Review Lessons from crystals grown in the Advanced Protein Crystallisation Facility for conventional crystallisation applied to structural biology Alessandro Vergara a,b , Bernard Lorber c , Claude Sauter c , Richard Giege ´ c, * , Adriana Zagari b,d a Dipartimento di Chimica, Universita ` di Napoli "Federico II", Monte S. Angelo, 80126, Napoli, Italia b Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134, Napoli, Italia c De ´partement FMachineries Traductionnelles_ Groupe de Cristallogene `se, UPR 9002, Institut de Biologie Mole ´culaire et Cellulaire du CNRS, 15 rue Rene ´ Descartes, F-67084 Strasbourg, France d Dipartimento delle Scienze Biologiche, Universita ´ di Napoli "Federico II", Via Mezzocannone 16, 80134, Napoli, Italia Received 26 May 2005; received in revised form 23 June 2005; accepted 23 June 2005 Available online 8 September 2005 Abstract The crystallographic quality of protein crystals that were grown in microgravity has been compared to that of crystals that were grown in parallel on earth gravity under otherwise identical conditions. A goal of this comparison was to assess if a more accurate 3D-structure can be derived from crystallographic analysis of the former crystals. Therefore, the properties of crystals prepared with the Advanced Protein Crystallisation Facility (APCF) on earth and in orbit during the last decade were evaluated. A statistical analysis reveals that about half of the crystals produced under microgravity had a superior X-ray diffraction limit with respect of terrestrial controls. Eleven protein structures could be determined at previously unachieved resolutions using crystals obtained in the APCF. Microgravity induced features of the most relevant structures are reported. A second goal of this study was to identify the cause of the crystal quality enhancement useful for structure determination. No correlations between the effect of microgravity and other system-dependent parameters, such as isoelectric point or crystal solvent content, were found except the reduced convection during the crystallisation process. Thus, crystal growth under diffusive regime appears to be the key parameter explaining the beneficial effect of microgravity on crystal quality. The mimicry of these effects on earth in gels or in capillary tubes is discussed and the practical consequences for structural biology highlighted. D 2005 Elsevier B.V. All rights reserved. Keywords: Protein structure; Crystal quality; Microgravity; Diffusion; Advanced Protein Crystallisation Facility Contents 1. Background........................................................... 103 2. Experimentation in the APCF ................................................. 103 3. Contribution of crystal growth experiments in the APCF to structural biology ........................ 104 4. Value of structures derived from Ag-grown crystals ...................................... 106 4.1. General considerations ................................................. 106 4.2. Collagen-like synthetic polypeptide ........................................... 106 0301-4622/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bpc.2005.06.014 Abbreviations: Ag, microgravity; ADH, alcohol dehydrogenase; APCF, Advanced Protein Crystallisation Facility; AspRS-1, aspartyl-tRNA synthetase (form 1 of eubacterial type); B, atomic displacement parameter; CcdB, poison of DNA-topoisomerase II complexes; d, resolution limit; ESA, European Space Agency; FWHM, Full Width at Half Maximum; g, ground; Hyp, hydroxyproline; I / r, intensity to standard deviation ratio; ISS, International Space Station; M r , molecular weight; DCAM, Diffusion Controlled Crystallisation Apparatus for Microgravity; PCDF, Protein Crystallisation Diagnostic Facility; PDB, Protein Data Bank; (PPG) 10 , synthetic polypeptide (Pro – Pro – Gly) 10 ; VDA, Vapour Diffusion Apparatus. * Corresponding author. Tel.: +33 3 88 41 70 58; fax: + 33 3 88 60 22 18. E-mail address: r.giege@ibmc.u-strasbg.fr (R. Giege ´). Biophysical Chemistry 118 (2005) 102 – 112 http://www.elsevier.com/locate/biophyschem