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Updated: Jan 28, 2026

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
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Long-wavelength Mesh&Collect native SAD phasing from microcrystals.

Michele Cianci1, Max Nanao2, Thomas R Schneider3

  • 1Department of Agricultural, Food and Environmental Sciences, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy.

Acta Crystallographica. Section D, Structural Biology
|March 2, 2019
PubMed
Summary
This summary is machine-generated.

This study demonstrates efficient macromolecular phase determination from microcrystals using anomalous scattering. By optimizing beam energy and utilizing a microbeam, researchers successfully obtained phasing information from naturally present ions like manganese and calcium.

Keywords:
genetic algorithmlong wavelengthmeshmicrocrystalsphasing

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Area of Science:

  • Structural Biology
  • Crystallography
  • Biophysics

Background:

  • Macromolecular crystallography often requires crystals larger than 10,000 µm³ for native phasing.
  • Harnessing anomalous signals from smaller microcrystals (<10,000 µm³) presents challenges in experimental planning.
  • Naturally present scatterers (sulfur, phosphorus, calcium) and crystal size influence experimental parameters.

Purpose of the Study:

  • To determine experimental phases from microcrystals using anomalous scattering.
  • To optimize data collection strategies for small crystals at synchrotron beamlines.
  • To leverage naturally occurring anomalous scatterers for phasing.

Main Methods:

  • Data collection using Mesh&Collect on concanavalin A microcrystals (∼20 µm) at the EMBL beamline P13, PETRA III.
  • Utilized a microbeam at 1.892 Å (6.551 keV), near the Mn K-edge, to enhance the Bijvoet ratio.
  • Serial data collection from flash-cooled microcrystals in micro-meshes, followed by data integration, scaling, and merging using a genetic algorithm.

Main Results:

  • Achieved an expected Bijvoet ratio of 2.1% at 6.551 keV, significantly higher than 0.7% at 12.6 keV.
  • Successfully determined experimental phases using anomalous signals from naturally present Mn²⁺ and Ca²⁺ ions.
  • Obtained a high-completeness, high-multiplicity data set enabling complete auto-tracing of the polypeptide chain.

Conclusions:

  • The optimized experimental approach allows accurate anomalous signal measurement from small crystals.
  • This method enables experimental phase determination, facilitating structure solution of proteins from microcrystals.
  • The entire experiment was completed within a practically acceptable timeframe of less than 2 hours.