Protocol

Bennett VHH design, E. coli expression and single-concentration SPR screen of 96 designs

Share of designed VHHs confirmed as binders by single-concentration SPR after E. coli expression, out of 96 designs screened per target.

3 evaluations · 6 results

Overview

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3 recorded evaluations, 6 metric rows. A comparison chart has not yet been validated for these results. The table retains the individual findings and their sources.

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Results

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All evaluations

3 evaluations · 6 results. Different protocols are not a single leaderboard.

Protocol: Bennett VHH design, E. coli expression and single-concentration SPR screen of 96 designs

Sorted by Count (SPR-confirmed binders; initial screening method SPR) (higher is better). The best value in each column is highlighted. Decimals are rounded for display; each value links to the printed value and its source.

Tested configuration	Protocol and dataset	Count (SPR-confirmed binders; initial screening method SPR)	Hit rate (SPR-confirmed binders out of designs screened; initial screening method SPR)
Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1Evaluation	Bennett VHH design target TcdB	2 (best in column)	2% (best in column)
Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1Evaluation	Bennett VHH design target Influenza Hemagglutinin	1	1%
Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1Evaluation	Bennett VHH design target IL-7 Receptor α	0	0%

All 6 result rows with coverage, uncertainty and sources

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Tested model or methodProtocolDatasetEvaluation setupMetricEvidence origin

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Exact evaluated configurations and original reported results
Tested configuration	Protocol and dataset	Finding	Evidence and details
Configuration: Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1	Protocol: Bennett VHH design, E. coli expression and single-concentration SPR screen of 96 designs Dataset: Bennett VHH design target Influenza Hemagglutinin	1 Count (SPR-confirmed binders; initial screening method SPR) count · higher Uncertainty: Not applicable: An exact count. Coverage: Not reported scored / Not reported eligible	Author-reported evaluation · Source checked Evidence concern: excluded from comparisons The main text says 'at lower throughput with Escherichia coli expression and single-concentration surface plasmon resonance (SPR; 95 designs per target; TcdB, IL-7Ra and influenza haemagglutinin)', but Supplementary Methods Table 6 prints 96 designs screened for TcdB, IL7R and H1 stem. Table 6's value is recorded because it is the chosen table; the main text figure is 95.Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Results paragraph 2; Supplementary Methods Table 6, column 'Designs screened' Methods, coverage and source Fine-tuned RFdiffusion VHH design against H1 stem, E. coli expression and single-concentration SPR uc-binder-20261022-protocol-bennett-vhh-ecoli-spr-96 Aggregation: Not reported Bennett 2025 Supplementary Information, including Supplementary Methods Tables 5 and 6; Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Supplementary Methods Table 6, row 'H1 stem' (Designs screened = 96), column 'SPR confirmed binders'
Configuration: Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1	Protocol: Bennett VHH design, E. coli expression and single-concentration SPR screen of 96 designs Dataset: Bennett VHH design target Influenza Hemagglutinin	1% Hit rate (SPR-confirmed binders out of designs screened; initial screening method SPR) percent · higher Uncertainty: Not reported by the source: No interval is printed. Coverage: Not reported scored / Not reported eligible	Author-reported evaluation · Source checked Evidence concern: excluded from comparisons The main text says 'at lower throughput with Escherichia coli expression and single-concentration surface plasmon resonance (SPR; 95 designs per target; TcdB, IL-7Ra and influenza haemagglutinin)', but Supplementary Methods Table 6 prints 96 designs screened for TcdB, IL7R and H1 stem. Table 6's value is recorded because it is the chosen table; the main text figure is 95.Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Results paragraph 2; Supplementary Methods Table 6, column 'Designs screened' Methods, coverage and source Fine-tuned RFdiffusion VHH design against H1 stem, E. coli expression and single-concentration SPR uc-binder-20261022-protocol-bennett-vhh-ecoli-spr-96 Aggregation: Not reported Bennett 2025 Supplementary Information, including Supplementary Methods Tables 5 and 6; Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Supplementary Methods Table 6, row 'H1 stem' (Designs screened = 96), column 'Success rate by SPR'
Configuration: Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1	Protocol: Bennett VHH design, E. coli expression and single-concentration SPR screen of 96 designs Dataset: Bennett VHH design target IL-7 Receptor α	0 Count (SPR-confirmed binders; initial screening method SPR) count · higher Uncertainty: Not applicable: An exact count. Coverage: Not reported scored / Not reported eligible	Author-reported evaluation · Source checked Evidence concern: excluded from comparisons The main text says 'at lower throughput with Escherichia coli expression and single-concentration surface plasmon resonance (SPR; 95 designs per target; TcdB, IL-7Ra and influenza haemagglutinin)', but Supplementary Methods Table 6 prints 96 designs screened for TcdB, IL7R and H1 stem. Table 6's value is recorded because it is the chosen table; the main text figure is 95.Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Results paragraph 2; Supplementary Methods Table 6, column 'Designs screened' Methods, coverage and source Fine-tuned RFdiffusion VHH design against IL7R, E. coli expression and single-concentration SPR uc-binder-20261022-protocol-bennett-vhh-ecoli-spr-96 Aggregation: Not reported Bennett 2025 Supplementary Information, including Supplementary Methods Tables 5 and 6; Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Supplementary Methods Table 6, row 'IL7R' (Designs screened = 96), column 'SPR confirmed binders'
Configuration: Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1	Protocol: Bennett VHH design, E. coli expression and single-concentration SPR screen of 96 designs Dataset: Bennett VHH design target IL-7 Receptor α	0% Hit rate (SPR-confirmed binders out of designs screened; initial screening method SPR) percent · higher Uncertainty: Not reported by the source: No interval is printed. Coverage: Not reported scored / Not reported eligible	Author-reported evaluation · Source checked Evidence concern: excluded from comparisons The main text says 'at lower throughput with Escherichia coli expression and single-concentration surface plasmon resonance (SPR; 95 designs per target; TcdB, IL-7Ra and influenza haemagglutinin)', but Supplementary Methods Table 6 prints 96 designs screened for TcdB, IL7R and H1 stem. Table 6's value is recorded because it is the chosen table; the main text figure is 95.Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Results paragraph 2; Supplementary Methods Table 6, column 'Designs screened' Methods, coverage and source Fine-tuned RFdiffusion VHH design against IL7R, E. coli expression and single-concentration SPR uc-binder-20261022-protocol-bennett-vhh-ecoli-spr-96 Aggregation: Not reported Bennett 2025 Supplementary Information, including Supplementary Methods Tables 5 and 6; Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Supplementary Methods Table 6, row 'IL7R' (Designs screened = 96), column 'Success rate by SPR'
Configuration: Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1	Protocol: Bennett VHH design, E. coli expression and single-concentration SPR screen of 96 designs Dataset: Bennett VHH design target TcdB	2 Count (SPR-confirmed binders; initial screening method SPR) count · higher Uncertainty: Not applicable: An exact count. Coverage: Not reported scored / Not reported eligible	Author-reported evaluation · Source checked Evidence concern: excluded from comparisons The main text says 'at lower throughput with Escherichia coli expression and single-concentration surface plasmon resonance (SPR; 95 designs per target; TcdB, IL-7Ra and influenza haemagglutinin)', but Supplementary Methods Table 6 prints 96 designs screened for TcdB, IL7R and H1 stem. Table 6's value is recorded because it is the chosen table; the main text figure is 95.Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Results paragraph 2; Supplementary Methods Table 6, column 'Designs screened' Methods, coverage and source Fine-tuned RFdiffusion VHH design against TcdB, E. coli expression and single-concentration SPR uc-binder-20261022-protocol-bennett-vhh-ecoli-spr-96 Aggregation: Not reported Bennett 2025 Supplementary Information, including Supplementary Methods Tables 5 and 6; Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Supplementary Methods Table 6, row 'TcdB' (Designs screened = 96), column 'SPR confirmed binders'
Configuration: Fine-tuned RFdiffusion VHH design with fine-tuned RoseTTAFold2 filtering at hotspot proportion 1	Protocol: Bennett VHH design, E. coli expression and single-concentration SPR screen of 96 designs Dataset: Bennett VHH design target TcdB	2% Hit rate (SPR-confirmed binders out of designs screened; initial screening method SPR) percent · higher Uncertainty: Not reported by the source: No interval is printed. Coverage: Not reported scored / Not reported eligible	Author-reported evaluation · Source checked Evidence concern: excluded from comparisons The main text says 'at lower throughput with Escherichia coli expression and single-concentration surface plasmon resonance (SPR; 95 designs per target; TcdB, IL-7Ra and influenza haemagglutinin)', but Supplementary Methods Table 6 prints 96 designs screened for TcdB, IL7R and H1 stem. Table 6's value is recorded because it is the chosen table; the main text figure is 95.Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Results paragraph 2; Supplementary Methods Table 6, column 'Designs screened' Methods, coverage and source Fine-tuned RFdiffusion VHH design against TcdB, E. coli expression and single-concentration SPR uc-binder-20261022-protocol-bennett-vhh-ecoli-spr-96 Aggregation: Not reported Bennett 2025 Supplementary Information, including Supplementary Methods Tables 5 and 6; Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Supplementary Methods Table 6, row 'TcdB' (Designs screened = 96), column 'Success rate by SPR'

Source checking is not independent reproduction. Release 2026-10-22-1b7cf027e4f6.

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Author-reported evaluations: 3

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Sources and history

Release 2026-10-22-1b7cf027e4f6 · Record review: source checked

2 source records and release history

Atomically accurate de novo design of antibodies with RFdiffusion (Bennett et al., Nature 2025) · Original source · Nature, version of record; Europe PMC JATS full text for PMC12727541
Bennett 2025 Supplementary Information, including Supplementary Methods Tables 5 and 6 · Original source · Supplementary Information PDF 41586_2025_9721_MOESM1_ESM.pdf, 129 pages, InDesign 17.4, ModDate 2026-09-15

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Technical metadata and extraction receipts

Stable ID: uc-binder-20261022-protocol-bennett-vhh-ecoli-spr-96

areas: proteins-complexes; protein-structure
contexts: research
denominator: 96
limitations: Developer-reported: the authors fine-tuned RFdiffusion, chose the targets, chose the filter and ran the screens.; The designs tested are those that passed the authors' own fine-tuned RoseTTAFold2 filter at hotspot proportion 1, which the Table 6 caption calls 'likely not the most predictive setting', so design and selection are coupled.; No conventional comparator (immunisation, hybridoma, phage or yeast library selection from a random library) was run on these targets.; No uncertainty is printed for any rate.; RSV-F site I and HIV Env appear in Supplementary Methods Table 5 but have no row in Table 6; the caption restricts Table 6 to 'the 5 targets for which de novo VHH binders were identified', so their outcomes are not printed.
metric: hit-rate
metric direction: higher
metric qualifier: SPR-confirmed binders out of designs screened, E. coli expression, single-concentration SPR
protocol: Computationally filtered VHH designs are expressed in Escherichia coli and screened at a single concentration by surface plasmon resonance. Table 6 reports the number of SPR-confirmed binders and the success rate out of 96 designs screened.
review: artifact sha256: d7876bc73c47f2cb3fcb0fa1ef917c89c9b67750a22f6402bf515f978cd1564a; method: source-hash-verification; independent-cell-check; ai-assisted-source-review; method note: Re-downloaded from Europe PMC (HTTP 200, 169,840 bytes) and the SHA-256 recomputed and matched. Own flattening of the JATS XML. The main text's '95 designs per target' was confirmed verbatim and the string '96 designs' does not occur, so the conflict with Supplementary Methods Table 6 is real.; note: Reviewed in docs/reviews/use-cases/binder-2026-10-22.md. Automated AI review of the source transcription only; it is not human scientific review and nothing here was reproduced experimentally.; retrieval url: https://www.ebi.ac.uk/europepmc/webservices/rest/PMC12727541/fullTextXML; reviewed at: 2026-10-22T15:00:00Z; reviewer: claude; reviewer note: Claude review agent, independent of the extraction agent; no human review claimed
selection: Designs passing the fine-tuned RoseTTAFold2 filter at hotspot proportion 1, from 200 backbones per target with the best of 8 ProteinMPNN sequences each.
selection bias: The filter is the authors' own.
source locator: Supplementary Methods Table 6, rows with 'Initial screening method' = SPR
success threshold: criterion: confirmed as a binder by single-concentration SPR; locator: Supplementary Methods Table 6, column 'SPR confirmed binders'; note: No numeric SPR response or affinity threshold is printed.
unit: percent
version: Nature 2025 version of record; Supplementary Methods Table 6

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