title: A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization.
abstract: Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.
authors.affiliation: Oxford Protein Production Facility, Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Headington, Oxford OX3 7BN, England.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Walter
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Diprose
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Mayo
authors.forename: Christian
authors.initials: C
authors.lastname: Siebold
authors.forename: Mike G
authors.initials: MG
authors.lastname: Pickford
authors.forename: Lester
authors.initials: L
authors.lastname: Carter
authors.forename: Geoff C
authors.initials: GC
authors.lastname: Sutton
authors.forename: Nick S
authors.initials: NS
authors.lastname: Berrow
authors.forename: James
authors.initials: J
authors.lastname: Brown
authors.forename: Ian M
authors.initials: IM
authors.lastname: Berry
authors.forename: Guillaume B E
authors.initials: GB
authors.lastname: Stewart-Jones
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Grimes
authors.forename: David K
authors.initials: DK
authors.lastname: Stammers
authors.forename: Robert M
authors.initials: RM
authors.lastname: Esnouf
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
authors.forename: Ray J
authors.initials: RJ
authors.lastname: Owens
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.forename: Karl
authors.initials: K
authors.lastname: Harlos
mesh_terms: D000818:Animals; D001331:Automation / Q000295:instrumentation / Q000379:methods; D018360:Crystallography, X-Ray / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins