Direct from the seed: an atomic resolution protein structure by ab initio MicroED

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: major_revision

Abstract
While purifying the seed protein crambin, we find that needles of pure protein nanocrystals form spontaneously during the drying of a simple ethanolic purification drop. These needles diffract X-rays weakly but are well-suited for microcrystal electron diffraction (MicroED). Merging data from 58 such nanocrystals yields diffraction to 0.85 Å resolution and solves the structure ab initio using a five-residue helical fragment to initiate density modification. The resulting map enables automated model building and resolves individual hydrogen atoms. This work reports an atomic resolution MicroED structure of the protein crambin solved ab initio. This study establishes a publicly available benchmark showing that sub-ångström ab initio MicroED can be achieved on standard 200 kV instrumentation without energy filtering or FIB milling, when serial merging is combined with anisotropy aware truncation to address preferred orientation. For this dataset, isotropic truncation alone did not produce a fragment placement suitable for phasing in this workflow, whereas applying anisotropy correction supported an ab initio solution.

Introduction
Crystallography, a cornerstone method for determining protein structures, has traditionally been limited by crystal size. X-ray crystallography, the gold standard for determining atomic resolution structures, requires relatively large, well-ordered crystals - typically tens to hundreds of micrometers across. Yet many proteins naturally form only extremely tiny crystals, far smaller than this threshold, leaving a significant fraction of important biological molecules structurally inaccessible. Single-particle cryo-electron microscopy (cryo-EM), another powerful approach, struggles to achieve high-resolution structures of proteins less than 100 kDa1. This limits its utility for many biologically important molecules, e.g., the median size of a human protein is only about 40 kDa2.

Microcrystal electron diffraction (MicroED) has emerged as an approach to overcome these limitations3. Instead of X-rays, MicroED uses electrons, which interact far more strongly with matter4. This interaction allows high-resolution structure determination from crystals thousands of times smaller by volume than those required for traditional X-ray methods. Although many crystals are invisible to the naked eye5 and often too small or delicate for standard crystallography, they can still yield atomic-resolution structures using MicroED. In favorable cases, direct analysis of nanocrystalline material is possible6,7.

Despite its promise, phasing protein structures by MicroED remains heavily reliant on pre-existing knowledge. Molecular replacement (MR) for MicroED, demonstrated with lysozyme in 2013–20148,9, is the workhorse method and has delivered important structures like Ca2+-ATPase10, R2lox11, MyD8812, and the human lens membrane protein MP2013. The arrival of AlphaFold214 has further expanded the reach of MR. However, this reliance on homologous models creates a fundamental limitation: it generates phase bias and leaves proteins with novel folds inaccessible. Establishing reliable ab initio phasing methods that remove this dependence is essential. While this has been routine for small-molecule electron crystallography for years6,15–17, progress for proteins has been incremental, moving from direct methods on small peptides18, to fragment-based searches19, and recently to density-modification-based bootstrapping from minimal ideal fragments20. Prior ab initio MicroED demonstrations established feasibility, and many implementations have operated in regimes that mitigate geometric incompleteness and anisotropy, for example, by reducing thickness or by using microscope and detector configurations that broaden the usable data range. Here, we ask whether an ab initio solution remains achievable on widely available 200 kV hardware without energy filtration or FIB milling, and we assess whether anisotropy-aware data treatment is a practical determinant of success in this dataset, a consideration that may be important in similarly anisotropic cases. Rather than introducing a new phasing algorithm, our goal is to provide a well-characterized, publicly available atomic resolution MicroED benchmark that complements existing lysozyme and proteinase K datasets and enables rigorous testing of electron-specific methods.

In this study, we bridge this methodological gap by using the classic benchmark protein crambin21. For decades, Crambin has served as a proving ground for the most advanced crystallographic methods. Its 1981 structure by Hendrickson and Teeter was a landmark in de novo phasing using sulfur anomalous scattering22, and it was later the protein solved by Hauptman and Weeks’ purely mathematical direct methods, a triumph developed at our home institutions23. Subsequent X-ray studies reached 0.48 Å24 and 0.54 Å25, and more recently, a 0.70 Å26 room-temperature X-ray structure provided an ultrahigh-resolution description of the crambin solvent network and disorder in the PL and SI isoforms. Furthermore, neutron diffraction has been reported to 1.1 Å resolution at room temperature27.

Despite this storied history in X-ray and neutron science, a completed de novo investigation of crambin by MicroED has remained unexplored. While a 1.07 Å MicroED structure solved by molecular replacement has been briefly described in a technical note (Rigaku Application note PX031)28, the full potential of using electrons to solve this benchmark protein from first principles has not been realized. We discovered that a simple ethanolic extraction from finely ground seeds spontaneously yields an abundant supply of nanocrystals ideal for such a test. Therefore, we set an ambitious goal: to determine if data from these naturally formed nanocrystals were of sufficient quality to not only break the sub-ångström barrier but to solve the structure ab initio from a minimal, non-homologous five-residue helical fragment29.

Here we report an electron ab initio structure of crambin at sub-ångström resolution obtained directly from spontaneously formed nanocrystals collected on standard 200 kV hardware without using a focused ion-beam to thin the crystals or an energy filter, with data merged from multiple crystals to mitigate preferred orientation. We position crambin as a reference for electron-specific methods and compare it with prior ab initio MicroED results.

Results
At a tale of two crystals: swapping fortunes in X-ray and electron diffraction

Our investigation began with a serendipitous discovery. During a simple ethanolic extraction of crushed Crambe abyssinica seeds (Supplementary Fig. S1), we found that a 1 µL droplet of the protein-rich solution nucleated a dense shower of sub-micron, needle-shaped crystals within seconds as the solvent evaporated (Fig. 1A). These crystals formed without a separate screening step. These needles proved unsuitable for X-ray diffraction. A single 14 µm needle diffracted poorly, only reaching 1.6 Å resolution and suffered from rapid radiation decay (Supplementary Fig. S2, Supplementary Table S1). In contrast, they proved ideal for MicroED. When loaded into the TEM, we noticed many tiny rod-shaped crystals on the grid (Fig. 1B). High-resolution imaging of these crystals showed clear lattice lines (Fig. 1C) that contained information to 3.6 Å by analysis of the Fourier transform of these images (Fig. 1D). These crystals consistently diffracted electrons to high resolution (Fig. 1E).

We also grew large, block-like crystals using traditional vapor diffusion (Supplementary Fig. S3). These crystals exhibited the opposite behavior: they produced excellent X-ray diffraction to beyond 0.8 Å but were too thick for MicroED. We mechanically crushed these blocks to obtain fragments for MicroED, but the crushed fragments produced markedly inferior electron diffraction data compared to the spontaneously formed needles (Supplementary Fig. S4). For electrons, the limiting dimension is thickness along the beam (below twice the inelastic mean free path, or about 500 to 600 nm)30; most crushed fragments remained too thick, increasing dynamical scattering and absorption. This system-specific outcome contrasts with reports where crushed fragments diffracted well, such as De La Cruz et al.31, where fragmentation produced abundant electron-transparent crystals; here the spontaneous needles provided higher-quality MicroED data.

This inversion of crystal quality between the two diffraction methods motivated our collection strategy. Rather than rely on non-ideal fragments to randomize orientation, we tackled the preferred orientation problem of the spontaneously formed needles directly using a serial crystallography approach. By merging data from multiple crystals, we aimed to overcome geometric data loss inherent to a single orientation and build a complete, high-redundancy dataset.

Achieving an atomic-resolution microED dataset by overcoming anisotropy

Having established the superiority of the spontaneously formed nanocrystals for MicroED experiments, we proceeded with data collection. The primary technical hurdle was, as anticipated, the severe preferred orientation of the needles on the grid32. This morphology introduces a fundamental challenge for MicroED: diffraction quality is highest at low tilt angles, where the electron beam passes through the thinnest dimension of the crystal, but degrades at high tilts as the effective crystal thickness increases30, compounding multiple-scattering and absorption effects. This inherent tilt-dependent resolution decay is a major source of anisotropy in data from plate- or needle-like crystals11.

To obtain a high-quality dataset, we merged data from 58 individual nanocrystals using a serial MicroED strategy (“Methods”: MicroED Data Collection). In practice, we treated data collection as a serial process in which many crystals were queued for low-fluence wedges and subsequently curated and merged, rather than prospectively optimizing the minimal number of crystals required for structure solution. To maximize data completeness and mitigate the missing data wedge effect inherent to single-crystal collection, we collected a 30° or 60° data wedge from each crystal. The starting angle for each wedge was varied, ensuring that data from different crystals sampled regions of the full stage-tilt range. Even with this comprehensive collection strategy, the merged dataset exhibited the expected anisotropy. A standard spherical resolution cutoff would therefore be suboptimal, either conservatively low, discarding valuable high-resolution data, or too high, including noise from the poorly ordered directions.

To produce the most physically meaningful and high-quality dataset, we addressed anisotropy explicitly. We processed the integrated intensities with the STARANISO server33, which determines the direction-dependent resolution limits and applies a corresponding elliptical truncation. This process removes the weak, high-resolution, impaired reflections from the poorly diffracting directions while preserving the strong, high-resolution data from the well-ordered directions. Principal-axis limits and statistics are reported in Supplementary Table S2; Table 1 reports ellipsoidal statistics after truncation. Our pipeline failed at the first step for both spherical cutoffs. Using the ideal five-residue polyalanine helical fragment, PHASER did not return a valid placement for either the conservative isotropic truncation at 1.432 Å or a spherical truncation to 0.85 Å. Because no fragment placement was obtained in either case, subsequent density modification and model building could not proceed. All raw datasets and the full STARANISO output for job bzQxhc_AELS71AjMa are deposited with the raw data so that alternative truncation or scaling strategies can be tested on the same underlying measurements.

The final dataset extended to 0.85 Å (Table 1, Ellipsoidal limits) and was a critical factor in producing the high signal-to-noise ratio within this well-defined resolution ellipsoid, aided by the high multiplicity obtained through serial merging (Table 1). Observed improvements are consistent with prior reports that multiplicity improves merged MicroED data34. Analysis of the data showed no obvious evidence of specific radiation damage35. No significant difference map peaks appeared at susceptible sites such as disulfide bonds or acidic residues.

Ab initio phasing and automated structure completion

A central achievement of this work was solving the structure without a homologous model. Our workflow, conceptually similar to the fragment-based Fragon36 pipeline developed for X-ray data, began with molecular replacement in PHASER37 using a generic five-residue polyalanine helix. This yielded a low-scoring solution (TFZ=7.1) with a non-interpretable potential map (Fig. 2A, Supplementary Video 1); however, these initial phases were sufficient to bootstrap the dynamic density modification algorithm in ACORN38. This fragment-based phasing approach is similar to the strategies recently used to solve the structures of lysozyme and Proteinase K by MicroED20, similar to the ARCIMBOLDO39 and SHELXE40 based workflows. This approach resolved the phase ambiguity and produced an interpretable map covering the chain (Fig. 2B, Supplementary Video 2). The density-modified map was sufficient for BUCCANEER41 to automatically build 100% of the crambin model in a single pass. The final structure was refined with the applied twin model and ellipsoidal truncation to Rwork/Rfree values of 15.54% / 16.41%, yielding a model with excellent stereochemistry (Table 1). While these values are higher than typical X-ray structures at this resolution, they are characteristic of MicroED refinements where dynamical scattering effects are not fully modeled by standard kinematic refinement20. A hydrogen- and disorder-refined model is provided in the Supplementary Information (Supplementary Files); these coordinates are not used for Table 1 statistics. The maps from REFMAC resolve all non-hydrogen atoms and many hydrogen atoms, consistent with atomic resolution (Fig. 1F, Supplementary Fig. S5).

Positioning crambin among ab initio MicroED benchmarks

We compare this result with prior ab initio MicroED structures of triclinic lysozyme and proteinase K in Supplementary Table S4, summarizing resolution, completeness, R values, phasing fragments, microscope voltage, detector mode, and FIB use. Crambin complements these benchmarks as a sub-ångström, 200 kV, no-FIB, no energy-filter case with natural isoform heterogeneity. Representative overlays of electron potential, X-ray electron density, and neutron scattering-length maps are shown in Supplementary Fig. S5, highlighting complementary features. Crambin thus provides a useful reference point across modalities for electron-specific method development.

Crystal habit and implications for MicroED

The inversion of crystal quality, where X-ray-ideal blocks failed in MicroED while X-ray-unsuitable needles excelled, is rooted in the physical limitations of electron diffraction. The primary determinant for high-quality MicroED data is crystal thickness along the beam path, ideally below twice the inelastic mean free path (about 500–600 nm)30, to minimize dynamical scattering and absorption. While the block-like crystals possessed high internal order for X-rays, mechanically crushing them did not reliably produce fragments this thin; most remained too thick, degrading the electron diffraction. The spontaneously formed needles, however, were inherently thin in one dimension. When lying flat on the grid, they presented an ideal, electron-transparent path for the beam. This favorable habit introduces a severe preferred orientation. We directly overcame this challenge by using a serial crystallography approach, merging data from 58 crystals to build the necessary completeness.

Complementary chemical information in electron potential maps

Electron potential maps differ from X-ray electron-density and neutron scattering length density maps and can be sensitive to charge; hydrogen visibility is illustrated in Supplementary Fig. S549–52. We anticipate use of these data and maps for modeling electron scattering, hydrogen visualization, and charge-density analysis in future studies. This high-quality electron-based model, when combined with existing ultra-high-resolution X-ray and neutron data, including the 0.70 Å room-temperature solvent structure of crambin, creates an opportunity for joint and cross-modality refinement that can probe hydrogen bonding, protonation, and dynamic solvent organization in a single benchmark system26.

We determined the 0.85 Å ab initio MicroED structure of crambin directly from spontaneously formed nanocrystals obtained during ethanolic extraction. Data were collected at 200 kV without focused-ion-beam milling or an energy filter, and serial merging mitigated preferred orientation. Ellipsoidal truncation addressed anisotropy (Table 1; Supplementary Table S2). Using a minimal five-residue helical fragment and density modification, we obtained phases and automatically built the complete model (Fig. 2). Mixed PL/SI isoforms at residues 22 and 25 were refined as alternates with occupancies summing to 1, supported by omit-map evidence and an additional X-ray structure (Supplementary Files). Key statistics are reported in Table 1.

Methods
Protein isolation and nanocrystal formation

Crambin was extracted from 230 g of manually de-husked and then crushed Crambe abyssinica seeds following a modified protocol53,54. The ground kernels were first defatted with hexane and then subjected to three rounds of extraction with 80% acetone in water (% v/v). After evaporation of the acetone under reduced pressure, the aqueous solution was stored at 4 °C for 18 h. This solution yielded a pale-yellow solid pellet upon centrifugation, which was redissolved in a 70% ethanol-water mixture (% v/v). During routine inspection, a 1 µL droplet of the ethanolic protein solution left to evaporate on a microscope slide rapidly nucleated a dense shower of sub-micron, needle-shaped crystals. These spontaneously formed nanocrystals were used for all MicroED experiments. The full detailed extraction protocol is available in the Supplementary Methods.

MicroED grid preparation and data collection

Holey-carbon copper grids (Quantifoil Cu300 R1.2/1.3) or lacey carbon grids (Cu200, Electron Microscopy Sciences) were glow-discharged (15 mA, 30 s). The grids were transferred to either a Leica GP2 plunge freezer or Thermo-Fisher Vitrobot, equilibrated to 4 °C and 95% relative humidity. A 3 µL aliquot of the crystalline slurry was applied, blotted for approximately 30 s, and plunge-frozen in liquid ethane. MicroED data were recorded on a Glacios TEM (200 kV, λ = 0.0251 Å) equipped with a Falcon 4 detector. Two sets of data were collected using SerialEM55,56: linear-mode datasets (0.2° per frame; 1.6 e− Å−2 total exposure) and counting-mode datasets (0.1° per frame; 0.8 e− Å−2 total exposure). Approximate absorbed dose estimates (MGy) for these datasets are given in Supplementary Table S4. This combination was used to capture robust low-resolution intensities (linear) and accurate high-resolution intensities (counting). This dual-mode strategy was designed to mitigate potential coincidence loss (CL) for intense low-resolution reflections, a known challenge for counting detectors in diffraction46. The total exposure per crystal that was used for MicroED data collection was kept below 2 e− Å−2. In both schemes, the starting angle of each wedge was incremented stepwise so that the combined datasets sample a stage tilt range of ±60°. Counting-mode and linear-mode datasets were collected on different crystals. We acquired 39 linear-mode datasets and used 30 of them in the final merged structure, and we acquired 117 counting-mode datasets and used 28 of them, after removing low-quality and non-merging datasets. While a lower number of crystals might be sufficient in an ideal case, the high redundancy from 58 crystals was critical for mitigating preferred orientation and achieving high signal-to-noise34. In practice, data collection followed a serial MicroED strategy analogous to single-particle workflows, where many crystals were queued for collection and subsequently curated and merged, rather than prospectively optimizing the minimal number of crystals required for structure solution. The low-flux conditions used here (~0.01–0.03 e−/Å²/s) are in a regime where CL has recently been confirmed to be present, albeit minimal, and can manifest as pseudo-twinning46. Our cautious approach was validated by this recent work, and the high quality of our merged dataset suggests the impact of CL was not severe in this case. A detailed summary of the datasets used for merging is provided in Supplementary Table S5.

Data processing and refinement

Raw movie frames were converted to the miniCBF format using a custom Python pipeline that performed 2×2 binning and a two-pass, smoothed beam-center correction (see Supplementary Methods for full details). The corrected images were processed with XDS (BUILD 20230630) and scaled with XSCALE. Datasets were curated with XSCALE_ISOCLUSTER prior to merging. The final merged dataset from 58 crystals was processed by the STARANISO server for direction-dependent resolution analysis and ellipsoidal truncation. Principal-axis limits and per-axis statistics are listed in Supplementary Table S2; ellipsoidal statistics are reported in Table 1. Phasing was performed using a combination of Phaser and ACORN, followed by automated model building in BUCCANEER. The final structure was refined using REFMAC5 with explicit modeling of a twin fraction. This twinning is consistent with pseudo-merohedry, though we note that coincidence loss in counting-detector data can also be registered as pseudo-twinning46. Isoform heterogeneity at residues 22 and 25 was modeled with alternate residue types (PL and SI) with occupancies constrained to sum to 1 using SHELXL. Final data collection and refinement statistics are provided in Table 1; Supplementary Table S6. Full .ins/.res/.fcf SHELX files are provided for both the X-ray and MicroED structures in Supplementary File 1 (hydrogen/disorder refinement).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Protein atomic structure is crucial across various disciplines, including microbiology, biochemistry, and pharmacy. The challenges in such research lie in the high requirements for spatial resolution and the extremely complex dynamics of proteins. This manuscript reports an important research outcome, demonstrating the potential of MicroED technology in resolving atomic-resolution protein structures. The research methods used in the article are both novel and rigorous. The manuscript is well-written and filled with passion. I would recommend the publication of this significant work in Nature Communications without hesitation, provided the following issues are addressed:\nMajor Issues:\n1. The manuscript focuses on the spontaneous formation of nanocrystals during ethanolic extraction. Have the authors explored the possibility of controlling the crystal size and morphology by varying extraction conditions (e.g., solvent type, concentration, temperature)? Such information could be valuable for optimizing MicroED experiments with other proteins. Why is needle-shaped crystal preferred compared with the block-like crystals?\n2. It is surprising to me that the crushed fragments of the bulk crystal could not preserve the internal atomic order. From Fig. S3c, I noticed that the crushed fragments still maintain a relatively large size (~100 μm), which should, in principle, preserve the structural information of the crystal.\n3. The authors should cite previous works regarding the crystallographic information of the crystal and compare them with the current study. I noticed that the reported lattice parameters in the following reference differ from those in this work. The authors should explain this discrepancy: (Ref. 42: Solvent organization in the ultrahigh-resolution crystal structure of crambin at room temperature, https://doi.org/10.1107/S2052252524007784)\n4. The crambin protein exists in two natural isomers, PL and SI, and a hybrid model with partial occupancies was successfully built. Did the authors perform further analysis and comparison of the structures and relationships of these two isomers?\nMinor Issues:\n1. Page 2, “Crystals invisible to the naked eye,”: Missing “are.”\n2. A citation should be added for this information: \"It was later the first protein solved by Hauptman and Weeks’ purely mathematical direct methods, a triumph developed at our home institutions.\"", "reviewer_2": "This study presents the first ab initio structure of crambin solved by MicroED, achieving the highest reported data quality to date in terms of resolution, completeness, and R values. As the authors note, this work establishes an important gold-standard reference for the future development of electron crystallography, including modelling electron scattering, analyzing charge density, and exploring hydrogen-bonding networks. The manuscript would be further strengthened by addressing the following points prior to publication.\n1. The authors repeatedly state that ‘Our workflow demonstrates that complex biological matrices can be mined directly for sub-ångström protein structures, establishing a practical and scalable pipeline from raw biomass to atomic-level models of previously intractable targets’. While this claim is compelling, a discussion is needed regarding the general applicability of this pipeline. Specifically, how broadly can high-resolution MicroED data be obtained directly from seeds for ab initio structure determination? For instance, is the proposed “low-tech” crystallization method generally effective for all small proteins? How frequently do crystals of suitable size and sufficient crystallinity arise for MicroED experiments using this approach? The authors should comment on these practical aspects to clarify the scope and limitations of their workflow.\n2. You note that previous ab initio structures have been obtained by MicroED for triclinic lysozyme and proteinase K (for example, see Nature Methods, DOI:10.1038/s41592-022-01485-4). It would be valuable for the authors to address how their crambin structure compares to these earlier achievements. Specifically:\nWhy might crambin serve as a more suitable gold-standard reference for advancing electron-specific methods?\nIs the choice driven primarily by superior data quality—such as finer resolution, improved completeness, better R values, or enhanced signal-to-noise?\nCould the data quality for triclinic lysozyme and proteinase K be further enhanced, and if not, what limitations prevent further improvement?\nA discussion along these lines would clarify the rationale for highlighting crambin and help frame its broader significance in the field.\n3. On lines 23–29, the authors state that “the crushed fragments produced markedly inferior electron diffraction data compared to the spontaneously formed needles.” This finding contrasts with the results reported by M. Jason de la Cruz and colleagues (Nat Methods, DOI:10.1038/nmeth.4178), who found that crushed crystals could yield high-quality diffraction.\nCould the authors elaborate on why this difference arises in the case of crambin? A comparison of methodological details and crystal behavior between crambin and other proteins would clarify why the crushed-fragment approach performed differently here and provide valuable insight into the limits of this technique.\n4. The authors applied anisotropy correction to the data using STARANISO, reporting an effective (ellipsoidal) completeness of 98.6%. It would be valuable to also provide the data statistics along the two principal axes of the ellipsoid for clarity. In addition, could the authors comment on whether ab initio structure determination would still be feasible without applying anisotropy correction, and if so, how large the differences in outcome (e.g., resolution limits, map interpretability, or refinement statistics) would be?\n5. A minor comment regarding Fig. 1E: it is not immediately clear that the highest observed reflection extends to 0.85 Å. The authors should highlight this more explicitly in the figure to aid clarity.", "reviewer_3": "Vasireddy et al. report on a method to solve the structure of proteins without a homologous model. In this opportunity, they used the spontaneously formed microcrystals of the seed protein crambin, avoiding the preparation of large size crystals for X-ray or the time-consuming FIB lamella preparation for MicroED. The ultra-high resolution (0.85Å) microED data allowed structure resolution using a five-alanine helical fragment to initiate density modification.\nGeneral comments:\nThe novelty of the report relies on being the first ab initio structure of the protein crambin determined by electron-diffraction. While validation of existing methods is valuable, the study does not introduce significant novelty or advance the field beyond confirming the applicability of ab initio phasing using minimal idealized fragments followed by density modification improvement [Martynowycz et al. Nature Methods 19, 724–729 (2022)]. The study's contribution to the field is incremental. It provides confirmatory evidence but does not present a major advance or novel insights.\nMajor Comments:\n1. Authors performed serial microED to try to reduce anisotropy. They collected on 58 crystals, each one with 30° or 60° wedge. The starting angle of each wedge was incremented stepwise so that the combined datasets sample a stage tilt range of +/- 60Å. Regarding this collection strategy:\n• 58 crystals could seem a large number of crystals, especially considering that the resulting merged data still displayed significant anisotropy. Assuming each crystal with at least 30° wedge, the stepwise increment between each crystal was ~2°, increasing the redundancy. I would like to see a discussion about the minimal number of crystals that would produce similar results.\n• Authors reported a combination of collection in linear and counting mode. What percentage of crystals were in linear mode? Previously, Martynowycz et al. collected only on counting mode with very low exposure rate. In the present report, authors combined linear and counting modes in “the hopes the low-resolution reflections in linear mode would be less prone to coincidence loss, and the high-resolution reflections in counting mode would be more accurately recorded”. What was the effect of using linear mode collection? How did data behaved if only counting mode datasets were used? What would be the recommended strategy for future projects, counting mode only or a combination of both?\n2. This report uses 0.85Å resolution data to performs molecular replacement in PHASER using a generic five-residue poly-alanine helix, and the resulting initial phases were sufficient to bootstrap the dynamic density modification algorithm in ACORN. Crambin is a protein of ~4.7kDa and 46 amino acids. While impressive, previously, Martynowycz et al. used data to 0.87Å (for lysozyme nanocrystals) and solved the structure with the same approach using a three poly-alanine fragment in PHASER followed by ACORN. The authors of the present report need to demonstrate what is novel about this work beyond what has already been published.\n3. Authors claim their map provides a complementary chemical picture of crambin to those available through X-ray or neutron techniques, highlighting that crambin is benchmark protein across crystallographic platforms. However, they did not show any comparison (figures) of those structures or the difference in the information obtained on the first structure of crambin solved by electrons.\n4. The statement at the end of the abstract ... “complex biological matrices can be mined directly for sub-ångström protein structures, establishing a practical and scalable pipeline from raw biomass to atomic-level models of previously intractable targets” it is an overstatement. It is very unlikely that will happen in general for other samples; unless the authors can prove otherwise.\n5. Comparisons with the 0.48 Å resolution structure would be important to show including overall structure, key residues.\n6. The authors should create difference maps where residues for the PI and SL isoforms are omitted from the calculation and show the map of the two possible conformations.\n7. To solve a structure by ab-initio methods the required resolution needs to be higher than 1.1 Å. This corresponds to less than 1% of all protein structures solved. Hence, it would be hard to sell it as a general method.\nMinor Comments:\n1. In Page 2, Line 31, authors wrote:\n“In subsequent years, synchrotron and neutron[23] sources pushed its resolution to an extraordinary 0.48 and 0.54 Å,[24,25] making it a key model for studying ultra-high-resolution features.”\nRef 23 reports neutron diffraction of crambin at 1.1Å. Both structures 0.48 (Ref 24) and 0.54 (Ref 25) are from X-Ray diffraction. Please rephrase the sentence to avoid confusion.\n2. The conclusion section seems largely as part of the discussion. Consider moving part of this section to the discussion section and focus on delivering a strong, shorter conclusion.", "reviewer_4": "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."}, "author_response": "We thank the reviewers and editor for their careful evaluations of our manuscript and their constructive feedback. We are pleased they found the work significant. We have addressed all points raised, which we believe has substantially strengthened the paper.\nWe note that during the revision, while addressing reviewer comments on anisotropy (R2.4), we identified an input error in our original STARANISO processing that resulted in the output statistics being reported to 0.858 Å resolution but having reflections out to 0.850 Å resolution which the final structure was refined to. This was confusing. We re-processed the entire dataset from scratch, which resulted in a model and statistics table that both extend to 0.850 Å resolutions that are essentially identical to the previous model. The conclusions remain unchanged, but all tables, figures, and statistics in the revised manuscript have been updated to reflect this.\nWe have also added three new Supplementary Tables (S2, S3, S4) and two new Supplementary Figures (S5, S6) to directly address the reviewers' points. All changes in the manuscript are marked with tracked changes with new or revised text also highlighted.\nBelow, we provide a point-by-point response to each comment.\nReviewer #1 (Remarks to the Author):\nProtein atomic structure is crucial across various disciplines, including microbiology, biochemistry, and pharmacy. The challenges in such research lie in the high requirements for spatial resolution and the extremely complex dynamics of proteins. This manuscript reports an important research outcome, demonstrating the potential of MicroED technology in resolving atomic-resolution protein structures. The research methods used in the article are both novel and rigorous. The manuscript is well-written and filled with passion. I would recommend the publication of this significant work in Nature Communications without hesitation, provided the following issues are addressed:\nRESPONSE: We thank the reviewer for the thoughtful assessment and for recommending publication. We address each point below and have made the indicated revisions.\nMajor Issues:\n1. The manuscript focuses on the spontaneous formation of nanocrystals during ethanolic extraction. Have the authors explored the possibility of controlling the crystal size and morphology by varying extraction conditions (e.g., solvent type, concentration, temperature)? Such information could be valuable for optimizing MicroED experiments with other proteins. Why is needle-shaped crystal preferred compared with the block-like crystals?\nRESPONSE: We did not systematically optimize the crystallization, as the spontaneously formed needles were already ideal for MicroED. We have added a new paragraph to the Discussion (\"Crystal habit and implications for MicroED\") that addresses this. It lists the variables that could plausibly tune habit (solvent, temperature, etc.) and explains precisely why needles are advantageous for MicroED (reduced effective thickness, lower dynamical scattering) and how serial merging mitigates the trade-off of preferred orientation.\n2. It is surprising to me that the crushed fragments of the bulk crystal could not preserve the internal atomic order. From Fig. S3c, I noticed that the crushed fragments still maintain a relatively large size (~100 μm), which should, in principle, preserve the structural information of the crystal.\nRESPONSE: This is an excellent point. We have clarified in the Results section that for electrons, the limiting factor is thickness along the beam, not lateral size. We explain that while the fragments were large laterally, most remained thicker than the ~0.5 μm useful limit, which degrades data quality via dynamical scattering. This is why the spontaneously formed, thin needles performed better. We also contrast this system-specific outcome with other reports where crushing was successful.\n3. The authors should cite previous works regarding the crystallographic information of the crystal and compare them with the current study. I noticed that the reported lattice parameters in the following reference differ from those in this work. The authors should explain this discrepancy: (Ref. 42: Solvent organization in the ultrahigh-resolution crystal structure of crambin at room temperature, https://doi.org/10.1107/S2052252524007784)\nRESPONSE: We have added Supplementary Table S2 (\"Crambin unit cell comparisons\"). This table compares the unit cell parameters (a, b, c, β), space group, temperature, and isoform composition from key prior X-ray and neutron structures (including the IUCrJ 2024 paper) with our new MicroED and X-ray structures. We also added text to the Discussion noting that apparent differences are largely due to axis settings.\n4. The crambin protein exists in two natural isomers, PL and SI, and a hybrid model with partial occupancies was successfully built. Did the authors perform further analysis and comparison of the structures and relationships of these two isomers?\nRESPONSE: We have. In the Results section, we now explicitly state that the PL/SI isoforms at residues 22 and 25 were refined as alternate conformers with occupancies constrained to sum to 1. The model converged to a 44(1):57(3) P/S ratio and 45(2):55(2) L/I ratio. This eliminated all difference map peaks. To demonstrate this, we have created Supplementary Figure S5, which shows the omit maps for this region, providing clear evidence for the mixed-occupancy model. We also added the refined 0.8 Å X-ray structure from the large block-like crystals to this manuscript and refined these residues, finding similar occupancies.\nMinor Issues:\n1. Page 2, “Crystals invisible to the naked eye,”: Missing “are.”\nRESPONSE: Thank you. This has been fixed.\n2. A citation should be added for this information: \"It was later the first protein solved by Hauptman and Weeks’ purely mathematical direct methods, a triumph developed at our home institutions.\"\nRESPONSE: This has been fixed. We have added the citation to Weeks et al. (1995) in the Introduction.\nReviewer #2 (Remarks to the Author):\nThis study presents the first ab initio structure of crambin solved by MicroED, achieving the highest reported data quality to date in terms of resolution, completeness, and R values. As the authors note, this work establishes an important gold-standard reference for the future development of electron crystallography, including modelling electron scattering, analyzing charge density, and exploring hydrogen-bonding networks. The manuscript would be further strengthened by addressing the following points prior to publication.\nRESPONSE: We appreciate the positive evaluation and address the requested clarifications below.\n1. The authors repeatedly state that ‘Our workflow demonstrates that complex biological matrices can be mined directly for sub-ångström protein structures, establishing a practical and scalable pipeline from raw biomass to atomic-level models of previously intractable targets’. While this claim is compelling, a discussion is needed regarding the general applicability of this pipeline. Specifically, how broadly can high-resolution MicroED data be obtained directly from seeds for ab initio structure determination? For instance, is the proposed “low-tech” crystallization method generally effective for all small proteins? How frequently do crystals of suitable size and sufficient crystallinity arise for MicroED experiments using this approach? The authors should comment on these practical aspects to clarify the scope and limitations of their workflow.\nRESPONSE: This is a critical point. We have tempered our claims in the Abstract and Results and added a new \"Scope and limitations\" paragraph. This paragraph explicitly states the conditions that favor success (e.g., electron-transparent crystals, high-quality diffraction) and the likely failure modes (e.g., thick blocks, weak diffraction, severe preferred orientation). We present this as a practical case that motivates checking for nanocrystals during routine extraction, not as a universally applicable solution.\n2. You note that previous ab initio structures have been obtained by MicroED for triclinic lysozyme and proteinase K (for example, see Nature Methods, DOI:10.1038/s41592-022-01485-4). It would be valuable for the authors to address how their crambin structure compares to these earlier achievements. Specifically:\nWhy might crambin serve as a more suitable gold-standard reference for advancing electron-specific methods?\nIs the choice driven primarily by superior data quality—such as finer resolution, improved completeness, better R values, or enhanced signal-to-noise?\nCould the data quality for triclinic lysozyme and proteinase K be further enhanced, and if not, what limitations prevent further improvement?\nA discussion along these lines would clarify the rationale for highlighting crambin and help frame its broader significance in the field.\nRESPONSE: To address this directly, we have added Supplementary Table S3 (\"Ab initio benchmark comparison\"). This table compares our crambin structure to the lysozyme and proteinase K results, focusing on key metrics like resolution, R-values, phasing fragment, microscope voltage, and, most importantly, sample preparation (FIB-milling vs. none). We also added a new paragraph (\"Positioning crambin among ab initio MicroED benchmarks\") that uses this table to frame our work's specific contribution: a sub-Å ab initio structure from spontaneously formed crystals on standard 200 kV hardware, without FIB-milling or an energy filter.\n3. On lines 23–29, the authors state that “the crushed fragments produced markedly inferior electron diffraction data compared to the spontaneously formed needles.” This finding contrasts with the results reported by M. Jason de la Cruz and colleagues (Nat Methods, DOI:10.1038/nmeth.4178), who found that crushed crystals could yield high-quality diffraction. Could the authors elaborate on why this difference arises in the case of crambin? A comparison of methodological details and crystal behavior between crambin and other proteins would clarify why the crushed-fragment approach performed differently here and provide valuable insight into the limits of this technique.\nRESPONSE: We have expanded the Results section to address this. We now explain that outcomes are system specific. Crushing can increase mosaicity and damage, but the primary issue for our crambin blocks was that fragmentation did not produce uniformly thin crystals. Most fragments remained too thick, amplifying dynamical and absorption effects. We explicitly state: \"This system-specific outcome contrasts with reports where crushed fragments diffracted well...\"\n4. The authors applied anisotropy correction to the data using STARANISO, reporting an effective (ellipsoidal) completeness of 98.6%. It would be valuable to also provide the data statistics along the two principal axes of the ellipsoid for clarity. In addition, could the authors comment on whether ab initio structure determination would still be feasible without applying anisotropy correction, and if so, how large the differences in outcome (e.g., resolution limits, map interpretability, or refinement statistics) would be?\nRESPONSE: We have done both. First, we have added Supplementary Table S4 (renamed from S2) which now includes the full statistics from the STARANISO server, including the resolution limits and data quality metrics along the principal axes. Additionally, we have supplied the STARANISO job ID in the revised manuscript, so that other researchers can readily inspect the data and have access to the exact files we used in our study. Second, we performed an ablation study and added the results to the Results section: \"Ab initio structure solution failed... Phaser produced no solutions in either case.\" This demonstrates that for our data, the anisotropy correction was essential for success.\n5. A minor comment regarding Fig. 1E: it is not immediately clear that the highest observed reflection extends to 0.85 Å. The authors should highlight this more explicitly in the figure to aid clarity.\nRESPONSE: We added arrows highlighting individual spots beyond 0.85 Å. We believe this was likely a poor print of the figure in the original manuscript. The PDF version of this figure has spots clearly visible to and beyond the ring.\nReviewer #3 (Remarks to the Author):\nVasireddy et al. report on a method to solve the structure of proteins without a homologous model. In this opportunity, they used the spontaneously formed microcrystals of the seed protein crambin, avoiding the preparation of large size crystals for X-ray or the time-consuming FIB lamella preparation for MicroED. The ultra-high resolution (0.85Å) microED data allowed structure resolution using a five-alanine helical fragment to initiate density modification.\nGeneral comments:\nThe novelty of the report relies on being the first ab initio structure of the protein crambin determined by electron-diffraction. While validation of existing methods is valuable, the study does not introduce significant novelty or advance the field beyond confirming the applicability of ab initio phasing using minimal idealized fragments followed by density modification improvement [Martynowycz et al. Nature Methods 19, 724–729 (2022)]. The study's contribution to the field is incremental. It provides confirmatory evidence but does not present a major advance or novel insights.\nRESPONSE: We have revised the Abstract, Introduction, and Results to state the distinct advance more clearly. As summarized in our response to R2.2, the novelty is the combination of (1) ab initio phasing at (2) sub-Å resolution from (3) spontaneously formed nanocrystals (no optimization), (4) on standard 200 kV hardware, (5) without FIB-milling or an energy filter. We have also created Supplementary Table S3 to explicitly compare our work to prior ab initio benchmarks to highlight these differences.\nMajor Comments:\n1. Authors performed serial microED to try to reduce anisotropy. They collected on 58 crystals, each one with 30° or 60° wedge. The starting angle of each wedge was incremented stepwise so that the combined datasets sample a stage tilt range of +/- 60Å. Regarding this collection strategy:\n• 58 crystals could seem a large number of crystals, especially considering that the resulting merged data still displayed significant anisotropy. Assuming each crystal with at least 30° wedge, the stepwise increment between each crystal was ~2°, increasing the redundancy. I would like to see a discussion about the minimal number of crystals that would produce similar results.\n• Authors reported a combination of collection in linear and counting mode. What percentage of crystals were in linear mode? Previously, Martynowycz et al. collected only on counting mode with very low exposure rate. In the present report, authors combined linear and counting modes in “the hopes the low-resolution reflections in linear mode would be less prone to coincidence loss, and the high-resolution reflections in counting mode would be more accurately recorded”. What was the effect of using linear mode collection? How did data behaved if only counting mode datasets were used? What would be the recommended strategy for future projects, counting mode only or a combination of both?\nRESPONSE: We have provided a detailed response to this in the Methods section under \"MicroED data collection.\" We now state the exact numbers: \"We acquired 39 linear-mode datasets and used 30... and we acquired 117 counting-mode datasets and used 28...\" We also state that we did not determine the strict minimal number of crystals but that high redundancy was key. Finally, we have added a detailed rationale for the dual-mode strategy, citing the new Vlahakis et al. (2025) paper. We explain that our cautious approach was designed to mitigate coincidence loss (CL), a concern that this new paper validates as real, even at low flux. Our data collection strategy deliberately emphasizes serial acquisition and retrospective curation, similar to single-particle cryo-EM, rather than minimizing the number of crystals, so we did not attempt to determine the theoretical minimum needed for ab initio solution.\n2. This report uses 0.85Å resolution data to performs molecular replacement in PHASER using a generic five-residue poly-alanine helix, and the resulting initial phases were sufficient to bootstrap the dynamic density modification algorithm in ACORN. Crambin is a protein of ~4.7kDa and 46 amino acids. While impressive, previously, Martynowycz et al. used data to 0.87Å (for lysozyme nanocrystals) and solved the structure with the same approach using a three poly-alanine fragment in PHASER followed by ACORN. The authors of the present report need to demonstrate what is novel about this work beyond what has already been published.\nRESPONSE: As noted in our responses to the General comments above and in R2.2, we have sharpened the novelty statement in the Abstract and Results and created Supplementary Table S3 to directly compare the key methodological differences (e.g., FIB-milling, 300 kV, energy filter) between this work and the prior lysozyme/proteinase K studies.\n3. Authors claim their map provides a complementary chemical picture of crambin to those available through X-ray or neutron techniques, highlighting that crambin is benchmark protein across crystallographic platforms. However, they did not show any comparison (figures) of those structures or the difference in the information obtained on the first structure of crambin solved by electrons.\nRESPONSE: We have now added Supplementary Figure S6 (\"X-ray, neutron, electron map comparison\"). This figure directly compares representative regions of our 0.85 Å electron-potential map with our 0.80 Å X-ray and the published 1.1 Å neutron maps. We also added a new paragraph (\"Complementary chemical information...\") to point to this figure and comment on the differences.\n4. The statement at the end of the abstract ... “complex biological matrices can be mined directly for sub-ångström protein structures, establishing a practical and scalable pipeline from raw biomass to atomic-level models of previously intractable targets” it is an overstatement. It is very unlikely that will happen in general for other samples; unless the authors can prove otherwise.\nRESPONSE: We agree. We have removed this sentence from the Abstract and replaced it with our more precise novelty statement. We have also tempered all related claims in the main text to \"in favorable cases.\"\n5. Comparisons with the 0.48 Å resolution structure would be important to show including overall structure, key residues.\nRESPONSE: To avoid model bias, we compared our MicroED structure to an isomorphous 0.80 Å X-ray dataset collected from the same batch of crystals and refined with an identical spherical-atom protocol (anisotropic non-H, riding H) in SHELXL. Charge-density studies of crambin employ multipole models and report deformation-density maps, which are not directly comparable to either MicroED electrostatic-potential maps or standard X-ray 2mFo-DFc maps. This yields a modality-controlled, apples-to-apples assessment of MicroED vs X-ray and anchors hydrogen positions to neutron data.\n6. The authors should create difference maps where residues for the PI and SL isoforms are omitted from the calculation and show the map of the two possible conformations.\nRESPONSE: This is addressed by our new Supplementary Figure S5 (see R1.4).\n7. To solve a structure by ab-initio methods the required resolution needs to be higher than 1.1 Å. This corresponds to less than 1% of all protein structures solved. Hence, it would be hard to sell it as a general method.\nRESPONSE: We agree that classical direct methods typically require very high resolution. We now cite fragment-based approaches (ACORN/ARCIMBOLDO/SHELXE) that have succeeded at lower resolutions than 1.2 Å in specific cases, and we avoid percentages. Indeed, several reports have been published using these methods on structures down to 3A, even in our prior work (Nat methods 2022) on proteinase K, we were able to bootstrap a solution at 1.5A, a much more common resolution. Our claim is calibrated accordingly: fragment-bootstrap ab initio can extend feasibility beyond the classical limit when high-quality MicroED data are available.\nMinor Comments:\n1. In Page 2, Line 31, authors wrote:\n“In subsequent years, synchrotron and neutron[23] sources pushed its resolution to an extraordinary 0.48 and 0.54 Å,[24,25] making it a key model for studying ultra-high-resolution features.”\nRef 23 reports neutron diffraction of crambin at 1.1Å. Both structures 0.48 (Ref 24) and 0.54 (Ref 25) are from X-Ray diffraction. Please rephrase the sentence to avoid confusion.\nRESPONSE: Thank you for catching this. We have corrected this sentence in the Introduction to clearly separate the X-ray and neutron resolution records and have updated the citations accordingly.\n2. The conclusion section seems largely as part of the discussion. Consider moving part of this section to the discussion section and focus on delivering a strong, shorter conclusion.\nRESPONSE: We have implemented this. The old Conclusions section has been removed. The forward-looking text (e.g., \"The robustness of this system...\") has been moved. The paper now ends with a single, concise summary paragraph.\nReviewer #4 (Remarks to the Author):\nI 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.\nRESPONSE: We thank the reviewer for their careful evaluation and for co-reviewing as part of the Nature Communications ECR initiative.\nWe thank the reviewers for their careful reading of the manuscript and for the detailed and constructive comments. We have revised the manuscript and the Supplementary Information accordingly. We have revised the manuscript and Supplementary Information to address the reviewers’ comments. The reviewer comments appear below in black unedited. Our responses follow in blue.\nReviewer #1 (Remarks to the Author)\nThe authors have carefully addressed all my comments, as well as those of the other referees. I would like to recommend the publication of this paper.\nRESPONSE: We thank the reviewer for their time and for recommending publication.\nReviewer #2 (Remarks to the Author)\nMost of the concerns and comments previously raised have been appropriately addressed.\nHowever, relating to my previous comments 1 and 2, I have a concern of the level of novelty relative to the journal’s threshold. I didn’t emphasize this point in my first review, but on re-reading the revised manuscript and the responses to comments, I continue to have the same concern. The work is technically sound, clearly presented, and of value to the MicroED community. However, the core advance appears incremental. This work reports the first ab initio structure of crambin solved by MicroED with high data quality, but the method ‘ab initio structure determination by MicroED’ has been reported previously (Nature Methods, DOI:10.1038/s41592-022-01485-4). The authors’ conclusion - ‘a sub-Å ab initio structure from spontaneously formed crystals on standard 200 kV hardware, without FIB-milling or an energy filter’ – is noteworthy but seems highly sample dependent and therefore not generalizable as a broadly applicable methodological advance.\nRESPONSE: We thank the reviewer for their assessment and for clarifying the novelty concern.\nWe agree that spontaneously formed microcrystals obtained during purification are not expected to be obtainable for every protein, and we have revised the manuscript to avoid implying broader generality than is supported here. Our intended novelty claim is instead that an atomic resolution ab initio MicroED protein structure can be obtained on widely available 200 kV instrumentation without an energy filter and without FIB milling, provided that data processing addresses preferred orientation and anisotropy. In this dataset, standard isotropic truncation did not yield a solution, whereas anisotropy correction using STARANISO enabled successful phasing. We present this as an empirically demonstrated requirement for this common MicroED failure mode, rather than as a universal rule.\nRelative to prior ab initio MicroED demonstrations, including Nature Methods (DOI:10.1038/s41592-022-01485-4), we have revised the Abstract, Introduction, and Discussion to state more precisely that our advance is the combination of accessible acquisition and processing choices that together achieve a sub Å structure on a 200 kV microscope configuration without energy filtering or FIB milling. We also clarify the specific elements required in this case, including extensive serial merging to address preferred orientation, anisotropy aware truncation, and a mixed acquisition strategy to extend usable dynamic range. These revisions are intended to make the scope of the methodological advance clear while avoiding claims beyond the demonstrated case.\nExtra two minor comments:\n1. The maps (B, D, and F) in Figure S6 should be displayed at the same magnification for proper comparison.\nRESPONSE: We have updated the figure to display all maps at identical magnification for direct comparison. The figure numbering has changed following the Supplementary reordering requested by Reviewer #3.\n2. The unit of ‘dose’ is Gray(Gy) rather than e- /Å2. The authors should either replace the term “dose” with “fluence” or convert the values to Gray. The latter option is more appropriate here, as Table S3 compares dose values obtained under different accelerating voltages. Please refer to a recent paper discussing this issue: https://doi.org/10.1107/S205979832500912X.\nRESPONSE: We report fluence (e−/Å²) as the directly measured exposure metric and provide estimated absorbed doses (Gy) for cross voltage comparison using stopping power based conversions as described in DOI:10.1107/S205979832500912X.\n3. A typer issue in line 64, ‘roomtemperature’ should be ‘room-temperature’\nRESPONSE: Corrected.\nReviewer #3 (Remarks to the Author):\nThe manuscript submitted by the authors is now adequate for publications.\nRESPONSE: We thank the reviewer for their constructive feedback and recommendation.\nMinor comments:\n1) Rfree seems high for this resolution; authors should comment on this.\nRESPONSE: We have added a brief comment in the main text. MicroED refinements often show higher R factors than X-ray structures at similar nominal resolution, in part because dynamical scattering is not fully captured by standard kinematic refinement models. We note that further reductions will likely require refinement approaches that better model dynamical scattering.\n2) Figure S3 caption is called in the text referring to block-like crystals using traditional vapor diffusion. Then the corresponding caption reads:\n“Supplementary Figure S3. Images of crambin crystal samples. (A) Crashed out needles of crambin formed by slow evaporation”\nAre crystals in (A) obtained as in Figure S1-L, by “fast” (within seconds, line 86) evaporation? Please avoid confusion.\nRESPONSE: We have revised the caption to state that the crystals shown are obtained by fast evaporation, consistent with the description in the main text.\n3) Consider updating order in Supplementary document”: Table S4 is called before Table S2, Fig. S6 before Fig S5, and Table S5 is never called.\nRESPONSE: We have reordered the Supplementary items so that tables and figures are cited in the same order in which they appear, and we have ensured that every Supplementary table and figure is called out in the manuscript text at least once.\n4) Line 138 states: “Ab initio structure solution failed at correctly placing the starting fragment in both the conservative isotropic truncation and the overly noisy optimistic spherical truncation to 0.85 Å”. Please explicitly add the resolution cutoff of the “conservative isotropic truncation”, is it 1.432A? Why do the authors believe that a solution at 1.4A was not feasible since in the case of proteinase K, a solution was bootstrap at 1.5A? Is the “starting fragment” the non-homologous five residue helical fragment? Try to be clear since specific this is the first time it is mentioned in the results section.\nRESPONSE: We have clarified this passage. We now state the conservative isotropic cutoff and define the starting fragment at its first mention as the ideal five residue poly alanine helix. We also clarify that the conservative truncation discards the highest resolution reflections that carried key phase information in this case, whereas a spherical truncation to the nominal best resolution incorporates substantially noisier data from weak directions.\n5) Line 164, “Figure 2A” is called, but it seems that it should be Figure 2B.\nRESPONSE: Corrected to Figure 2B.\n6) “our results suggest that extraction conditions which favor needle or plate-like habits (e.g., by varying solvents, concentration, or temperature) could be a practical strategy for obtaining MicroED-ready samples of other proteins, provided serial merging is used to overcome the resulting anisotropy.” Suggesting as a practical strategy to look for conditions that grow needles or plate-like habits in other target proteins for microED would require a specific study to prove that this is the case. Please correct this sentence.\nRESPONSE: We agree and have removed this sentence.\n7) Line 64: Change “roomtemperature” to room temperature.\nRESPONSE: Corrected.\n8) Regarding the number of datasets that made the final cut to the merged data, 30 out of 39 in linear mode, and 28 out of 117 in counting mode, any reason/explanation why counting mode seems to be less favored? In addition, the previously raised question about how data behaved if only counting mode datasets were used was not answered. Authors emphasized that collecting in linear mode was a strategy to mitigate CL, and cited a paper about how CL is real, even at low flux. However, considering that previous lysozyme/Proteinase K study reported only counting mode, it would be interesting to see how counting +linear vs counting only data compare.\nRESPONSE: We agree that the different utilization rates are of interest. For the final merge, we used the automated clustering procedure in XSCALE (XSCALE_ISOCLUSTER) to select the most internally consistent subset for merging, and we did not manually exclude datasets by acquisition mode. In our acquisition conditions, counting mode datasets were more frequently identified as outliers by the clustering statistics, which is consistent with known count rate limitations in counting detectors for the strongest reflections. A full, systematic comparison of “counting only” versus “counting plus linear” merges across alternative clustering thresholds and scaling choices would substantially expand the scope of the manuscript beyond demonstrating an ab initio solution from the highest-quality merged dataset. To enable deeper investigation by interested readers, we have made all raw data and processing outputs publicly available, so that alternative merging strategies, including counting only, can be explored without ambiguity about data access or selection.\nReviewer #4 (Remarks to the Author):\nI 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.\nRESPONSE: We thank the reviewer for co-reviewing the manuscript as part of the early career peer review initiative."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have carefully addressed all my comments, as well as those of the other referees. I would like to recommend the publication of this paper.", "reviewer_2": "Most of the concerns and comments previously raised have been appropriately addressed.\nHowever, relating to my previous comments 1 and 2, I have a concern of the level of novelty relative to the journal’s threshold. I didn’t emphasize this point in my first review, but on re-reading the revised manuscript and the responses to comments, I continue to have the same concern. The work is technically sound, clearly presented, and of value to the MicroED community. However, the core advance appears incremental. This work reports the first ab initio structure of crambin solved by MicroED with high data quality, but the method ‘ab initio structure determination by MicroED’ has been reported previously (Nature Methods, DOI:10.1038/s41592-022-01485-4). The authors’ conclusion - ‘a sub-Å ab initio structure from spontaneously formed crystals on standard 200 kV hardware, without FIB-milling or an energy filter’ – is noteworthy but seems highly sample dependent and therefore not generalizable as a broadly applicable methodological advance.\nExtra two minor comments:\n1. The maps (B, D, and F) in Figure S6 should be displayed at the same magnification for proper comparison.\n2. The unit of ‘dose’ is Gray(Gy) rather than e- /Å2. The authors should either replace the term “dose” with “fluence” or convert the values to Gray. The latter option is more appropriate here, as Table S3 compares dose values obtained under different accelerating voltages. Please refer to a recent paper discussing this issue: https://doi.org/10.1107/S205979832500912X.\n3. A typer issue in line 64, ‘roomtemperature’ should be ‘room-temperature’", "reviewer_3": "The manuscript submitted by the authors is now adequate for publications.\nMinor comments:\n1) Rfree seems high for this resolution; authors should comment on this.\n2) Figure S3 caption is called in the text referring to block-like crystals using traditional vapor diffusion. Then the corresponding caption reads:\n“Supplementary Figure S3. Images of crambin crystal samples. (A) Crashed out needles of crambin formed by slow evaporation”\nAre crystals in (A) obtained as in Figure S1-L, by “fast” (within seconds, line 86) evaporation? Please avoid confusion.\n3) Consider updating order in Supplementary document”: Table S4 is called before Table S2, Fig. S6 before Fig S5, and Table S5 is never called.\n4) Line 138 states: “Ab initio structure solution failed at correctly placing the starting fragment in both the conservative isotropic truncation and the overly noisy optimistic spherical truncation to 0.85 Å”. Please explicitly add the resolution cutoff of the “conservative isotropic truncation”, is it 1.432A? Why do the authors believe that a solution at 1.4A was not feasible since in the case of proteinase K, a solution was bootstrap at 1.5A? Is the “starting fragment” the non-homologous five residue helical fragment? Try to be clear since specific this is the first time it is mentioned in the results section.\n5) Line 164, “Figure 2A” is called, but it seems that it should be Figure 2B.\n6) “our results suggest that extraction conditions which favor needle or plate-like habits (e.g., by varying solvents, concentration, or temperature) could be a practical strategy for obtaining MicroED-ready samples of other proteins, provided serial merging is used to overcome the resulting anisotropy.” Suggesting as a practical strategy to look for conditions that grow needles or plate-like habits in other target proteins for microED would require a specific study to prove that this is the case. Please correct this sentence.\n7) Line 64: Change “roomtemperature” to room temperature.\n8) Regarding the number of datasets that made the final cut to the merged data, 30 out of 39 in linear mode, and 28 out of 117 in counting mode, any reason/explanation why counting mode seems to be less favored? In addition, the previously raised question about how data behaved if only counting mode datasets were used was not answered. Authors emphasized that collecting in linear mode was a strategy to mitigate CL, and cited a paper about how CL is real, even at low flux. However, considering that previous lysozyme/Proteinase K study reported only counting mode, it would be interesting to see how counting +linear vs counting only data compare.", "reviewer_4": "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."}, "author_response": "We thank the reviewers for their careful reading of the manuscript and for the detailed and constructive comments. We have revised the manuscript and the Supplementary Information accordingly. We have revised the manuscript and Supplementary Information to address the reviewers’ comments. The reviewer comments appear below in black unedited. Our responses follow in blue.\nReviewer #1 (Remarks to the Author)\nThe authors have carefully addressed all my comments, as well as those of the other referees. I would like to recommend the publication of this paper.\nRESPONSE: We thank the reviewer for their time and for recommending publication.\nReviewer #2 (Remarks to the Author)\nMost of the concerns and comments previously raised have been appropriately addressed.\nHowever, relating to my previous comments 1 and 2, I have a concern of the level of novelty relative to the journal’s threshold. I didn’t emphasize this point in my first review, but on re-reading the revised manuscript and the responses to comments, I continue to have the same concern. The work is technically sound, clearly presented, and of value to the MicroED community. However, the core advance appears incremental. This work reports the first ab initio structure of crambin solved by MicroED with high data quality, but the method ‘ab initio structure determination by MicroED’ has been reported previously (Nature Methods, DOI:10.1038/s41592-022-01485-4). The authors’ conclusion - ‘a sub-Å ab initio structure from spontaneously formed crystals on standard 200 kV hardware, without FIB-milling or an energy filter’ – is noteworthy but seems highly sample dependent and therefore not generalizable as a broadly applicable methodological advance.\nRESPONSE: We thank the reviewer for their assessment and for clarifying the novelty concern.\nWe agree that spontaneously formed microcrystals obtained during purification are not expected to be obtainable for every protein, and we have revised the manuscript to avoid implying broader generality than is supported here. Our intended novelty claim is instead that an atomic resolution ab initio MicroED protein structure can be obtained on widely available 200 kV instrumentation without an energy filter and without FIB milling, provided that data processing addresses preferred orientation and anisotropy. In this dataset, standard isotropic truncation did not yield a solution, whereas anisotropy correction using STARANISO enabled successful phasing. We present this as an empirically demonstrated requirement for this common MicroED failure mode, rather than as a universal rule.\nRelative to prior ab initio MicroED demonstrations, including Nature Methods (DOI:10.1038/s41592-022-01485-4), we have revised the Abstract, Introduction, and Discussion to state more precisely that our advance is the combination of accessible acquisition and processing choices that together achieve a sub Å structure on a 200 kV microscope configuration without energy filtering or FIB milling. We also clarify the specific elements required in this case, including extensive serial merging to address preferred orientation, anisotropy aware truncation, and a mixed acquisition strategy to extend usable dynamic range. These revisions are intended to make the scope of the methodological advance clear while avoiding claims beyond the demonstrated case.\nExtra two minor comments:\n1. The maps (B, D, and F) in Figure S6 should be displayed at the same magnification for proper comparison.\nRESPONSE: We have updated the figure to display all maps at identical magnification for direct comparison. The figure numbering has changed following the Supplementary reordering requested by Reviewer #3.\n2. The unit of ‘dose’ is Gray(Gy) rather than e- /Å2. The authors should either replace the term “dose” with “fluence” or convert the values to Gray. The latter option is more appropriate here, as Table S3 compares dose values obtained under different accelerating voltages. Please refer to a recent paper discussing this issue: https://doi.org/10.1107/S205979832500912X.\nRESPONSE: We report fluence (e−/Å²) as the directly measured exposure metric and provide estimated absorbed doses (Gy) for cross voltage comparison using stopping power based conversions as described in DOI:10.1107/S205979832500912X.\n3. A typer issue in line 64, ‘roomtemperature’ should be ‘room-temperature’\nRESPONSE: Corrected.\nReviewer #3 (Remarks to the Author):\nThe manuscript submitted by the authors is now adequate for publications.\nRESPONSE: We thank the reviewer for their constructive feedback and recommendation.\nMinor comments:\n1) Rfree seems high for this resolution; authors should comment on this.\nRESPONSE: We have added a brief comment in the main text. MicroED refinements often show higher R factors than X-ray structures at similar nominal resolution, in part because dynamical scattering is not fully captured by standard kinematic refinement models. We note that further reductions will likely require refinement approaches that better model dynamical scattering.\n2) Figure S3 caption is called in the text referring to block-like crystals using traditional vapor diffusion. Then the corresponding caption reads:\n“Supplementary Figure S3. Images of crambin crystal samples. (A) Crashed out needles of crambin formed by slow evaporation”\nAre crystals in (A) obtained as in Figure S1-L, by “fast” (within seconds, line 86) evaporation? Please avoid confusion.\nRESPONSE: We have revised the caption to state that the crystals shown are obtained by fast evaporation, consistent with the description in the main text.\n3) Consider updating order in Supplementary document”: Table S4 is called before Table S2, Fig. S6 before Fig S5, and Table S5 is never called.\nRESPONSE: We have reordered the Supplementary items so that tables and figures are cited in the same order in which they appear, and we have ensured that every Supplementary table and figure is called out in the manuscript text at least once.\n4) Line 138 states: “Ab initio structure solution failed at correctly placing the starting fragment in both the conservative isotropic truncation and the overly noisy optimistic spherical truncation to 0.85 Å”. Please explicitly add the resolution cutoff of the “conservative isotropic truncation”, is it 1.432A? Why do the authors believe that a solution at 1.4A was not feasible since in the case of proteinase K, a solution was bootstrap at 1.5A? Is the “starting fragment” the non-homologous five residue helical fragment? Try to be clear since specific this is the first time it is mentioned in the results section.\nRESPONSE: We have clarified this passage. We now state the conservative isotropic cutoff and define the starting fragment at its first mention as the ideal five residue poly alanine helix. We also clarify that the conservative truncation discards the highest resolution reflections that carried key phase information in this case, whereas a spherical truncation to the nominal best resolution incorporates substantially noisier data from weak directions.\n5) Line 164, “Figure 2A” is called, but it seems that it should be Figure 2B.\nRESPONSE: Corrected to Figure 2B.\n6) “our results suggest that extraction conditions which favor needle or plate-like habits (e.g., by varying solvents, concentration, or temperature) could be a practical strategy for obtaining MicroED-ready samples of other proteins, provided serial merging is used to overcome the resulting anisotropy.” Suggesting as a practical strategy to look for conditions that grow needles or plate-like habits in other target proteins for microED would require a specific study to prove that this is the case. Please correct this sentence.\nRESPONSE: We agree and have removed this sentence.\n7) Line 64: Change “roomtemperature” to room temperature.\nRESPONSE: Corrected.\n8) Regarding the number of datasets that made the final cut to the merged data, 30 out of 39 in linear mode, and 28 out of 117 in counting mode, any reason/explanation why counting mode seems to be less favored? In addition, the previously raised question about how data behaved if only counting mode datasets were used was not answered. Authors emphasized that collecting in linear mode was a strategy to mitigate CL, and cited a paper about how CL is real, even at low flux. However, considering that previous lysozyme/Proteinase K study reported only counting mode, it would be interesting to see how counting +linear vs counting only data compare.\nRESPONSE: We agree that the different utilization rates are of interest. For the final merge, we used the automated clustering procedure in XSCALE (XSCALE_ISOCLUSTER) to select the most internally consistent subset for merging, and we did not manually exclude datasets by acquisition mode. In our acquisition conditions, counting mode datasets were more frequently identified as outliers by the clustering statistics, which is consistent with known count rate limitations in counting detectors for the strongest reflections. A full, systematic comparison of “counting only” versus “counting plus linear” merges across alternative clustering thresholds and scaling choices would substantially expand the scope of the manuscript beyond demonstrating an ab initio solution from the highest-quality merged dataset. To enable deeper investigation by interested readers, we have made all raw data and processing outputs publicly available, so that alternative merging strategies, including counting only, can be explored without ambiguity about data access or selection.\nReviewer #4 (Remarks to the Author):\nI 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.\nRESPONSE: We thank the reviewer for co-reviewing the manuscript as part of the early career peer review initiative."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have carefully addressed my concerns as well as other comments. The revisions to the Abstract, Introduction, and Discussion enhance the novelty of this work in relation to previous studies. I therefore recommend this manuscript for publication."}, "author_response": ""}]

Decision letter