The cotranslational cycle of the ribosome-bound Hsp70 homolog Ssb.
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓No relevant deviation in data/preprocessing
- ✓No authors-side cause for any deviation
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- ✓Overall, the reproduction was clean
- Every checked point held up.
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Single-particle cryo-EM paper (ribosome-bound Hsp70 Ssb). Described well enough to reproduce the headline pipeline output: the gold-standard FSC=0.143 map resolution of both deposited maps. Reproduction is essentially 1:1 (different/independent route): I recomputed the FSC directly from the deposited EMDB half-maps (half_map_1 x half_map_2, masked with the deposited solvent mask msk_1) using a transparent numpy/mrcfile implementation on «our HPC» («job», n095). Result: 2.819 A (EMD-53860, reported 2.8) and 2.985 A (EMD-53861, reported 3.0) -- within 0.02 A of the reported values, and matching the authors' own deposited cryoSPARC GSFSC corrected-mask curve (2.817 / 2.980 A) almost exactly. The unmasked FSC is correctly worse (3.6 / 4.1 A), confirming the resolution genuinely derives from the masked half-map signal rather than manipulated data -> NO fabrication signal; the deposits are internally consistent with the paper. NOTE: minor metadata discrepancy -- EMDB header rounds S1 to 2.9 A while the deposited curve and the paper both give 2.8 A. NOT attempted (the hard 80%, by design): full reconstruction from raw data (EMPIAR-12933 -> map via cryoSPARC/crYOLO/cryoDRGN/RELION5/DeepEMhancer; GPU-weeks, TB-scale, under-specified hyperparameters); atomic model building/refinement (9R9O/9R9P); and running the paper's 'Code' link cs2star (a cryoSPARC .cs -> RELION .star converter) -- no .cs intermediate is deposited, so it cannot be exercised on this paper's data and would reproduce no reported number. Also note the brief's data accession (zenodo 3576630) was a mis-enrichment: it is the pyem v0.5 SOFTWARE (cs2star's dependency), not the paper data; the real data is EMDB/PDB/EMPIAR.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessment Score 85assessed: 2026-06-16 ⛓ 855a3332c090
✎ I am an author of this paper
Updated or fixed a deposit, or is there an erratum? Ask us to re-run the metrics. We verify by email first; the new result is published as a new version with full history — nothing is overwritten.
Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusHow does the ribosome-bound yeast Hsp70 Ssb interact with translating ribosomes and nascent chains, and how is its chaperone cycle coordinated with translation via its cochaperone RAC? The paper tests where Ssb binds on the ribosome and how its substrate binding domain is positioned to receive nascent chains.
- ★ Rpl25/uL23 is the primary ribosomal attachment site of Ssb, contacted via the Ssb-αD RKKR-motif (R596, K597, K603, R604) binding the Rpl25 EDD-motif (E77, D131, D134) and C-terminus. finding
- ★ Two cryo-EM structures (Ssb-ADP S1 and S2) show ribosome-bound Ssb with its SBD in the closed ADP-bound conformation and its peptide-binding cleft positioned close to the tunnel exit engaged with a nascent chain. finding
- ★ The Ssb-Rpl25 interaction is electrostatic, demonstrated by charge-reversal complementation between Ssb-DD1-DD2 and Rpl25-KKK ribosomes. mechanism
- ★ Ssb and SRP compete for an overlapping ribosomal attachment site involving the Rpl25 EDD-motif at the tunnel exit. finding
- ★ Salt-resistant ribosome-binding of Ssb is mediated by interaction with the nascent chain rather than the direct Rpl25 contact. mechanism
- ★ Ribosome-binding of both Ssb-ADP and Ssb-ATP depends on the Rpl25 EDD-motif. finding
- Cryo-EM structure determination of Ssb on translating ribosome-nascent chain complexes via a yeast in vitro translation system with FLAG-Pgk1-70 RNCs. method
- Mutations impairing direct (Ssb RKKR) and Ssz1-mediated ribosome-binding of Ssb act synergistically, causing synthetic growth defects. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| cryo-EM single-particle structure determination | yeast ribosome-nascent chain complexes (RNCs) with FLAG-Pgk1-70, P-site tRNA Met-e | in vitro translation arrest on truncated mRNA in presence of Ssb (ADP-bound) | atomic structure / map of ribosome-bound Ssb (S1 and S2 conformations) | — |
| ribosome co-sedimentation / binding assay (immunoblotting) | yeast total cell extract (wild type, Rpl25-AAA, Rpl25-KKK) | Rpl25 EDD-motif mutations (AAA, KKK) | fraction of Ssb cosedimenting with ribosomes under low/high salt | — |
| ribosome-binding assay with purified components (immunoblotting) | purified non-translating wild type or Rpl25-KKK ribosomes + recombinant Ssb | Rpl25-KKK mutation; high vs low salt; RAC as control | Ssb in supernatant vs ribosomal pellet | — |
| charge-reversal ribosome-binding assay (immunoblotting) | purified Rpl25-KKK or wild type ribosomes + recombinant Ssb-DD1-DD2 | Ssb RKKR→DD charge reversal combined with Rpl25 KKK | restored/enhanced ribosome-binding in pellet vs supernatant | — |
| ribosome-binding assay for nucleotide states (immunoblotting) | purified wild type or Rpl25-KKK ribosomes + Ssb (ADP) or Ssb-T207A (ATP) | nucleotide preincubation (ADP/ATP); Rpl25-KKK | ribosome-binding dependence on Rpl25 EDD-motif | — |
| site-specific crosslinking | yeast nascent chains Pgk1 and Dap2 + Ssb | presence/absence of SRP (Δsrp54) | Ssb crosslink to nascent chain | — |
| in vivo growth/expression assay | yeast strains (Rpl25-KKK, Δsrp54, Ssb mutants) | Rpl25 EDD mutations; combined Ssb ribosome-binding mutations; paromomycin | Dap2 expression level; serial-dilution colony growth | — |
- – Ssb-ADP S1 and S2 cryo-EM structures resolved with SBD in closed ADP conformation; SBDβ 19° closer to ribosomal surface in S2 than S1 S1 2.8 Å, S2 3.0 Å; 19° difference
- – Side chains of a large fraction of the 17 C-terminal nascent chain residues were assignable within the proximal exit tunnel 17 C-terminal residues
- – Approximately 50% of wild type Ssb was ribosome-bound, ~two-thirds of which exhibited salt-resistant binding ~50% bound; ~2/3 salt-resistant
- ▼ Ribosome-binding of Ssb reduced more than 2-fold in Rpl25-AAA and Rpl25-KKK extracts under low- and high-salt conditions >2-fold
- ▼ Binding of Ssb to purified Rpl25-KKK ribosomes strongly reduced while RAC binding unaffected; high salt fully released wild type Ssb from non-translating ribosomes
- – Ssb-DD1-DD2 binding strongly reduced and fully salt-resistant in extracts, completely abolished with purified non-translating ribosomes; binding to Rpl25-KKK ribosomes enhanced vs wild type Ssb
- ▼ Dap2 expression significantly reduced in Rpl25-KKK strain, resembling Δsrp54; Ssb crosslinked to nascent Dap2 only in absence of SRP
- – Combined Ssb mutations impairing direct and Ssz1-mediated ribosome-binding act synergistically causing severe growth defects
- other 2.8 Å (Ssb-ADP S1), 3.0 Å (Ssb-ADP S2) (cryo-EM resolution of the two structures)
- other 19° (SBDβ rotation closer to ribosomal surface in S2 vs S1)
- count ~50% ribosome-bound, ~2/3 salt-resistant (wild type Ssb cosedimentation; two-way ANOVA, 4 experiments)
- fold_change >2-fold reduction (Ssb ribosome-binding in Rpl25-AAA/KKK extracts)
- count 17 C-terminal residues (nascent FLAG-Pgk1-70 residues modeled in exit tunnel)
- count 3 independent experiments (two-way ANOVA for Ssb-DD1-DD2 and nucleotide-state binding assays)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The paper combines cryo-EM structural analysis with quantitative biochemical ribosome-binding assays. Cryo-EM structures are characterized by stated nominal resolutions (2.8 Å and 3.0 Å) and described through atomic model geometry and contact distances rather than inferential statistics. Ribosome-binding experiments comparing wild type and mutant yeast strains/proteins used two-way ANOVA on 3–4 independent biological replicates, with results displayed as box or range plots overlaid with individual data points. A subset of experiments was performed twice without formal statistical testing, reported qualitatively as 'yielding similar results'.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Two-way ANOVA | Fraction of Ssb cosedimenting with ribosomes across wild type, Rpl25-AAA, and Rpl25-KKK strains under low- and high-salt conditions (Fig. 2b) | four independent experiments | not stated |
| Two-way ANOVA | Ribosome-binding of Ssb-DD1-DD2 vs wild type Ssb in total cell extract under low- and high-salt conditions (Fig. 3c) | three independent experiments | not stated |
| Two-way ANOVA | Ribosome-binding of wild type Ssb-ADP and Ssb-T207A (ATP-locked) to wild type or Rpl25-KKK ribosomes (Fig. 3f) | three independent experiments | not stated |
| Two-way ANOVA | Ribosome-binding of Ssb variants carrying combined mutations impairing direct and Ssz1-mediated ribosome-binding (Fig. 3h) | three independent experiments | not stated |
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Two-way ANOVA was applied to groups of 3–4 biological replicates for all quantitative binding assays↳ Could also: A non-parametric aligned ranks transformation ANOVA (ART-ANOVA) or Scheirer-Ray-Hare test could also be used when n per cell is small and normality cannot be empirically verified — With 3–4 replicates per group the Shapiro-Wilk test has very low power to detect non-normality; rank-based alternatives make fewer distributional assumptions while still accommodating a two-factor design
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Post-hoc pairwise comparisons following the two-way ANOVAs are not described in the text provided↳ Could also: An explicitly stated post-hoc procedure (e.g., Tukey HSD for all pairwise comparisons, or Dunnett's test for comparisons vs. wild type only) could also follow each omnibus ANOVA — Declaring which specific pairwise contrasts are significant—and controlling the family-wise error rate within each ANOVA—makes inferential conclusions more precise and directly interpretable
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Several key experiments (Figs. 2c, 2d, 3d, 3e) were 'independently performed twice, yielding similar results' without formal statistical analysis↳ Could also: Adding a third biological replicate and applying a simple two-sample t-test or Mann-Whitney U test could also be done; alternatively, quantification with all replicates shown as individual points provides more transparent communication of consistency — n = 2 does not support formal hypothesis testing; a third replicate enables even minimal inferential analysis and allows readers to judge effect magnitude and consistency independently
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Dispersion is reported inconsistently: box plots (25th–75th percentile with median) in Fig. 2b versus min–mean–max range lines in Figs. 3c, 3f, and 3h↳ Could also: A uniform dispersion metric across all figures—such as SD or 95% CI overlaid on individual data points—could also be used throughout — Consistent dispersion reporting simplifies cross-figure comparison of variability and aligns with transparency recommendations in Nature Portfolio reporting guidelines
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Effect magnitudes were communicated qualitatively (e.g., 'reduced more than 2-fold') rather than with formal effect-size statistics↳ Could also: Reporting partial eta-squared (η²p) from each ANOVA, or fold-change with a bootstrap 95% CI, could also accompany each result — Standardized effect sizes allow readers to evaluate biological relevance independently of sample size and are increasingly requested to support meta-analyses and replication
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No a priori power analysis or sample-size justification is provided for the choice of n = 3 or n = 4 independent experiments↳ Could also: A brief power calculation based on an estimated effect size from pilot data, or citation of a precedent n in comparable yeast biochemistry literature, could also be provided — Documenting the basis for the chosen replication level helps readers assess whether the study was sufficiently powered to detect the reported differences, particularly for comparisons yielding borderline significance
Citation network
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Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-41545346
Paper: Zhang Y, et al. "The cotranslational cycle of the ribosome-bound Hsp70 homolog Ssb." Nat Commun 2026. PMID 41545346 · PMCID PMC12847954 · DOI 10.1038/s41467-025-67685-6.
This is a single-particle cryo-EM structural-biology paper. The "Code" link
in the brief (github.com/brisvag/cs2star) is a third-party format-conversion
utility (cryoSPARC .cs → RELION .star, wrapping pyem); the authors used
it as one glue step in their pipeline. The brief's "Data" accession
(zenodo 10.5281/zenodo.3576630) is a mis-enrichment — that DOI is
asarnow/pyem v0.5, the software dependency of cs2star, not this paper's
data. The paper's real depositions are in EMDB / PDB / EMPIAR.
Reported computational depositions
| State | EMDB map | PDB model | Reported resolution | Particles (EMDB) | pixel |
|---|---|---|---|---|---|
| Ssb-ADP S1 | EMD-53860 | 9R9O | 2.8 Å (paper) / 2.9 Å (EMDB hdr) | 32,921 | 1.19 Å |
| Ssb-ADP S2 | EMD-53861 | 9R9P | 3.0 Å | 14,049 | 1.19 Å |
Raw: EMPIAR-12933 (motion-corrected micrographs). Resolution method (EMDB header + FSC file): FSC 0.143 cut-off, "cryoSPARC GSFSC Corrected Mask" (gold-standard, phase-randomization-corrected, masked).
Image-processing pipeline (Methods): cryoSPARC live (v4.2.1/4.4.1/4.5.1) →
crYOLO 1.9 (picking) → cryoDRGN (sorting) → RELION 5.0 (refine/classify) →
DeepEMhancer (sharpen) → ChimeraX/coot/phenix (model build/refine).
cs2star/pyem for cryoSPARC↔RELION conversion.
IN SCOPE (reproduced) — the headline pipeline output: map resolution
The single clearly-specified, low-cost pipeline-derived number is the gold-standard FSC=0.143 resolution of each map. It is independently recomputable from deposited half-maps + mask (EMDB), with standard methods, no proprietary software, in minutes. Two clean data points (S1, S2).
Three levels of evidence per map:
- Parse the deposited FSC curve (
emd_*_fsc.xml) → 0.143 crossing. Reproduces the reported number directly from deposited metadata. - Recompute masked half-map FSC from
half_map_1/2× depositedmsk_1(numpy+mrcfile, «our HPC») → independent check that the deposited curve is actually derived from the deposited half-maps (anti-fabrication test). - Recompute unmasked half-map FSC → honest baseline (expected worse; masking + phase-randomization is what recovers the reported value).
OUT OF SCOPE (not attempted — the hard 80%) — and why
- Full reconstruction EMPIAR-12933 → 2.8/3.0 Å map. Requires the full raw movie/micrograph set + crYOLO + cryoDRGN + RELION 5 GPU refinement (GPU-weeks, TB-scale, many under-specified hyper-parameters). Not 80/20.
- Atomic model building / phenix real-space refinement (9R9O/9R9P) — manual, iterative, not a deterministic pipeline output.
- cs2star
.cs→.starconversion on this paper's data — the intermediate cryoSPARC.csparticle files are not deposited (EMPIAR has micrographs, EMDB has maps), so cs2star cannot be exercised on this paper's data. The tool itself is trivially a format converter; running it on a synthetic.cswould not reproduce any reported paper value. - Wet-lab results (FRET/smFRET, growth assays, biochemistry) — out of scope by definition (non-pipeline).
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Independent recomputation of the gold-standard FSC=0.143 resolution from the authors' deposited half-maps + mask reproduces both reported values (2.8 A → 2.819 A; 3.0 A → 2.985 A) to within 0.02 A, matching the deposited cryoSPARC curve almost exactly; the unmasked FSC is correctly worse, confirming the signal is genuine. The only flags are a rounding-level numerical deviation and a benign EMDB-header rounding mismatch (2.9 vs 2.8 A) — no fabrication signal, no authors'-side or methodology defect. Note the hard 80% (raw reconstruction from EMPIAR-12933, atomic model building, wet-lab biophysics) was not attempted by design, so this judgement covers only the reproducible headline pipeline output, which holds 1:1.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.