biostudies-literaturePachl PFrench Ministry for Higher Education and ResearchInstitut des sciences biologiquesFrench Ministry for Europe and Foreign AffairsFrench Centre National de la Recherche Scientifique (CNRS)Laboratoire d'Excellence NetRNAInstitut des Sciences Biologiques (10.13039/501100017210 - CNRS biologie)Centre National de la Recherche ScientifiqueCzech Ministry for Education, Youth and SportsNextGenerationEUMinistère de l'Europe et des Affaires ÉtrangèresLaboratoire d'Excellence NetRNAUniversité de Strasbourg532-541https://www.ebi.ac.uk/biostudies/studies/S-EPMC11961392biostudies-literatureUnknown15(4)The production of high-quality crystals is a key step in crystallography in general, but control of crystallization conditions is even more crucial in serial crystallography, which requires sets of crystals homogeneous in size and diffraction properties. This protocol describes the implementation of a simple and user-friendly microfluidic device that allows both the production of crystals by the counter-diffusion method and their in situ analysis by serial crystallography. As an illustration, the whole procedure is used to determine the crystal structure of three proteins from data collected at room temperature at a synchrotron radiation source.FEBS open bioProtein crystallization and structure determination at room temperature in the CrystalChip.PMC11961392LX22NPO5102USIAS‐W21RSAUT49306SCANR‐10‐LABX‐0036_NETRNAUSIAS-W21RSAUT8J23FR035ANR-10-LABX-0036_NETRNANilles LScheer HRezacova PCoudray LFejfarova ARitzenthaler CEngilberge SPachl PSauter CVincent RfalseProtein crystallization and structure determination at room temperature in the CrystalChip.The production of high-quality crystals is a key step in crystallography in general, but control of crystallization conditions is even more crucial in serial crystallography, which requires sets of crystals homogeneous in size and diffraction properties. This protocol describes the implementation of a simple and user-friendly microfluidic device that allows both the production of crystals by the counter-diffusion method and their in situ analysis by serial crystallography. As an illustration, the whole procedure is used to determine the crystal structure of three proteins from data collected at room temperature at a synchrotron radiation source.2025-01-01T00:00:00Z2025 Apr2026-04-08T19:22:01.441Z2026-04-08T13:22:02.582ZS-EPMC119613923957288610.1002/2211-5463.13932