A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization.
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.
Walter
Thomas S
TS
Oxford Protein Production Facility, Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Headington, Oxford OX3 7BN, England.
Diprose
Jonathan M
JM
Mayo
Chris J
CJ
Siebold
Christian
C
Pickford
Mike G
MG
Carter
Lester
L
Sutton
Geoff C
GC
Berrow
Nick S
NS
Brown
James
J
Berry
Ian M
IM
Stewart-Jones
Guillaume B E
GB
Grimes
Jonathan M
JM
Stammers
David K
DK
Esnouf
Robert M
RM
Jones
E Yvonne
EY
Owens
Ray J
RJ
Stuart
David I
DI
Harlos
Karl
K
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*
D011506:Proteins