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TitleBreaking Barriers: Transitioning from X-ray Crystallography to Cryo-EM for Structural Studies of ATAD2B.
Journal, issue, pagesbioRxiv, Year 2025
Publish dateOct 14, 2025
AuthorsHassan Zafar / Kiera L Malone / Ajit K Singh / Michael A Cianfrocco / Karen C Glass /
PubMed AbstractCryo-electron microscopy (cryo-EM) has transformed structural biology by enabling near-atomic resolution of large macromolecular complexes without the need for crystallization. Here, we describe our ...Cryo-electron microscopy (cryo-EM) has transformed structural biology by enabling near-atomic resolution of large macromolecular complexes without the need for crystallization. Here, we describe our laboratory's transition from X-ray crystallography to single-particle cryo-EM to investigate the ATPase family AAA+ domain-containing protein 2B (ATAD2B), a chromatin regulator implicated in epigenetic signaling. We outline the challenges encountered during protein expression, purification, and sample preparation, including co-purification of the chaperonin GroEL, and strategies employed to overcome these obstacles. Our workflow highlights critical steps in sample optimization, grid vitrification, and data processing using CryoSPARC, cisTEM, and Topaz, as well as computational requirements for high-resolution reconstructions. We also discuss model building, refinement, and validation approaches, emphasizing best practices for new cryo-EM users. This work provides practical insights for structural biologists adopting cryo-EM, particularly for large, flexible protein complexes, and underscores the importance of integrated approaches combining biochemical, computational, and imaging strategies.
External linksbioRxiv / PubMed:41279824 / PubMed Central
MethodsEM (single particle)
Resolution3.3 - 4.2 Å
Structure data

73044

9ykc

EMDB-73044, PDB-9ykc:
Cryo-EM structure of GroEL-gammaATP
Method: EM (single particle) / Resolution: 3.3 Å

73045

9yke

EMDB-73045, PDB-9yke:
GroEL Apoenzyme
Method: EM (single particle) / Resolution: 3.7 Å

73200

9ynj

EMDB-73200, PDB-9ynj:
Cryo-EM structure of GroEL-ADP
Method: EM (single particle) / Resolution: 4.2 Å

Chemicals

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

Source
  • escherichia coli (E. coli)
KeywordsCHAPERONE / Chaperonin / Tetradecamer / Nucleotide-binding / Allosteric-change / nucleotide binding / apoenzyme / protein folding / molecular chaperone

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