Cryo-EM

Cryo-electron microscopy (Cryo-EM)

一行要約

Cryo-EM は急速凍結された生体分子を低温電子顕微鏡で撮像し、計算的に 3D 再構築する構造生物学手法。resolution revolution (2013-) で near-atomic resolution (2-3 Å) が routine 化し、X-ray crystallography が困難な membrane protein, large complex (ribosome, spliceosome), antibody-antigen complex, bispecific antibody / ADC linker 構造を高解像度で決定。腫瘍領域では IO 標的構造 (PD-1/PD-L1, CTLA-4), bispecific T-cell engager geometry, ADC payload conjugation site, receptor activation 機構の構造的合理化に貢献。

原理

(1) 試料を液体エタン (~-180°C) で plunge freeze → vitreous ice 中に分子を保存 (結晶化不要)。(2) Titan Krios 等の高解像度 EM で direct electron detector (K3 / Falcon) を用い数千-数万枚の micrograph 取得。(3) RELION / cryoSPARC pipeline で粒子検出 → 2D classification → 3D reconstruction → refinement → model building (Coot / Phenix)。(4) AlphaFold2 predicted structure を初期モデルに hybrid refinement する手法が標準化。

Cryo-electron tomography (cryo-ET) は cellular context で in situ 構造を取得 (細胞内 organelle / membrane assembly)。

主要エビデンス / 適用領域

• IO 標的構造: PD-1/PD-L1, CTLA-4/B7, TIGIT/PVR の binding interface
• Bispecific antibody geometry: T-cell engager (BiTE / DART) の dual-arm orientation, dual-affinity, T-cell synapse 形成 capacity
• ADC: payload conjugation site / DAR (drug-antibody ratio) 効果、linker 安定性 / bystander effect の構造的合理化
• EGFR / HER2 / MET activation: ligand binding → dimerization → kinase activation の構造遷移
• GPCR / ion channel drug binding: novel druggable pocket discovery

適用分野と限界

• 強み: 結晶化不要、large / membrane / heterogeneous complex、native conformation 保存、AlphaFold2 hybrid で新規分子可
• 限界: 試料量 (数 µg-mg)、preferred orientation 問題、small molecule (<100 kDa) は単独再構築困難、cost (Krios ~$5M)、computational throughput 大、専門技術依存

Open Questions

• In situ structural biology: cryo-ET で細胞内 cancer-relevant complex を直接観察
• Time-resolved cryo-EM: drug binding kinetics / conformational dynamics
• AI-driven model building: AlphaFold3 / RoseTTAFold-AA との hybrid 標準化
• Throughput / cost reduction: clinical structural biology への展開

関連エンティティ

• 補完手法: Proteomics-mass-spec / Proximity-labeling-BioID-APEX / Machine-learning-AI
• 関連薬剤: ADC / BiTE-bispecific / PD-1-inhibitor / PD-L1-inhibitor / CTLA-4-inhibitor / EGFR-MET-bispecific / HER2-directed-therapy
• 関連遺伝子: EGFR / HER2 / MET / PD-L1

関連論文

直近 30 件のみ表示中 (関連 47 件)。 全件は MOC ページ または Obsidian (source Markdown) から辿れます。

Basic-Extracellular vesicles

• Pegtel et al. AnnuRevBiochem 2019 — Exosomes
• Kudo et al. CellMetab 2022 — Secreted phospholipase A2 modifies extracellular vesicles and accelerates B cell lymphoma
• Sun et al. CellMolLifeSci 2020 — The SF3b complex Splicing and beyond
• Cocozza et al. EMBOJ 2023 — Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic
• Ochkasova et al. IntJMolSci 2023 — Two edges in our knowledge on the functions of ribosomal proteins the revealed contributions of their regions to
• Crescitelli et al. JExtracellVesicles 2025 — Recommendations for studying in situ extracellular vesicles from solid tissue
• Du et al. JNanobiotechnology 2026 — Plant-derived nanovesicles the intelligent nanoplatforms for therapeutics and drug delivery
• Tian et al. NatBiomedEng 2026 — Skeletal-muscle-targeted non-viral delivery of full-length DMD mRNA for duchenne muscular dystrophy
• Chen et al. NatCellBiol 2019 — Extracellular vesicle-packaged HIF-1alpha-stabilizing lncRNA from tumour-associated macrophages regulates aerobic
• Bertin et al. NatCommun 2020 — Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation
• An et al. NatCommun 2021 — An active tethering mechanism controls the fate of vesicles
• El-Shennawy et al. NatCommun 2022 — Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2
• Gong et al. NatMater 2024 — Tumour-derived small extracellular vesicles act as a barrier to therapeutic nanoparticle delivery
• DAcunzo et al. SciAdv 2021 — Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in down syndrome

Basic-Immunotherapy

• Corrigan et al. SciImmunol 2026 — In vivo reprogramming of cytotoxic effector CD8 T cells via fractalkine-conjugated mRNA-LNPs
• Zhou et al. Science 2026 — Intermetallic nanoassemblies potentiate systemic STING activation

Basic-Neutrophil and myeloid cell

• Burn et al. Nature 2025 — Myeloperoxidase transforms chromatin into neutrophil extracellular traps

Basic-Oncogene and genetics

• Li et al. NatCommun 2026 — Molecular glue that stabilizes the LRPPRC-MET-G4 interaction complex to drive MET downregulation
• Sroka et al. Nature 2021 — An epigenetic fulcrum tipped in cancer

Basic-Others

• Wilkinson et al. AnnuRevBiochem 2020 — RNA splicing by the spliceosome
• Wen et al. Blood 2022 — Molecular mechanisms of leukocyte beta2 integrin activation
• Yan et al. ColdSpringHarbPerspectBiol 2019 — Molecular mechanisms of pre-mRNA splicing through structural biology of the spliceosome
• Burley et al. NEnglJMed 2021 — Predicting proteome-scale protein structure with artificial intelligence
• Jiang et al. NatNanotechnol 2026 — Efficient prime editing in vivo and in vitro using lipid nanoparticles
• Pike et al. NatRevChem 2026 — Lessons learned in linking PROTACs from discovery to the clinic
• Lu et al. Nature 2026 — Molecular glue degraders of HuR suppress BRAF-mutant colorectal cancer
• Zhang et al. Nature 2026 — The mutational landscape of STING-induced immunity

Clinical-NSCLC-ALK

• Pailler et al. ClinCancerRes 2019 — Acquired resistance mutations to ALK inhibitors identified by single circulating tumor cell sequencing in

Clinical-NSCLC-Driver-Other

• Liu et al. CancerDrugResist 2020 — RET kinase alterations in targeted cancer therapy

Clinical-NSCLC-EGFR

• Brown et al. ClinCancerRes 2019 — On-target resistance to the mutant-selective EGFR inhibitor osimertinib can develop in an allele-specific manner