks laborious, the procedures outlined in Fig. 1 yielded consistent results without loss of crystals. The weakest point in our hands was the transfer of the milled crystals into cryo-TEMs during the summer months when humidity was high in the laboratory and ice microcrystals contaminated some of the lamellae during transfer (e.g., Supplementary Fig. 9c). While this problem cannot be completely avoided, there are generally accepted strategies to mitigate it and maintain high success rates, e.g., by using fresh liquid nitrogen, filtering it through tissue or paper, and working in a low humidity environment.

3D ED structure of MgHEX-1
The electrostatic potential maps were refined for the 3D ED micro- and nano-volume data sets at 1.9 Å and 2.2 Å resolution (Fig. 4a, b and Table 1) and provided a high level of detail, sufficient to rebuild the non-flexible part of the main MgHEX-1 chain and to model most of the side chains. No interpretable electrostatic potential density is observed for the N-terminal residues 1–35, which have already been classified as disordered for the NcHEX-1 structure37, and the three C-terminal residues 179-181. The maps of Gly153, Arg154 and Gly155 that are located in a flexible loop at the C-terminus of helix 1 are not fully resolved, as well as the side chains of residues Gln117 and Arg148. The final micro- and nano-volume models show a protein backbone Cα root mean square deviation (RMSD) of 0.341 Å and 0.340 Å, respectively, compared to the 134 equivalent Cα atoms of the molecular replacement search model, which is in the expected range for proteins of this sequence identity level. Moreover, the micro- and nano-volume ED models are almost superimposable with an overall RMSD of 0.166 Å for the 123 Cα atoms. Deviations of more than 0.5 Å are restricted to residues Glu151 and Ser152 located in the flexible loop mentioned above. Even for the side chains the RMSD does not exceed 0.75 Å, except for the three residues Asn133, Glu144, Arg148 that are not fully resolved in both maps.

X-ray crystal structure of MgHEX-1 and comparison to 3D ED structures
Next, we compared the structural information obtained by 3D ED from a single micro- or nano-volume MgHEX-1 crystal to that obtained by applying the well-established approach of serial synchrotron X-ray diffraction (SSX) on the crystal-containing insect cells2. Visual inspection of test X-ray patterns revealed diffraction up to 2 Å, allowing the collection of a complete, serial line scan data set. Data processing using CrystFEL48 and molecular replacement (MR) using again the NcHEX-1 structure (PDB 1KHI) as a search model yielded an electron density map at 1.8 Å resolution, which allowed refinement of the structure model to 0.213/0.236 of Rwork/Rfree (Table 1 and Fig. 4c). The final MgHEX-1 structure included residues 35 to 174, again without the flexible N-terminus. In contrast to the 3D ED maps, no interpretable electron density is observed for residues 153 and 154 located in the previously mentioned flexible loop, and for the C-terminal residues 175 to 178. This might result from averaging thousands of crystals. Likewise, the side chains of residues Arg112 and Gln117 are not fully resolved. The calculated RMSD of 0.520 Å compared to the 134 equivalent Cα atoms of the MR search model is increased compared to that of 0.341 and 0.340 Å calculated for the micro- and nano-volume 3D ED structures. However, it is still in the expected range.

Comparison of the 3D ED micro- (RMSD 0.546 Å for 134 equivalent Cα atoms) and nano-volume MgHEX-1 structures (RMSD 0.509 Å for 134 equivalent Cα atoms) obtained from single crystals with that elucidated by SSX using 62,496 intracellular crystals revealed no major differences in the overall conformation of the MgHEX-1 monomer. Deviations of more than 1 Å are restricted to residues Ser66 to Gly70 located in a flexible loop connecting β-strands 3 and 4 in the N-terminal domain (Fig. 5a). Also, the calculated overall RMSD for the side chain atom positions compared to the micro- (0.738 Å) and the nano-volume (0.756 Å) model indicated an almost identical MgHEX-1 structure obtained by 3D ED and SSX. Similar RMSD values (about 1 Å) were obtained by comparing the Cα atom positions of the 3D ED micro-volume and the SSX structures with that of a predicted AlphaFold3 model49 for MgHEX-1 (Supplementary Fig. 10).

The observed difference in the c-axes of the unit cells between the SSX structure and the 3D ED structures (Table 1) is likely attributable to the impact of distinct sample handling conditions. For SSX, cells were overlaid with PEG200 for cryo-protection during freezing in liquid nitrogen2, which causes dehydration. For 3D ED experiments, cells were plunge-frozen in liquid ethane, which does not require cryo-protection due to a faster freezing process. A similar effect was previously observed for serial femtosecond X-ray data collection at an XFEL at room temperature8.

All MgHEX-1 models revealed the typical two-domain structure that was previously reported for NcHEX-137, consisting of mutually perpendicular antiparallel β-barrels (Fig. 5b). The N-terminal domain is formed by six antiparallel β-strands, while the C-terminal domain consists of a five-stranded β-barrel and an α-helix. This indicates a high degree of structural conservation among HEX-1 proteins from different ascomycetes, especially in the “common region” comprising the 148 C-terminal residues. However, for residues 89–94, representing the most divergent part between the NcHEX-1 and the MgHEX-1 structure, the side chains fit well into the 2Fo-Fc map of the MgHEX-1 3D ED micro-volume structure, in contrast to the corresponding residues of NcHEX-1 (Fig. 5c). This rules out model bias imposed by phase determination applying the molecular replacement approach with a search model similar to the elucidated MgHEX-1 structure, confirming the correct interpretation of the map.

Three types of intermolecular surface interactions were previously identified that are responsible for the self-assembly of NcHEX-1 into a crystalline state in Woronin bodies37, representing the physiological function of HEX-1 proteins in ascomycetes36. All involved residues are conserved in terms of sequence and structure between MgHEX-1 and NcHEX-1 (Supplementary Fig. 11a). Again, a combination of salt bridges and hydrogen bonds forms the interactions of monomers, previously denoted as group I and group II interactions, which build up a structural HEX-1 polymer that establishes a helical spiral. Each filament is in contact with six identical neighbouring polymers via hydrogen bonds (group III interactions), finally producing the six-fold symmetry of the MgHEX-1 crystals, identical to that reported for NcHEX-137 (Supplementary Fig. 11b).

In conclusion, the comparable level of biologically relevant information obtained by the IncelluloED approach presented in this study and by well-established SSX validate 3D ED as a suitable technique to obtain in situ high-resolution structural information from a single protein crystal in the very low micrometer size range. IncelluloED is an additional tool in the methodological toolbox, one that could be advantageous in situations where other techniques are more demanding.

Methods
InCellCryst, a streamlined approach for in cellulo crystallization of recombinant proteins in living insect cells2, was applied to MgHEX-1, involving vector construction, virus stock production, virus stock titration, and the detection of protein crystals within insect cells.

MgHEX-1 vector construction
Cloning of the Hexagonal peroxisome 1 (HEX-1) gene from ascomycetous fungus M. grisea (NCBI Accession AY044846, codon optimized for Trichoplusia ni expression) into the pFastBac 1 V2 cyto vector under the control of the polyhedrin promotor2,83 was performed by PCR amplification using primers 5′-GATCGGTACCTACTATGAAGACGATCGTGAGACC-3′ and 5′-GATCGCTAGCCAGCCTGCTGCCGTG-3′, adding KpnI and NheI restriction sites at 5′ and 3′ ends of the MgHEX-1 gene without start and stop codon and HiFi DNA polymerase (highQu). After digestion of the PCR product with FastDigest NheI and KpnI (Thermo Fisher Scientific), the insert was ligated into the equally digested pFB1 V2 cyto vector by T4 DNA ligase (Thermo Fisher Scientific), yielding pFB1 MgHEX-1 V2 cyto plasmid. Competent Escherichia coli DH5α cells (NEB C2987) were transformed with ligation mixture and amplified pFB1 MgHEX-1 V2 cyto plasmid DNA was extracted using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) and sequence verified (LGC Genomics).

Recombinant baculovirus stock production and titration
For rBV production E. coli DH10EmBacY cells (Geneva Biotech) were transformed with pFB1 MgHEX-1 V2 cyto according to the Bac-to-Bac manual (Invitrogen). Recombinant bacmid DNA, isolated by applying the ZRBac DNA Miniprep Kit (Zymo Research), was verified for the presence of the transposed pFB1 sequence by PCR using pUC/M13 primers. In a 12-well cell culture plate, 5 × 105 Spodoptera frugiperda Sf9 insect cells (11496015, Thermo Fisher Scientific) were grown in serum- and protein-free ESF921 cell culture medium (Expression Systems), supplemented with penicillin/streptomycin (Sigma Aldrich), at 27 °C and transfected with recombinant bacmid DNA using Escort IV reagent (Sigma Aldrich). After four days of incubation at 27 °C, the supernatant was harvested (P1 virus stock) and used for infection of 2 × 106 Sf9 cells/mL in a T-25 mL cell culture flask that was incubated for another 4 days at 27 °C with constant shaking (100 rpm), followed by harvesting the supernatant (P2 stock). The virus titre of the P2 stock was calculated using the TCID50 (tissue-culture infectious dose84) in a serial dilution assay as previously described2,38,83. The efficiency of transfection and viral stock production as well as the virus titre was determined by detection of the fluorescence of the EYFP marker encoded in the bacmid2 using a wide field inverted X81 (Olympus) or Ts2R-FL (Nikon) microscope.

Intracellular crystallization of MgHEX-1
For intracellular crystallization, 0.9 × 106 High Five insect cells (B85502, Thermo Fisher Scientific) were plated in 2 mL ESF921 medium (Expression Systems) in one well of a six-well cell culture plate and infected with the MgHEX-1 encoding rBV using a multiplicity of infection (MOI) of 1. The required amount of P2 stock was calculated using the following formula: virus stock [mL] = (MOI · cell number) / (0.69 · TCID50/mL)85. After 4 days of incubation at 27 °C, the High Five cells were examined for the presence of MgHEX-1 protein crystals by light microscopy via bright-field imaging2 using Ts2R-FL (Nikon) and DMi8 (Leica Microsystems) inverted microscopes equipped with 20× and 40× objectives, respectively. Median intensity Z-projection of bright-field Z-stacks were performed by ImageJ86 to better visualize the crystals. Cells containing MgHEX-1 crystals were carefully harvested by gentle pipetting for subsequent electron or X-ray crystallography experiments. Note that the excitation of EYFP fluorescence (FL), which is co-expressed in the cells, provided an additional way of detecting in cellulo crystals as regularly shaped areas lacking the fluorescent signal (Fig. 2). At room temperature, the latter was performed with a confocal laser scanning FV1000 microscope (Olympus) using a 488 nm laser and a 100× objective (UPlanSApo, oil-immersion, NA 1.4, Olympus). 3D model visualization based on the FL stack was created using Amira software (Thermo Fisher Scientific). In Amira, the Volume Rendering function and the 2D Median filter were used, and the blue model of the crystals was semi-automatically generated (Fig. 2).

In cellulo crystal preparation for ED experiments
The sample preparation workflow for 3D ED data collection is depicted in Fig. 1, and the individual steps are detailed in separate sections below.

Cell collection and freezing on TEM grids. After verification by light microscopy (Fig. 2) a 3 μL aliquot of cells containing MgHEX-1 crystals was carefully collected with a pipette from the bottom of a well in a six-well cell culture plate. Then, an EM GP2 plunge freezer (Leica Microsystems) was used, with its sample environment chamber set to 20 °C and 95% relative humidity. The cell aliquot was deposited onto a holey gold film covered TEM grid (UltrAuFoil® R2/2, 200 gold mesh, Quantifoil Micro Tools). The grid was then blotted with filter paper (#1, Whatman) from the back side for 12 s, directly plunge-frozen into liquid ethane cooled by liquid nitrogen bath, transferred to liquid nitrogen and clipped into an Autogrid assembly (Thermo Fisher Scientific) for further handling. The grid with the cells was stored in liquid nitrogen until further use. Note that the holey gold film covered TEM grids had better mechanical stability during the blotting procedure than holey carbon film covered grids (QUANTIFOIL®, Quantifoil Micro Tools), and this resulted in larger intact film areas with retained cells in our hands. The frozen samples were transferred, under cryogenic conditions, using a VCM/VCT500 cryo-transfer system (samples were kept below -170 °C, Leica Microsystems), into an ACE600 cryo-coater (Leica Microsystems). The samples were sputter-coated with a ~20 nm platinum conductive protection layer41,87, while maintained below -153 °C.

Determining regions of interest and crystal coordinates by cryo-light microscopy (cryo-LM). The coated samples were transferred to a cryo-fluorescence upright wide-field microscope (THUNDER Imager EM CryoCLEM, Leica Microsystems) equipped with a 50× objective possessing a high NA of 0.90 (HC PL APO, CRYO CLEM, Leica Microsystems; ~373 nm XY-resolution and ~679 nm Z-resolution at 550 nm wavelength) and a cryo-stage (temperature set to -180 °C), which accommodates up to two TEM Autogrids. Within the THUNDER microscope, the sample grid plane is oriented perpendicular to the optical axis during cryo-light microscopy (cryo-LM) imaging.

Grid overviews (Supplementary Fig. 2a) in reflected light (RL; using a LED3 fluorescence light source (Lumencor) and a longpass filter >425 nm) and FL (with 450-490 nm excitation and 500–550 nm emission filters) channels were obtained within the THUNDER cryo-LM microscope using the embedded Navigator module of LAS X software package (Leica Microsystems) by stitching individual fields of view (271.08 µm × 271.08 µm). Zones with cells exhibiting EYFP FL and an intact support film were investigated in RL, FL and transmitted light (TL; using light of a halogen lamp) regimes and regions of interest (ROIs) containing suitable crystals were identified (see Supplementary Fig. 2b).

For each ROI containing a crystal of interest, a 3D volume was imaged as a ~40 µm dual-channel Z-stack using RL and FL channels (Supplementary Fig. 3) and a system-optimized Z-step size of 0.445 µm. THUNDER deconvolution88 technique embedded in the LAS X software was applied to the FL Z-stack data to improve the contrast of the FL images. This dual-channel RL/FL Z-stack was used to locate the 3D center of the crystal (Xc, Yc, Zc), as shown in Supplementary Fig. 3. Using the FL data of the Z-stack, the position (Xc, Yc, Zc) was determined as the center of the negative FL signal of the crystal (Supplementary Fig. 3f). The Z-depth of the crystal’s center (Zc) was calculated relative to the Z-position of the top surface of the sample (Zs) at (Xc, Yc) location. The Z-position was determined using the RL Z-stack data and set as the Z=0 reference (Supplementary Fig. 3e). The determined lateral position of the crystal center (Xc, Yc) was marked with a dot on the maximum intensity Z-projection of the RL channel of the ROI containing the crystal (see Supplementary Fig. 3c and Supplementary Fig. 4b). This RL image and the Z-depth of the crystal’s center (Zc) were used for subsequent image correlation and crystal localization procedures as depicted in Supplementary Fig. 4b and Supplementary Fig. 5.

Note that the metallic Pt sputter coating of the samples, in addition to mitigating charging and protecting against unwanted ion beam damage during subsequent FIB/SEM imaging41,87, also amplified the light reflection from the coated top surface of the samples. This enhanced the surface imaging contrast for RL microscopy and improved FIB/SEM surface imaging, thereby facilitating both the determination of the Z-position of the sample top surface (Supplementary Fig. 3c, e, g) and subsequent correlation between FIB and RL images inside the FIB/SEM microscope (Supplementary Fig. 4b). We observed that ~20 nm thick Pt sputter layers provided good light reflection while remaining sufficiently transparent for TL and FL signals, while thicker layers (e.g., ~40 nm) significantly dampened FL imaging.

Localization of crystals in cryo-FIB/SEM and generation of fiducial markers. Grids containing the crystals of interest were unloaded from the cryo-LM microscope and, using a VCM/VCT500 cryo-transfer system (< -170 °C, Leica Microsystems), placed into a 40° pre-tilted cryo-FIB/SEM grid holder (Leica Microsystems), which accommodates up to two TEM Autogrids per session, and inserted into an Amber cryo-FIB/SEM microscope (Tescan) equipped with a passively cooled cryo-stage (maintained at < -143 °C, Leica Microsystems). The angle between the ion and electron beams is 55° for the Amber FIB/SEM microscope. To avoid unwanted sample damage during navigation and monitoring of the milling process, SEM and FIB imaging were performed with 0.5 kV electrons of 10 pA current and with 30 kV Ga+ ions of 10 pA current throughout the described cryo-FIB milling procedure.

In the cryo-FIB/SEM microscope, an overview SEM image of a TEM grid was acquired with the electron beam at a 65° angle to the grid plane (the default angle for the Autogrid-compatible sample holder used). To enable rapid localization of ROIs containing crystals of interest, this overview SEM image was correlated with the combined RL and FL maps derived from cryo-LM imaging (Supplementary Fig. 4a). This was performed using a three-point image correlation procedure (based on image affine transformations) of the CORAL module within the Essence software (Tescan), which controls the FIB/SEM instrument.

Subsequently, for each ROI containing a target crystal, the following steps were performed. An ROI with a crystal of interest was positioned at the intersection of the SEM and FIB beam foci, and the sample was rotated such that the ion beam was at a 90° angle to the sample grid plane. The ROI was then imaged by FIB, and the resulting image was correlated, using the three-point correlation procedure, with the previously obtained LM image (containing the marked lateral position of the crystal’s center (Xc, Yc); see Supplementary Fig. 4b). Using the Draw Beam module of the Essence software, fiducial markers were drawn typically as two 2 × 5 µm rectangles separated by a 12 µm gap on the correlated overlay of FIB and LM images, positioning the crystal’s center midway between the fiducials (Supplementary Fig. 5a, b). These fiducial markers were milled (Supplementary Fig. 5c) by raster scanning over the drawn pattern using a 30 kV ion beam with a 50 pA current to a depth of ~1 µm. For the calculations herein, we used an experimentally derived cryo-FIB milling rate of ~4 µm3/nA/s. Subsequently, the ROI was positioned such that the ion beam was at a 20° angle to the sample plane and then imaged by FIB. The position of the crystal’s center at the 20° angle FIB view was estimated to be Zc × cos 20° lower than the top surface above the crystal center (Zc = Zs) defined at this FIB view by the previously milled two fiducial markers (see Supplementary Fig. 5d–f). The third fiducial marker (a 3 × 12 µm rectangle) was drawn 3.25 µm lower than the estimated position of the crystal’s center and FIB-milled at 150 pA current to a depth of ~2 µm. All three fiducial markers per each crystal served as 3D locators of estimated crystal center positions for subsequent procedures.

Crystal lamella production by cryo-FIB milling. After creating all the fiducial markers for all the crystals of interest, the samples were coated with a protective (see e.g., refs. 28, 31, 41) organoplatinum layer using a gas injection system (PtGIS, 60 °C, 45 s, ~400 nm/min layer deposition rate) integrated into the Amber cryo-FIB/SEM microscope. The resulting protective PtGIS layer was ~300 nm thick. For each crystal of interest, the following FIB milling steps were performed.

A center of a crystal of interest was relocalized at different FIB view angles using the previously FIB-marked fiducials. “Rough” cryo-FIB milling was performed via raster scanning with a 30 kV Ga+ ion beam, first, at 90° and then at 20° to the TEM grid plane using high currents of 2.5 nA and 1 nA, respectively, as depicted in Supplementary Fig. 6. The bulk of the sample was removed to provide a wide rotation range accessible for transmission of an electron beam in subsequent 3D ED measurements. This high-current milling produced a ~4.5 µm thick lamella at a 20° angle to the grid plane. Subsequently, a “gentle” cryo-FIB milling procedure was performed by raster scanning with the 30 kV Ga+ FIB at 20° to the TEM grid plane in a stepwise manner, reducing the beam current as the lamella was thinned to its final thickness, as shown in Supplementary Fig. 7. Milling currents of 150, 50, and 10 pA were used (in this order) to generate the final lamella by decreasing the thickness of the sample to ~1.5 µm, ~0.8 µm, and the final thickness, respectively. Such low currents were used for the gentle procedure to retain the maximum possible degree of crystalline order of the in cellulo crystal (see e.g. refs. 31, 87). Lamellae produced for 200 and 300 kV ED experiments were ~4 µm wide and with final thicknesses, corresponding to the reported optimum28,29, of ~250 nm and ~300 nm, respectively, and were angled at 20° with respect to the TEM grid plane.

Using the VCM/VCT500 cryo-transfer system, the grids were then transferred from the cryo-FIB/SEM microscope to cryo-EM grid boxes (Thermo Fisher Scientific) for storage in liquid nitrogen until further use in ED experiments.

ED data collection, data processing and structure determination
For 200 kV ED experiments, we used a JEM-F200 cryo-TEM (JEOL) equipped with a cold cathode field-emission electron gun, a side-entry dual Autogrid cryo-holder (-177 °C, Model 210, Simple Origin), and a scintillator-based CMOS detector (4096 × 4096, 15.5 × 15.5 µm2 pixel, TemCam-XF416, TVIPS). Crystal lamellae were identified in imaging mode (Fig. 3d), and ED experiments were performed with parallel electron beam illumination at a flux of ~0.0435 e−/Å2/s. ED data were collected either from the parallel beam illumination area, which was ~4.8 µm in diameter on the sample plane, or from a crystal lamella zone of ~1 µm in diameter, as defined by a selected area aperture. ED data were recorded as still images with the XF416 camera, using 2 × 2 binning and a 2 s exposure (corresponding to a fluence of ~0.087 e−/Å2). The effective sample-to-detector distance was ~2310 mm, as calibrated with a standard evaporated aluminum grid (Ted Pella). To evaluate radiation sensitivity of a MgHEX-1 crystal lamella, the sample was irradiated with the ~4.8 µm diameter 200 kV electron beam (Fig. 3d), and a series of 40 still ED images was collected sequentially. The resulting total cumulative fluence was ~3.5 e−/Å2. The intensities of high-resolution ED peaks (1.9–2.5 Å range) were then summed for each image and analyzed as a function of the cumulative electron dose (Supplementary Fig. 8). ED data visualization and analysis were performed using Adxv89, ImageJ86 software and an in-house Python 3.12.4 script. Absorbed radiation doses were estimated with the RADDOSE-ED software42.

For 300 kV ED experiments, we used a Titan Krios G1 cryo-TEM (Thermo Fisher Scientific), equipped with a Schottky field emission gun (XFEG), a cryogenic sample Autoloader (~-193 °C), a GIF Bio-Quantum energy filter (Gatan/Ametek) and a K3 direct electron detector (5760 × 4092, 5 × 5 µm2 pixel, Gatan/Ametek). The sample grids were cryo-transferred from storage to the Autoloader cassette, ensuring the microscope’s rotation (α-tilt) axis was orthogonal to the FIB milling direction. In the cryo-TEM, the crystal lamellae were identified in imaging mode (Supplementary Fig. 9). ED data acquisition employed a parallel electron beam with a 14 µm diameter illumination area on the specimen plane and a low electron flux of ~0.0009 e−/Å2/s. The energy filter slit was set to select only electrons with energy loss less than 10 eV. Following the manufacturer’s recommendations (Gatan/Ametek) for 3D ED data collection, the K3 detector was operated in standard electron-counting mode (without correlated double sampling; internal frame rate of 1.5 kHz) at a dose rate below 40 e−/pixel/s for ED peaks (to avoid significant coincidence loss90,91). This dose rate was achieved by setting up the low flux of the parallel electron beam (~0.0009 e−/Å2/s) and validated by acquiring 1 s exposure ED still images from a crystal lamella prior to 3D ED collection. The effective sample-to-detector distance was ~905 mm, as calibrated with a standard evaporated aluminum grid (Ted Pella). 3D ED data were recorded in a continuous rotation mode collecting ED signal from crystal lamella zones with diameters of 2.5 µm (referred to as “microvolume”) or 1.75 µm (referred to as “nanovolume”) as defined by selected area apertures of the cryo-TEM. The K3 camera recorded at 1 fps during the data collection. Microvolume data were collected during 325 s exposure at a rotation speed of 0.249°/s, covering a total angular wedge of 80.9° (from -40.4° to +40.5° α-tilt) at a fluence of ~0.3 e−/Å2. Nanovolume data were collected during 660 s exposure at a rotation speed of 0.1224°/s, covering a total angular wedge of 80.8° (from -40.4° to +40.4° α-tilt) at a fluence of ~0.6 e−/Å2. Absorbed radiation doses were estimated using RADDOSE-ED software42. The electron-counting data images were saved as 8-bit unsigned Tagged Image File Format stack without detector gain normalization being applied. For further processing, the images were gain-normalized and converted to the Crystallographic Binary File format using an in-house developed Python 3.12.4 script and 2cbf program of XDS software package92.

The 3D ED data were indexed and integrated using XDS software92, and then scaled and merged using AIMLESS software93. Initial phases were obtained by molecular replacement with Phaser94 using the NcHEX-1 structure, previously obtained using crystals grown by a sitting drop vapor diffusion method, as a search model (PDB 1KHI). MgHEX-1 crystal structure refinements for all collected datasets were carried out using REFMAC595 (version 5.5.0051; electron scattering factors were set with the keyword “source ECMB”) and iterative cycles of manual model building in Coot96 (version 0.9.7). PyMOL Molecular Graphics System (version 4.5.0, Schrödinger, Inc.) was used for graphical illustrations.

Serial X-ray diffraction data collection, data processing and structure determination
Sample preparation for X-ray diffraction measurements was performed as previously described83. In brief, High Five cells carrying MgHEX-1 protein crystals were harvested into a 1.5 mL tube and allowed to settle by gravitation. In a 90% humidity air stream 0.5 µL cells from the loose pellet were loaded onto a MicroMesh (700/25; MiTeGen) previously mounted on a B1A goniometer base (MiTeGen), which was positioned by a custom-made holder in the optical focus of a standard upright cell culture light microscope. The excess medium was removed using an extra fine liquid wick (MiTeGen). For cryo-protection, 0.35 µL of a 40% PEG 200 solution diluted in ESF921 cell culture medium were pipetted onto the cells, again followed by removal of the excess liquid. Cell-loaded MicroMeshes were flash frozen and stored in liquid nitrogen until further use in X-ray diffraction experiments.

X-ray diffraction data collection was performed at the EMBL microfocus beamline P14 at the PETRA III storage ring (DESY, Hamburg) using the mxCuBE v2 user interface97. The cell-loaded MicroMeshes were mounted on a mini-Kappa goniostat attached to an MD3 diffractometer using the MARVIN Sample Changer. Using a 7 × 3 µm² double focused X-ray beam characterized by an energy of 12.7 keV and a flux of 1.71 × 1013 ph/s MicroMeshes were scanned employing a previously established serial helical grid scan strategy2 to collect complete datasets using the EIGER 16M detector (DECTRIS) at 100 K. During 20 ms irradiation at each position the MicroMesh was rotated by 1°. Absorbed radiation doses were estimated using RADDOSE-3D software42. Data collection parameters are presented in Table 1.

To process X-ray diffraction data using CrystFEL48, a geometry file specifying information about the sample to detector distance (clen), the wavelength (photon_energy), the size of the detector (max_fs and max_ss) as well as the beam position on the detector relative to the detector corner (corner_x and corner_y) is required. Then, peak detection parameters can be specified using the graphical user interface of CrystFEL version 10.0/10.1 or the check-peak-detection script of version 9.1 and older versions using peak finding algorithm peakfinder8. In general, for this study, a threshold of 0, a local_bg_radius of 3, min_res of 50, a max_res of 2200 and a max_pix_value of 50 were a suitable starting point. The min_snr and min_pix were adjusted to allow proper peak detection for each sample. Afterwards, all frames were indexed by Mosflm, and the correct lattice and unit cell parameters were optimized, and cycles of beam position refinement were performed by executing the detector_shift script (version 9.1) on the obtained stream files. If the optimized beam position did not differ more than 0.1 mm from the input, the beam position was accepted and all frames were indexed invoking mosflm-latt-cell, mosflm-latt-cell, xds and xgandalf. Then the peakogram-stream script was executed to set the maximum allowed intensity for each resolution range to separate salt reflections from protein reflections applying a filtering script. Finally, the filtered intensities were scaled and merged. The resolution limit was set where CC* is above 50%, SNR above 0.5 and the completeness over 95%. Mtz-files for subsequent modeling were generated using get_hkl. To set up peak detection parameters CrystFEL version 10.0 was applied, for indexing CrystFEL version 10.1 or 10.2, while for merging and scaling CrystFEL version 9.1 or 10.2 were used.

Phases were retrieved by molecular replacement with Phaser94 using the NcHEX-1 structure as a search model (PDB 1KHI). Structure refinements for all generated datasets were carried out using PHENIX98 (version 1.19.2-4158) and iterative cycles of manual model building in Coot96 (version 0.9.7). Simulated-annealing omit maps were calculated in PHENIX. Applying the FastFourierTransform program, electron density maps with CCP4 extensions were saved and loaded in the PyMOL Molecular Graphics System (version 4.5.0, Schrödinger, Inc.) for graphical illustrations of contoured omit maps.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Redecke and co-workers present an adaptation of the InCellCryst concept, (i.e. a pipeline based on crystallization directly within living insect cells to produce a sufficient amount of homogeneous microcrystals of a target protein for structure determination by serial X-ray, thereby avoiding the purification step), to electron diffraction (InCellED), a technique that is gaining momentum but is still in its infancy in the context of protein crystallography.\nThey chose HEX-1 from Magnaporthe grisea (MgHEX-1) crystals, grown in insect cells to preserve their native state, to demonstrate that electron diffraction data collected from a single crystal can yield enough information to determine the previously unknown 3D structure at 1.9 Å resolution. They also compare these results with a similar dataset obtained by serial X-ray crystallography, which used tens of thousands of crystals and reached a resolution limit of 1.8 Å. Because electron diffraction requires far fewer cell-containing crystals, this method could help overcome the bottleneck of low efficiency in in cellulo crystal growth. Nevertheless, sample production, preparation, and adaptation for 3D microED data collection require specialized know-how, which is still under development.\nThe chosen system, MgHEX-1, is consistent with their previously characterized Neurospora crassa HEX-1 (NcHEX-1), since both naturally self-assemble into a hexagonal crystalline material. The new MgHEX-1 structure could shed light on the mechanism underlying the self-assembly process that drives cellular stress but further analysis seems to be need to find the mechanism behind its formation naturally.\nThe procedure involves cryogenic focused ion beam milling (cryo-FIB milling) coupled to scanning electron microscopy (SEM) to adjust sample thickness after locating crystals within the cell using fluorescence (FL) microscopy, as well as cryo-light microscopy and correlative light–electron microscopy. The sample preparation process is well described; however, fine details (beyond the scope of the present work) are not provided, and reproducibility may be challenging even for experienced technicians.\nOverall, the manuscript is well written, and the methodology, its main focus, is clearly explained, even from the perspective of a non-expert in electron diffraction. Therefore, I believe this article will be significant for the structural biology community, although I have one major concern regarding its framing.\nMajor concerns:\nAlthough this approach for InCellED clearly has high potential and could become a viable alternative technique, the data presented in the manuscript do not allow one to envision its general applicability. Looking at the authors’ own previous work in this journal, it is evident that they have several other systems available that could be used to showcase the broader potential of this method/pipeline. The current study demonstrates that a single crystal can yield a complete high-resolution dataset; however, to properly frame the methodology’s potential, other lower-symmetry samples should be analyzed and presented.\nTherefore, I recommend a major revision to include a broader set of systems, at least two more, that clearly represent a variety of potential scenarios, such as low-symmetry crystals requiring the collection and merging of multiple datasets, as well as crystals of different morphologies.\nMinor concerns:\nBefore the discussion, the authors state that the level of biologically relevant information obtained by the novel InCellED approach is comparable to that obtained by the well-established SSX. While this may be true, it is also somewhat obvious, its validity will always depend on the resolution limit, and it has already been shown in previous studies using microED. I suggest rephrasing this point to emphasize instead that InCellED provides an additional tool in the methodological toolbox, one that could be advantageous in situations where other techniques are more demanding.\nAlso, in line with my earlier comments, the discussion includes the statement: “In this study, we established and validated InCellED, a workflow for high-resolution structure determination by 3D ED using only a single intracellular protein crystal in the micrometer size range.” The present results only demonstrate that the approach is possible. To substantiate the claim that it constitutes a workflow pipeline, it would need to be shown that it can be successfully applied to multiple systems.", "reviewer_2": "The manuscript titled “In situ protein crystallography: a single-crystal electron diffraction pipeline for structure determination inside living cells” by Bílá, Pinkas, Khakurel et al. presents a novel and technically significant approach for determining protein structures within living cells using micro-electron diffraction (microED). This work represents a valuable contribution not only to the field of in vivo crystallization, but also to the broader electron diffraction and structural biology communities. This robust in situ approach offers a powerful platform for advancing structural studies of proteins in their native cellular milieu.\nThat said, the study is innovative and of high potential impact. While the manuscript is generally well written and the methodology is clearly described, I recommend a number of minor revisions to further strengthen its clarity and presentation. My comments and suggestions listed below are intended to support the authors in refining the manuscript and ensuring its readiness for further publication in Nature Communications.\nIntroduction\n-->The referee recommends including a graphical abstract in the Introduction section to enhance clarity and accessibility. If possible, the authors may consider repositioning Figure 2 as Figure 1 and integrating it directly into this section to provide an early visual overview of the workflow or methodology.\nResults\nIntracellular crystallization of MgHEX-1\nLine 22: ‘The crystallization efficiency and low-micrometre size range position HEX-1 crystals as ideal candidates for IncellED method development’\n--> The referee recommends that the authors specify the expected size range of the HEX-1 crystals and provide an appropriate citation to support this, referencing prior studies where applicable. Quantitative detail would strengthen this statement and contextualize it within existing literature.\nIn cellulo crystal preparation for ED experiments\n--> The referee notes that the novelty of the sample preparation method as described is somewhat limited. Upon further examination of the existing literature, similar sample preparation workflows have already been reported for related systems. The primary difference in this study appears to be the use of entire cells as samples, rather than isolated crystals.\nGiven the close methodological overlap, the referee suggests that this section could be merged with the previous one, to streamline the presentation and avoid redundancy.\nAdditionally, the authors are encouraged to indicate whether alternative grid types were tested during the sample preparation phase. Comparing results across different grids might improve sample distribution and provide further methodological insight.\nElectron diffraction data collection.\n-->The referee is interested to know whether, in addition to the previously reported NcHEX-1 structure, the authors have considered using an AlphaFold-predicted model for molecular replacement. Including such an approach could offer an informative structural comparison between the in situ HEX-1 structure, the previously published NcHEX-1, and the AlphaFold model. If feasible, the authors are encouraged to present RMSD values among the three structures, ideally in a tabulated form or as an additional figure, to quantitatively assess structural similarities and deviations.\nFurthermore, the referee suggests that the authors address the potential issue of radiation damage, particularly given the use of electron diffraction and the presumed sensitivity of in cellulo samples. A brief discussion—supported by any available data or observations—on how radiation damage was minimized or evaluated during data collection would be helpful.\nLastly, the authors are requested to specify the angular wedge (tilt range) used during collection of diffraction data. Since small angular wedges at elevated fluence from multiple crystals can help enhance high-resolution signal.\nSerial X-ray diffraction data collection, data processing and structure determination & Discussion\n-->The referee would be particularly interested to know whether the authors have considered, or have an opinion on, the use of a serial data collection approach in electron diffraction. This strategy would be highly relevant in the context of minimizing radiation damage, especially when using high-fluence exposure on single crystals or cells without rotation.\nIncorporating such an approach (e.g., serialED) could offer several advantages, including enhanced data quality and a more systematic evaluation of beam-induced damage. Furthermore, if such datasets are or become available, it would be valuable to perform comparative analyses between different collection modes—for example, 3DED vs. serialED—to quantitatively assess radiation effects. Similarly, comparisons could be made to explore vitrification-related artifacts, analogous to what's studied in ED-SSX workflows.\nWhile the referee understands that implementing this methodology may require additional experiments and specific instrumentation, it would be highly appreciated if the authors could address these possibilities in the Discussion section, and provide a brief outlook or commentary on the feasibility and potential impact of such approaches.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The manuscript “In Situ protein crystallography: a single crystal electron diffraction pipeline for structure determination inside living cells,” by Bílá et al. provides a thorough description of a pipeline for determining crystal structures of proteins grown inside of cells. This process builds upon the InCell Cryst pipeline previously established for serial X-ray crystallography. The authors use the homologous MgHEX-1 protein to demonstrate the process, whereas the original X-ray work had used NcHEX-1. The critical advancement presented here is that the structure could be determined using a single crystal inside a cell, greatly reducing the number of cells and crystals required. The total illuminated crystalline volume between the MicroED and serial X-ray methods is orders of magnitude different, a clear advantage. Although the 1.8 Å resolution obtained by serial synchrotron radiation was slightly higher than the 1.9 Å from MicroED, this served as an excellent validation of the method's accuracy. It was also demonstrated that single diffraction patterns from these crystals could reach resolutions comparable to those obtained by SSX. I believe this paper serves two functions: it broadens the applicability of MicroED to a challenging set of targets, and it demonstrates a workflow for routinely approaching such targets. As such, I believe this work is of high significance to the community and should be accepted for publication.\nHowever, I believe there are several improvements the authors could make that would make the work more approachable, factually accurate, and appealing to those looking to solve protein structures inside of cells.\n1. Context/Editorial\nThe largest criticism I have of this work is not technical, it is editorial. The authors relegate a foundational earlier work from Yang et al. to a single sentence in the discussion. Since planting a flag as the ‘first’ has little bearing on the quality of this excellent work, the authors should address the Yang et al. study in the introduction. Their work is about using MicroED to solve protein crystal structures from proteins expressed and crystallized in cellulo. The work by Yang et al. used the same core techniques (cryo-FIB milling and MicroED) to determine the structures of proteins that naturally crystallize in human cells. Critically, Yang et al. also correlated their structural data with cryo-electron tomography (cryo-ET), allowing them to map the crystal's 3D location and interactions within the unperturbed cellular landscape. The \"IncellED\" pipeline, by contrast, is a pure structure-determination method.\nProperly discussing this does not damage the current paper; rather, it strengthens it by placing it in the context of other major advances (e.g., milling viscous LCP to study membrane proteins). It would demonstrate to the reader that these in situ approaches are maturing rapidly and have diverse, powerful applications. The authors could also note that cryo-ET could be readily integrated into their workflow if desired.\n2. Emphasis on the Single-Crystal Advantage\nI encourage the authors to more forcefully emphasize the profound advantages of their single-crystal approach. Solving a high-quality structure from a single crystal lamella and a mere ~1µm³ of illuminated volume is a monumental achievement, especially when contrasted with the >60,000 crystals required for the SSX experiment. The manuscript could be strengthened by explicitly discussing the significant drawbacks inherent to merging such a vast number of microcrystals. The risks of polymorphism, the averaging of partial occupancies that can obscure important structural details, low hit rates, and the immense challenges in data processing and scaling, etc. Their approach bypasses these issues by providing a \"clean\" dataset from a single, homogenous crystalline entity. This is easy to process and deal with. This is a truly outstanding result, and I feel its impact is somewhat understated in the current text. A more direct comparison would better highlight the transformative power of this work for the structural biology community.\n3. Claims\nI would be careful with claims about \"living cells.\" Vitrified cells are preserved in a near-native state but are not metabolically active. The fact that protein crystals are grown in living cells is spectacular, but for technical accuracy during data collection, simply saying ‘in cells’ or ‘in situ’ is sufficient.\nClaims that the crystal structure is more physiological should be made with care. HEX-1 is a key component of Woronin bodies in filamentous fungi. Here, these crystals are grown inside insect cells, a heterologous host. A statement in the discussion could address this as a known limitation and a fascinating area for future research, such as comparing structures from different expression systems.\n4. Clarity\nThe abstract reads more like a brief introduction. The results are strong enough to speak for themselves. Focusing the abstract more on the quantitative achievements would be more appealing to a broad audience.\nThe authors alternate between \"3D ED,\" \"MicroED,\" and \"3D ED/MicroED.\" I do not care what you use, but choosing one and using it consistently throughout the manuscript would improve clarity.\nThe manuscript could benefit from a brief discussion on generalizability. HEX-1 and its homologues might be a \"best-case scenario.” Acknowledging that the pipeline's performance on more challenging targets remains an open question would add valuable perspective.\nMinor note. I don’t have an unawkward way of bringing this up, but ‘InCellED’ is likely to be heard as ‘incel ED’ in English. You might consider an alternative to avoid an unintended association. This is editorial and does not affect my scientific assessment.\nI hope the authors find our feedback useful, and I look forward to seeing their work in print.\nBest,\n-Mike Martynowycz"}, "author_response": "Reply to reviewers’ comments\nWe are grateful to the referees for their exceptionally thorough and thoughtful comments. In response, we\nrevised several parts of the manuscript. Our replies are inserted in blue into the referees’ comments below.\nReviewer #4 noted that the abbreviation “InCellED” could have unintended associations in English, and we\nchanged the name to “IncelluloED”. This name is used throughout the manuscript and in our responses below.\nReviewer #1:\nIn this manuscript, Redecke and co-workers present an adaptation of the InCellCryst concept, (i.e. a pipeline\nbased on crystallization directly within living insect cells to produce a sufficient amount of homogeneous\nmicrocrystals of a target protein for structure determination by serial X-ray, thereby avoiding the purification\nstep), to electron diffraction (InCellED), a technique that is gaining momentum but is still in its infancy in the\ncontext of protein crystallography.\nThey chose HEX-1 from Magnaporthe grisea (MgHEX-1) crystals, grown in insect cells to preserve their\nnative state, to demonstrate that electron diffraction data collected from a single crystal can yield enough\ninformation to determine the previously unknown 3D structure at 1.9 Å resolution. They also compare these\nresults with a similar dataset obtained by serial X-ray crystallography, which used tens of thousands of crystals\nand reached a resolution limit of 1.8 Å. Because electron diffraction requires far fewer cell-containing crystals,\nthis method could help overcome the bottleneck of low efficiency in in cellulo crystal growth. Nevertheless,\nsample production, preparation, and adaptation for 3D microED data collection require specialized know-how,\nwhich is still under development.\nRESPONSE: This is correct, and our present work contributes directly to building the requisite knowledge\nbase. IncelluloED provides a direct means to monitor and analyse intracellular crystallization processes right\nthere where the crystallization occurs. Such studies could offer insights into fundamental aspects, including\nthe mechanisms of in cellulo crystallization, the fraction of proteins that crystallize in cells, and the proportion\nof crystals that diffract. While the methodology requires skilled personnel and specialist knowledge, the\nIncelluloED pipeline is robust, and no crystals were lost during our studies. To emphasize this point, we added\nan additional comment on page 9 (first paragraph) regarding the performance of the method: “During the\ndevelopment and optimization steps of the IncelluloED sample preparation workflow, we used 14 cells\ncontaining MgHEX-1 microcrystals, selected by RL/FL imaging. All crystals were successfully milled into\nlamellae, and all lamellae provided high-resolution ED signals.”\nThe chosen system, MgHEX-1, is consistent with their previously characterized Neurospora crassa HEX-1\n(NcHEX-1), since both naturally self-assemble into a hexagonal crystalline material. The new MgHEX-1\nstructure could shed light on the mechanism underlying the self-assembly process that drives cellular stress\nbut further analysis seems to be need to find the mechanism behind its formation naturally.\nmicroscopy (SEM) to adjust sample thickness after locating crystals within the cell using fluorescence (FL)\nmicroscopy, as well as cryo-light microscopy and correlative light–electron microscopy. The sample\npreparation process is well described; however, fine details (beyond the scope of the present work) are not\nprovided, and reproducibility may be challenging even for experienced technicians.\nRESPONSE: While the methodology requires skilled personnel and specialist knowledge, the IncelluloED\npipeline is robust, and we lost no crystals during our studies. Following the advice of Referee #2, we introduced\na graphical abstract in the Introduction section (new Figure 1) to enhance clarity and accessibility. We also\nexpanded and restructured the Results section with a detailed description of the procedures under the heading\n“Workflow of in cellulo crystal preparation for ED experiments” on pages 6-7 to highlight the key steps of\nsample preparation and the expected results. The Methods section was also restructured accordingly (pages\n17-21). We believe these changes will facilitate the adoption of the IncelluloED method by new users.\nOverall, the manuscript is well written, and the methodology, its main focus, is clearly explained, even from\nthe perspective of a non-expert in electron diffraction. Therefore, I believe this article will be significant for\nthe structural biology community, although I have one major concern regarding its framing.\nMajor concerns:\nAlthough this approach for InCellED clearly has high potential and could become a viable alternative\ntechnique, the data presented in the manuscript do not allow one to envision its general applicability.\nLooking at the authors’ own previous work in this journal, it is evident that they have several other systems\navailable that could be used to showcase the broader potential of this method/pipeline. The current study\ndemonstrates that a single crystal can yield a complete high-resolution dataset; however, to properly frame the\nmethodology’s potential, other lower-symmetry samples should be analyzed and presented.\nTherefore, I recommend a major revision to include a broader set of systems, at least two more, that clearly\nrepresent a variety of potential scenarios, such as low-symmetry crystals requiring the collection and merging\nof multiple datasets, as well as crystals of different morphologies.\nRESPONSE: The aim of this paper is to demonstrate that it is possible to obtain a high-resolution structure\nfrom a single protein crystal within a single cell, using the new IncelluloED pipeline. Our results show that in\ncellulo electron crystallography can be performed many orders of magnitude more efficiently than synchrotron\nserial X-ray crystallography with the help of the IncelluloED pipeline. For quantitative data on efficiency see\nthe end of para 3, page 12 and Supplementary Text 1.\nWe agree with Reviewers #1 and #4 that the generalisability and limitations of the method should be discussed\nmore explicitly. In response, we have substantially revised the Discussion section. On page 12, para 2 we\ndiscuss heterologous protein expression, a point raised by Referee #4. On page 13, para 1, we describe data\ncollection strategies for low symmetry crystals (point raised by Referee #1). We explain that “For all space\ngroups except triclinic P1, a 90° wedge can achieve >90% completeness, desired for reliable structure\n2\nrefinement and model building60, provided the crystal is favourably oriented59.” We discuss options to improve\ncompleteness either by manually rotating the frozen sample grid in the cryo-FIB/SEM holder prior to milling\nto bring crystals into a favorable orientation based on their morphology and orientation on the frozen grid\n(which can be observed by cryo-light microscopy), or by using a dual-axis (double-tilt) cryo-holder. P1 crystals\nare special and require 180° rotation of the sample grid, and this is not possible in cryo-TEMs due to practical\nconstraints like increased lamella thickness at high tilts and obstruction by grid bars (Supplementary Figure\n2), which restrict the usable tilt range to about 110°–120°, despite nominal hardware limits of −70° to +70°.\nIf high completeness cannot be achieved easily, data from multiple crystals can be merged, as is common\npractice in X-ray and electron crystallography. All these points are incorporated into the revised Discussion\nsection with appropriate references.\nMinor concerns:\nBefore the discussion, the authors state that the level of biologically relevant information obtained by the novel\nInCellED approach is comparable to that obtained by the well-established SSX. While this may be true, it is\nalso somewhat obvious, its validity will always depend on the resolution limit, and it has already been shown\nin previous studies using microED. I suggest rephrasing this point to emphasize instead that InCellED provides\nan additional tool in the methodological toolbox, one that could be advantageous in situations where other\ntechniques are more demanding.\nRESPONSE: We introduced the following sentence on page 11, para 3 to address this concern: “IncelluloED\nis an additional tool in the methodological toolbox, one that could be advantageous in situations where other\ntechniques are more demanding.”\nAlso, in line with my earlier comments, the discussion includes the statement: “In this study, we established\nand validated InCellED, a workflow for high-resolution structure determination by 3D ED using only a single\nintracellular protein crystal in the micrometer size range.” The present results only demonstrate that the\napproach is possible. To substantiate the claim that it constitutes a workflow pipeline, it would need to be\nshown that it can be successfully applied to multiple systems.\nRESPONSE: We agree. We only demonstrate here that the approach is possible and produces excellent results\non MgHEX-1 crystals in insect cells. Para 1 of the newly rewritten Discussion section states “We introduced\nand validated the IncelluloED workflow for high-resolution structure determination of intracellularly\ncrystallized recombinant proteins, using 3D electron diffraction from a single crystal inside a single cell. As a\nproof of concept, we determined the previously unknown structure of the HEX-1 protein from Magnaporthe\ngrisea, selected as a pilot target. The IncelluloED pipeline achieved comparable resolution and identical\nstructural information to serial synchrotron X-ray crystallography (SSX) while reducing the required sample\nvolume by seven orders of magnitude to about 1 µm3, and potentially less. Its performance on other targets\nremains to be established, and experimental validation across a broader range of proteins will be the focus of\nfuture work.” (page 12)\nIn this context, it may perhaps be appropriate - though excessive - to invoke Newton’s advice: “To explain all\nnature is too difficult a task for any one man or even for any one age. ’Tis much better to do a little with\n3\ncertainty & leave the rest for others that come after than to explain all things by conjecture without making\nsure of any thing.” (Isaac Newton, The Correspondence of Isaac Newton. Edited by H. W. Turnbull, vol. 5,\nCambridge University Press, 1961. Draft of a letter to Roger Cotes, June 1712).\nReviewer #2:\nThe manuscript titled “In situ protein crystallography: a single-crystal electron diffraction pipeline for structure\ndetermination inside living cells” by Bílá, Pinkas, Khakurel et al. presents a novel and technically significant\napproach for determining protein structures within living cells using micro-electron diffraction (microED).\nThis work represents a valuable contribution not only to the field of in vivo crystallization, but also to the\nbroader electron diffraction and structural biology communities. This robust in situ approach offers a powerful\nplatform for advancing structural studies of proteins in their native cellular milieu.\nThat said, the study is innovative and of high potential impact. While the manuscript is generally well written\nand the methodology is clearly described, I recommend a number of minor revisions to further strengthen its\nclarity and presentation. My comments and suggestions listed below are intended to support the authors in\nrefining the manuscript and ensuring its readiness for further publication in Nature Communications.\nIntroduction\n-->The referee recommends including a graphical abstract in the Introduction section to enhance clarity and\naccessibility. If possible, the authors may consider repositioning Figure 2 as Figure 1 and integrating it directly\ninto this section to provide an early visual overview of the workflow or methodology.\nRESPONSE: We fully agree, and we added a graphical abstract to the Introduction section (new Figure 1) to\nimprove clarity and accessibility. We also expanded and reorganised the Results section, adding a detailed\ndescription of the procedures: “Workflow of in cellulo crystal preparation for ED experiments” (pages 6–7)\nto highlight the key steps of sample preparation and the expected outcomes. The Methods section has also\nbeen restructured accordingly (see the very detailed subsection on “In cellulo crystal preparation for ED\nexperiments” on pages 17-21). We believe these changes will facilitate the adoption of the IncelluloED method\nby new users.\nResults\nIntracellular crystallization of MgHEX-1\nLine 22: ‘The crystallization efficiency and low-micrometre size range position HEX-1 crystals as ideal\ncandidates for InCellED method development’\n--> The referee recommends that the authors specify the expected size range of the HEX-1 crystals and provide\nan appropriate citation to support this, referencing prior studies where applicable. Quantitative detail would\nstrengthen this statement and contextualize it within existing literature.\nRESPONSE: We agree and we added the requisite information on page 5 (first paragraph) including\nappropriate citations: “A Neurospora crassa HEX-1 (NcHEX-1) was found to produce regular, hexagonal\n4\ncrystals with average dimensions of 9.1 ± 3.2 µm (± standard deviation) in length and 3.5 ± 0.7 µm in width\nwhen overexpressed in insect cells using the recombinant baculovirus (rBV) system8,38.”\nIn cellulo crystal preparation for ED experiments\n--> The referee notes that the novelty of the sample preparation method as described is somewhat limited.\nUpon further examination of the existing literature, similar sample preparation workflows have already been\nreported for related systems. The primary difference in this study appears to be the use of entire cells as\nsamples, rather than isolated crystals. Given the close methodological overlap, the referee suggests that this\nsection could be merged with the previous one, to streamline the presentation and avoid redundancy.\nRESPONSE: As mentioned above, we rewrote the section “Workflow of in cellulo crystal preparation for ED\nexperiments” (pages 6-7) in the Results section in a way that its subsections follow the schematic overview in\nthe new Figure 1. In addition, there is now a subsection “Cell collection and freezing on TEM grids” (page 6)\nwhich is introduced to address the point of the referee.\nAdditionally, the authors are encouraged to indicate whether alternative grid types were tested during the\nsample preparation phase. Comparing results across different grids might improve sample distribution and\nprovide further methodological insight.\nRESPONSE: There were no systematic comparisons of different grid types. We reported the observation in\nthe section “In cellulo crystal preparation for ED experiments” of Methods that holey gold-film grids\n(UltrAuFoil® R2/2, 200 gold mesh, Quantifoil Micro Tools) performed well, and the gold film appeared to be\nmore sturdy than the holey carbon film (QUANTIFOIL®, Quantifoil Micro Tools), and we continued using\nthe gold grids. To avoid implying generality, we added the phrase “in our hands” to the relevant sentence (page\n18, para 1) to indicate that this is a practical advice rather than a systematic conclusion.\nElectron diffraction data collection.\n-->The referee is interested to know whether, in addition to the previously reported NcHEX-1 structure, the\nauthors have considered using an AlphaFold-predicted model for molecular replacement. Including such an\napproach could offer an informative structural comparison between the in situ HEX-1 structure, the previously\npublished NcHEX-1, and the AlphaFold model. If feasible, the authors are encouraged to present RMSD\nvalues among the three structures, ideally in a tabulated form or as an additional figure, to quantitatively assess\nstructural similarities and deviations.\nRESPONSE: This is an interesting suggestion and yes, we calculated an Alphafold 3 prediction for the\nMgHEX-1 structure and used it for molecular replacement trials, and it worked well. However, it turned out\nthat the subsequent refinement went faster in our hands when we used the experimental NcHEX-1 (PDB:\n1KHI) structure as a search model. The structural comparison of the predicted MgHEX-1 AlphaFold model\nand our experimental 3D ED microvolume and the serial X-ray structures shows highly comparable protein\nfolds with only minor deviations, characterized by RMSD values of in the range of 1 Å. This is expected,\nbecause HEX-1 proteins are comparatively small (molecular mass around 20 kDa), and they are well-folded\nproteins closely related to the NcHEX-1 structure, already deposited in PDB and thus part of the training data\nset for AlphaFold 3.\ncomparison of our experimental MgHEX-1 structures with the AlphaFold 3 model in the new Supplementary\nFigure 10. The requested RMSD values are now presented in the legend of this figure: “The Cα atoms of the\nAlphaFold 3 model show an average RMSD of 1.17 Å and of 1.09 Å relative to the equivalent atoms of the\n3D ED microvolume and the SSX structure, respectively, indicating no significant structural differences.” We\nalso added a sentence to Results (page 10, para 2): “Similar RMSD values (about 1 Å) were obtained by\ncomparing the Cɑ atom positions of the 3D ED microvolume and the SSX structures with that of a predicted\nAlphaFold 3 model49 for MgHEX-1 (Supplementary Fig. 10).”\nFurthermore, the referee suggests that the authors address the potential issue of radiation damage, particularly\ngiven the use of electron diffraction and the presumed sensitivity of in cellulo samples. A brief discussion—\nsupported by any available data or observations—on how radiation damage was minimized or evaluated during\ndata collection would be helpful.\nRESPONSE: We have added extensive information on radiation damage to the paper. This includes\nquantitative data in Results on pages 7-8 coupled with new Supplementary Figure 8 and its legend, and\ndescription of radiation damage experiments in Methods (first paragraph in the section of “ED data collection,\ndata processing and structure determination”; page 21). The new Discussion section gives further information\non radiation damage (page 13, para 2).\nLastly, the authors are requested to specify the angular wedge (tilt range) used during collection of diffraction\ndata. Since small angular wedges at elevated fluence from multiple crystals can help enhance high-resolution\nsignal.\nRESPONSE: We specified the tilt range in Table 1.\nSerial X-ray diffraction data collection, data processing and structure determination & Discussion\n-->The referee would be particularly interested to know whether the authors have considered, or have an\nopinion on, the use of a serial data collection approach in electron diffraction. This strategy would be highly\nrelevant in the context of minimizing radiation damage, especially when using high-fluence exposure on single\ncrystals or cells without rotation.\nIncorporating such an approach (e.g., serialED) could offer several advantages, including enhanced data\nquality and a more systematic evaluation of beam-induced damage. Furthermore, if such datasets are or\nbecome available, it would be valuable to perform comparative analyses between different collection modes—\nfor example, 3DED vs. serialED—to quantitatively assess radiation effects. Similarly, comparisons could be\nmade to explore vitrification-related artifacts, analogous to what's studied in ED-SSX workflows.\nWhile the referee understands that implementing this methodology may require additional experiments and\nspecific instrumentation, it would be highly appreciated if the authors could address these possibilities in the\nDiscussion section, and provide a brief outlook or commentary on the feasibility and potential impact of such\napproaches.\n“SerialED” by Bücker et al. [Nat Commun 11, 996 (2020), https://doi.org/10.1038/s41467-020-14793-0]. A\nrecent bioRxiv preprint by Gallagher-Jones et al. [bioRxiv (2025), https://doi.org/10.1101/2025.06.24.661318]\nshows that high-resolution protein structures can be obtained using more that ten thousand crystals grown in\ncellulo and then extracted out of the cells for data collection in a cryo-TEM. Serial crystallography gives\nstructures with extremely low-doses (page 12, paras 1 and 3). The main problem is that the efficiency of\nintracellular crystallization can be very low (frequently less than 0.1%) and this limits the use of serial\ncrystallography with intracellularly grown crystals in general. Another limiting factor is that during the\nextraction process, conditions change and small-molecule ligands may diffuse out of microcrystals. Merging\ndata from thousands of crystals can obscure heterogeneity, mask twinning, impose artificial symmetry, and\nsmoothen out conformational variations, dynamics, and rare states (page 12, paragraph 3). For SerialED,\ncrystals must be sufficiently thin and the grids well blotted to ensure adequate electron transmission during\ncryo-TEM ED experiments. In contrast, IncelluloED allows optimization of crystal thickness through cryo-\nFIB milling and provides high-resolution structures with the same level of structural detail as serial\nsynchrotron crystallography, while using a seven-million-fold smaller sample volume and a remarkably low\ndose. An ~80° data wedge from a MgHEX-1 crystal required only ~1.1 MGy for a structure at 1.9 Å resolution,\nand this is far from the generally observed dose limits for high-resolution studies by electron diffraction\n(Supplementary Figure 8; Table 1; new Discussion on page 13, para 2 with refs. therein).\nSingle-crystal diffraction methods such as IncelluloED offer additional possibilities for radiation-damage\ncontrol and dose reduction, compared with serial crystallography. Instead of using a large number of crystals\nand taking still or small-rotation exposures, a strategy based on an idea described in Berglund et al. [Nature\n417, 463–468 (2002), https://doi.org/10.1038/417463a] uses 3D data sets from a small number of crystals,\nabout a dozen or so. This number is in the ball park of the daily production capabilities of cryo-FIB lamella\nmilling with current technologies (page 14, para 2). Data collection from each lamella by electron diffraction\nwould start at systematically offset crystal orientations as illustrated in Figure 3a of the Berglund et al. paper,\nand proceed until full 3D data sets are recorded from all lamellae. Note that the method also works with a\nsomewhat larger number of randomly oriented crystals. By selecting and combining defined sections of each\n3D data set, the method redistributes the absorbed dose to create a number of “composite 3D data sets,” from\nwhich a number of “composite structures” can be calculated each at a different dose level. At the low-dose\nend, 3D structures with doses in the kGy range can be obtained, while the high-dose end shows structures with\nseveral MGy doses. Each “composite structure” represents a frame from a damage movie, showing radiation-\ninduced changes in the structure as a function of the absorbed dose.\nThe method is flexible, and key parameters can be changed. This includes choosing the number of lamellae to\nbe used, the total dose a lamella receives, and the angular range used for collecting the individual data sets.\nBy reducing the angular range, “small-wedge” data acquisition can be done, and by going further, a series of\nstill diffraction patterns can be recorded. A fascinating aspect of this method is that most of these operations\ncan be performed computationally after data collection, provided that enough data are available. For instance,\nposthumous resolution extension is possible from the 3D data sets by selecting and combining those data\nwedges where the signal-to-noise ratios are the highest, the relative diffraction intensity changes the smallest,\nthe unit cell dimensions remain unchanged, and data statistic parameters such as CC and CC* are optimal.\n1/2\nReviewer #3:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nRemarks to the Author:\nThe authors’ exploration of an alternative method, MicroED/3D ED, for the direct analysis of protein crystals\ngrown in cells represents a fascinating and valuable contribution. I believe that the MicroED approach is\nparticularly advantageous in cases where obtaining large protein crystals is challenging. The authors have also\nappropriately discussed the limitations of their study and proposed possible directions for future work.\nOne question for consideration: are these crystals separable from the cells? If so, such separation might enable\nfuture crystallization through seeding, which could be beneficial in cases where cell preparation is not feasible\nfor repetitive processes.\nRESPONSE: We thank the referee for this suggestion. It depends on the individual protein if intracellular\ncrystals can be isolated from cells. Some proteins form highly stable crystals, others form crystals that\nimmediately dissolve after cell lysis. MgHEX-1 crystals can be isolated, but a change of the environment might\nimpact the crystal quality, even if it is not directly visible. Thus, we highly recommend not to isolate crystals\nthat have been grown in a cell for diffraction. However, we agree that seeding with isolated fragments of these\ncrystals can be beneficial for conventional crystallization of proteins in vitro. Such experiments are currently\nongoing.\nHowever, I believe the manuscript would benefit from addressing the following points:\n1. Abstract focus – The abstract primarily emphasizes the importance of this work and its contribution to\nthe field of in-cell protein crystallography, rather than highlighting the key findings and specific results of this\nstudy. Consider revising to give more weight to the main achievements.\nRESPONSE: We agree. We revised the abstract accordingly, also considering the suggestions of Reviewer #4.\n2. Abbreviations and materials – The large number of abbreviations and parenthetical material sources\nwithin the main text disrupts the reading flow. Moving abbreviations to a dedicated “Abbreviations” section\nand listing material sources in the “Methods” section would improve clarity.\nRESPONSE: We have substantially revised the Introduction and Discussion, and improved other sections as\nwell, using abbreviations less frequently to enhance readability and flow. We believe these changes improve\nthe clarity of the manuscript. We would prefer to leave the decision on including an abbreviation list and\n8\ndetailed source listings to the editor, as this practice does not appear to be consistent with most papers\npublished in the journal.\n3. Clarifications in results:\no Line 102: “MgHEX-1” appears to be the protein code but is placed directly after the fungus name\nMagnaporthe grisea. This placement may be confusing. o Line 114: In the results section, “MgHEX-1” is\nplaced next to the approach name— please confirm this usage is correct.\nRESPONSE: MgHEX-1 is indeed the abbreviation for “HEX-1 protein from [the filamentous fungus]\nMagnaporthe grisea”. Thus, the placement of the abbreviation (MgHEX-1) right after the name of the fungus\nis correct in our opinion. On page 5, para 1, the following sentence “To establish the IncelluloED approach,\nMgHEX-1, a structurally uncharacterized HEX-1 protein derived from the filamentous fungus M. grisea, has\nbeen selected. ” is correct, since the approach name and the protein abbreviation are separated by a comma.\no Line 127: The text states that three crystals per cell were identified; however, Supplementary Fig. 1 shows\nonly one crystal per cell. Please reconcile this discrepancy.\nRESPONSE: Text from page 5, para 2: “Applying the InCellCryst approach, UP TO three hexagonal-shaped\nstructures per cell were detected by light microscopy…”. This means that we have cells with none, one, two,\nor three crystals inside. We have not seen more. See also Figure 2 (previously Figure 1).\n4. Line 177 – The sentence “ED datasets were characterized by highly similar data processing statistics,\nand one of them was selected…” lacks details on what statistical criteria were used. A reference or specific\nvalues would improve clarity.\nRESPONSE: We agree with the referee. To address this, we included the text “These two microvolume ED\ndata sets gave very similar data processing statistics (Supplementary Table 1).” in page 8, para 2, and the\nnew Supplementary Table 1 was added into Supplementary Information.\n5. Lines 175 and 185 – Two different fluences (0.3 and 0.6 e⁻/Å²) were used for similar experiments\ninvolving different crystal volumes. The rationale for selecting these different fluences should be explained.\nRESPONSE: We added the following sentence on page 8, para 3: “Three-dimensional ED data were collected\nfrom one lamella at a total fluence of ~0.6 e−/Å2, which was double of the fluence used for the microvolume\nED data collection to compensate for the reduced sample volume.”\n6. Line 224 – RMSD values are mentioned here but are not referenced in the “3D ED Structure” section.\nConsider cross-referencing for consistency.\nRESPONSE: We included the RMSD values of the 3D ED structures to the MR search model in the\ncorresponding sentence at page 10, para 1: “The calculated RMSD of 0.520 Å compared to the 134 equivalent\nCα atoms of the MR search model is increased compared to that of 0.341 Å and 0.340 Å calculated for the\n9\nmicro- and nano-volume 3D ED structures.” These values were already presented in the “3D ED structure of\nMgHEX-1” section of the Results.\n7. Sentence length – Some sentences, such as the one on line 224, are overly long and difficult to follow.\nBreaking them into shorter sentences would improve readability.\nRESPONSE: We agree that some sentences might be a bit too long and thus difficult to understand. We revised\nthe manuscript in this context. For example, we split the sentence mentioned by the reviewer into two\nsentences.\n8. Gold vs. carbon film grids – Line 406: When stating that gold film grids have better mechanical stability,\nit would be valuable to note whether any comparative data were collected using carbon film grids and, if so,\nwhether any differences in data quality were observed.\nRESPONSE: There were no systematic comparisons of different grid types. The holey gold-film grids\n(UltrAuFoil® R2/2, 200 gold mesh, Quantifoil Micro Tools) performed well in our hands and the gold film\nappeared to be more sturdy than the holey carbon film (QUANTIFOIL®, Quantifoil Micro Tools), and we so\ncontinued using the gold grids (page 18, para 1).See also reply to Referee #2 about the same subject.\n9. Lines 410 and 412 – The expressions “keeping the sample at <-170°C” and “while being kept at <-\n153°C” should be revised to “kept below” or “maintained below” for clarity and grammatical correctness.\nRESPONSE: We incorporated the changes as suggested.\nOverall, this is a strong piece of work with significant potential impact, and addressing these points would\nfurther strengthen the manuscript’s clarity and scientific rigor.\nReviewer #4:\nThe manuscript “In Situ protein crystallography: a single crystal electron diffraction pipeline for structure\ndetermination inside living cells,” by Bílá et al. provides a thorough description of a pipeline for determining\ncrystal structures of proteins grown inside of cells. This process builds upon the InCell Cryst pipeline\npreviously established for serial X-ray crystallography. The authors use the homologous MgHEX-1 protein to\ndemonstrate the process, whereas the original X-ray work had used NcHEX-1. The critical advancement\npresented here is that the structure could be determined using a single crystal inside a cell, greatly reducing\nthe number of cells and crystals required. The total illuminated crystalline volume between the MicroED and\nserial X-ray methods is orders of magnitude different, a clear advantage. Although the 1.8 Å resolution\nobtained by serial synchrotron radiation was slightly higher than the 1.9 Å from MicroED, this served as an\nexcellent validation of the method's accuracy. It was also demonstrated that single diffraction patterns from\nthese crystals could reach resolutions comparable to those obtained by SSX. I believe this paper serves two\nfunctions: it broadens the applicability of MicroED to a challenging set of targets, and it demonstrates a\n10\nworkflow for routinely approaching such targets. As such, I believe this work is of high significance to the\ncommunity and should be accepted for publication.\nHowever, I believe there are several improvements the authors could make that would make the work more\napproachable, factually accurate, and appealing to those looking to solve protein structures inside of cells.\n1. Context/Editorial\nThe largest criticism I have of this work is not technical, it is editorial. The authors relegate a foundational\nearlier work from Yang et al. to a single sentence in the discussion. Since planting a flag as the ‘first’ has little\nbearing on the quality of this excellent work, the authors should address the Yang et al. study in the\nintroduction. Their work is about using MicroED to solve protein crystal structures from proteins expressed\nand crystallized in cellulo. The work by Yang et al. used the same core techniques (cryo-FIB milling and\nMicroED) to determine the structures of proteins that naturally crystallize in human cells. Critically, Yang et\nal. also correlated their structural data with cryo-electron tomography (cryo-ET), allowing them to map the\ncrystal's 3D location and interactions within the unperturbed cellular landscape. The \"InCellED\" pipeline, by\ncontrast, is a pure structure-determination method.\nProperly discussing this does not damage the current paper; rather, it strengthens it by placing it in the context\nof other major advances (e.g., milling viscous LCP to study membrane proteins). It would demonstrate to the\nreader that these in situ approaches are maturing rapidly and have diverse, powerful applications. The authors\ncould also note that cryo-ET could be readily integrated into their workflow if desired.\nRESPONSE: We agree with the referee. To address the points, we have significantly revised the whole\nIntroduction. We added a paragraph on page 4 to highlight the work of Yang et al.[In situ crystalline structure\nof the human eosinophil major basic protein-1. bioRxiv (2024), https://doi.org/10.1101/2024.10.09.617336 -\nref. #25]: “The possibility of collecting ED data sets from multiple intracellular crystals in frozen cells was\nrecently demonstrated for the major basic protein-1 (MBP-1)25, which naturally forms numerous nanocrystals\nin the cytoplasmic secretory granules of eosinophils. The study also showed that combining cryo-electron\ntomography26 with 3D ED enables analysis of physiologically relevant structural changes associated with the\nrelease of MBP-1 in situ and on multiple length scales, from molecular structure to nanometer-scale changes\nin the crystal packing during MBP-1 release.”\nMilling viscous lipidic cubic phase (LCP) samples to study membrane proteins was first introduced by some\nof the authors of the present manuscript [Polovinkin et al. Demonstration of electron diffraction from\nmembrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling\nat cryogenic temperatures. J. Appl. Cryst. 53, 1416–1424 (2020);\nhttps://doi.org/10.1107/S1600576720013096]. This work served as an important stimulus for the development\nof the IncelluloED concept. We cite this paper [ref. #31] on page 6, paragraph 1: \"Building upon the\ndevelopment of cryo-FIB milling procedures for crystals31,33,41 and also on the idea of using 3D negative\nfluorescence imaging for accurate 3D localization of target crystals inside a cryo-FIB/SEM microscope, we\nestablished the protocol for ED sample preparation. \"\n2. Emphasis on the Single-Crystal Advantage\nI encourage the authors to more forcefully emphasize the profound advantages of their single-crystal approach.\nmonumental achievement, especially when contrasted with the >60,000 crystals required for the SSX\nexperiment. The manuscript could be strengthened by explicitly discussing the significant drawbacks inherent\nto merging such a vast number of microcrystals. The risks of polymorphism, the averaging of partial\noccupancies that can obscure important structural details, low hit rates, and the immense challenges in data\nprocessing and scaling, etc. Their approach bypasses these issues by providing a \"clean\" dataset from a single,\nhomogenous crystalline entity. This is easy to process and deal with. This is a truly outstanding result, and I\nfeel its impact is somewhat understated in the current text. A more direct comparison would better highlight\nthe transformative power of this work for the structural biology community.\nRESPONSE: We absolutely agree with the referee. We have revised the entire Discussion to underline the\nsingle-crystal \"advantage\" of the IncelluloED approach and to address related points from other reviewers,\nwhile also ensuring the Discussion section remains concise.\n3. Claims\nI would be careful with claims about \"living cells.\" Vitrified cells are preserved in a near-native state but are\nnot metabolically active. The fact that protein crystals are grown in living cells is spectacular, but for technical\naccuracy during data collection, simply saying ‘in cells’ or ‘in situ’ is sufficient.\nRESPONSE: The referee is right. We were using cells that were alive at the point of freezing for lamella\npreparation. We changed the text as suggested by the referee. We also changed the title of the manuscript from\n“In situ protein crystallography: a single-crystal electron diffraction pipeline for structure determination inside\nliving cells” to “In situ protein crystallography: a single-crystal electron diffraction pipeline for direct structure\ndetermination inside cells”.\nClaims that the crystal structure is more physiological should be made with care.\nRESPONSE: We agree that claims that a structure from an intracellular crystal is physiological should be\nmade with care. However, our manuscript does not contain such a claim. We state at the beginning of the\nIntroduction (first paragraph): “In certain cases, the quasi-native cellular environment enables binding and\nidentification of physiologically relevant ligands and cofactors2,3, which AI-based structure prediction cannot\nyet replicate.” This is a statement that refers to our previous work, where we could clearly show that cofactors\npresent in the cellular environment are bound to the enzymes and are visible in the electron density maps after\nstructure determination. And since the native environment of a cell contains the entire set of cofactors, there\nis a selection for the one with highest binding affinity, which is usually the physiologically relevant cofactor\nor inhibitor. We do not state that the crystal structure is more physiological, only that the cellular environment\nprovides the opportunity for cofactor selection and binding at near-physiological effector concentrations.\nHEX-1 is a key component of Woronin bodies in filamentous fungi. Here, these crystals are grown inside\ninsect cells, a heterologous host. A statement in the discussion could address this as a known limitation and a\nfascinating area for future research, such as comparing structures from different expression systems.\nbroadly applicable IncelluloED will be. We studied crystals of a fungal protein grown inside an insect cell, a\nheterologous host to HEX-1. Heterologous expression can present challenges, but an analysis of proteins in\nthe PDB with identical sequences produced in native versus recombinant hosts found no cases where the same\nsequence adopted a significantly different global fold50. Post-translational modifications, host-specific\nchaperones, or the assembly of multi-component complexes may affect applicability and will need to be\nexplored.”\n4. Clarity\nThe abstract reads more like a brief introduction. The results are strong enough to speak for themselves.\nFocusing the abstract more on the quantitative achievements would be more appealing to a broad audience.\nRESPONSE: We agree and improved the abstract accordingly.\nABSTRACT: Intracellular crystallisation is an emerging approach in structural biology that bypasses the need\nfor protein purification. In 2024, the InCellCryst pipeline was introduced for structural studies of intracellular\ncrystals by serial X-ray crystallography. Serial crystallography requires the exposure of tens of thousands of\ncells containing intracellular crystals, precluding high-resolution studies on proteins that crystallize only in a\nfew cells. Here we introduce IncelluloED, a method that combines intracellular crystallization with in situ 3D\nelectron diffraction in cells and achieves high-resolution structures from just one crystal inside one cell.\nExperiments on a microcrystal of the HEX-1 protein from Magnaporthe protein from Magnaporthe grisea, grown inside an insect cell,\ngave a structure at 1.9 Å resolution from a volume of ~1.6 µm3 as compared to 1.8 Å resolution achieved by\nserial X-ray crystallography from a combined volume exceeding eleven million µm3. IncelluloED uses widely\navailable cryo-EM tools and brings high-resolution structural biology into home laboratories while also\nadvancing a vision for a “single-cell structural laboratory”.\nThe authors alternate between \"3D ED,\" \"MicroED,\" and \"3D ED/MicroED.\" I do not care what you use, but\nchoosing one and using it consistently throughout the manuscript would improve clarity.\nRESPONSE: We agree with the referee, and we are now using 3D ED throughout the manuscript.\nThe manuscript could benefit from a brief discussion on generalizability. HEX-1 and its homologues might be\na \"best-case scenario.” Acknowledging that the pipeline's performance on more challenging targets remains\nan open question would add valuable perspective.\nRESPONSE: We agree with the referee. This comment is similar to one from Reviewer #1. The significantly\nrevised Discussion now addresses “generalizability”. We also ensured that the Discussion section was concise.\nWe acknowledge “that the pipeline's performance on more challenging targets remains an open question”\n(page 12, para 1): “Its performance on other targets remains to be established, and experimental validation\nacross a broader range of proteins will be the focus of future work.”\nED’ in English. You might consider an alternative to avoid an unintended association. This is editorial and\ndoes not affect my scientific assessment.\nRESPONSE: We thank the referee for pointing this out and changed the name of the pipeline from InCellED\nto IncelluloED.\nI hope the authors find our feedback useful, and I look forward to seeing their work in print.\n14"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I believe the manuscript merits publication in its present form. I have no further comments or suggestions for the authors.\nI would also like to respond to the authors by quoting Albert Einstein: “The important thing is not to stop questioning. Curiosity has its own reason for existing.”\n— Albert Einstein, Life Magazine, May 2, 1955.", "reviewer_2": "I appreciate the authors’ comprehensive and well-integrated revisions. The updated manuscript reflects a clear effort to address all comments raised during the review process. The authors have provided additional data and clarifications where requested, improved the methodological description, and strengthened the interpretation of their results. Importantly, the concerns raised by all reviewers have been carefully considered and are now satisfactorily resolved in the revised version.\nIn light of these substantial improvements, I find the manuscript to be significantly strengthened and scientifically sound. I therefore support its publication in Nature Communications.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nRemarks to the Author:\nThe authors have satisfactorily addressed all of my questions and concerns and have revised the manuscript appropriately. Their exploration of MicroED/3D ED as an alternative approach for the direct analysis of protein crystals grown in cells constitutes a significant and intriguing contribution to the field.", "reviewer_4": "The authors have addressed my comments. Their edits and responses to both mine and the other reviewer's comments have resulted in a much stronger, easier to read manuscript. I look forward to seeing it in press. -MWM"}, "author_response": "Reply to reviewers’ comments\nWe are grateful to the four reviewers for their exceptionally useful and insightful comments, which\nsignificantly strengthened the paper.\nReviewer #1:\nI believe the manuscript merits publication in its present form. I have no further comments or suggestions for\nthe authors.\nI would also like to respond to the authors by quoting Albert Einstein: “The important thing is not to stop\nquestioning. Curiosity has its own reason for existing.” — Albert Einstein, Life Magazine, May 2, 1955.\nReviewer #2:\nI appreciate the authors’ comprehensive and well-integrated revisions. The updated manuscript reflects a clear\neffort to address all comments raised during the review process. The authors have provided additional data\nand clarifications where requested, improved the methodological description, and strengthened the\ninterpretation of their results. Importantly, the concerns raised by all reviewers have been carefully considered\nand are now satisfactorily resolved in the revised version.\nIn light of these substantial improvements, I find the manuscript to be significantly strengthened and\nscientifically sound. I therefore support its publication in Nature Communications.\nReviewer #3:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nRemarks to the Author:\nThe authors have satisfactorily addressed all of my questions and concerns and have revised the manuscript\nappropriately. Their exploration of MicroED/3D ED as an alternative approach for the direct analysis of\nprotein crystals grown in cells constitutes a significant and intriguing contribution to the field.\nReviewer #4:\nThe authors have addressed my comments. Their edits and responses to both mine and the other reviewer's\ncomments have resulted in a much stronger, easier to read manuscript. I look forward to seeing it in press. -\nMWM\n1"}]

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