Home › Biochemistry › Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect
Biochemistry JoVE (Open Access) Citable · DOI

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

DOI: 10.3791/58594-v
What you'll learn
  • ✓ Prepare and load protein crystals for synchrotron beamline experiments
  • ✓ Execute MeshAndCollect workflow to collect partial diffraction datasets
  • ✓ Assemble complete diffraction data from multiple small crystals for structure determination
Protocol

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

Difficulty
advanced
Total time
~4–8 hours (beamline session); preparation ~1–2 days prior

Steps

1
Prepare synchrotron experiment offline

Plan and organize experimental parameters, crystal mounting strategies, and data collection protocols before arrival at the beamline facility.

▶ 00:51
2
Load sample onto synchrotron beamline

Mount prepared protein crystal samples onto the beamline goniometer and align them for X-ray exposure.

▶ 01:31
3
Execute MeshAndCollect automated workflow

Run the MeshAndCollect protocol to automatically collect partial diffraction data from multiple small crystals in a single automated session.

▶ 03:49
4
Determine fluorescent protein structure

Merge and process collected partial datasets to assemble complete diffraction data for downstream structure solution of Cerulean.

▶ 05:02
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