title: An approach to automated acquisition of cryoEM images from lacey carbon grids.
abstract: An approach to automated acquisition of cryoEM image data from lacey carbon grids using the Leginon program is described. Automated liquid nitrogen top up of the specimen holder dewar was used as a step towards full automation, without operator intervention during the course of data collection. During cryoEM studies of actin labelled with myosin V, we have found it necessary to work with lacey grids rather than Quantifoil or C-flat grids due to interaction of myosin V with the support film. Lacey grids have irregular holes of variable shape and size, in contrast to Quantifoil or C-flat grids which have a regular array of similar circular holes on each grid square. Other laboratories also prefer to work with grids with irregular holes for a variety of reasons. Therefore, it was necessary to develop a different strategy from normal Leginon usage for working with lacey grids for targeting holes for image acquisition and suitable areas for focussing prior to image acquisition. This approach was implemented by using the extensible framework provided by Leginon and by developing a new MSI application within that framework which includes a new Leginon node (for a novel method for finding focus targets).
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, LS2 9JT, UK. william_v_nicholson@yahoo.com
authors.forename: William V
authors.initials: WV
authors.lastname: Nicholson
authors.forename: Howard
authors.initials: H
authors.lastname: White
authors.forename: John
authors.initials: J
authors.lastname: Trinick
mesh_terms: D002244:Carbon; D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*
chemical_list: D002244:Carbon
references.citation: J Struct Biol. 1999 Dec 1;128(1):3-14
references.pmid: 10600552
references.citation: J Struct Biol. 1997 Dec;120(3):309-19
references.pmid: 9441934
references.citation: Ultramicroscopy. 1999 Jul;77(3-4):153-61
references.pmid: 10406132
references.citation: Cell. 1996 Oct 18;87(2):241-51
references.pmid: 8861908
references.citation: Q Rev Biophys. 1995 May;28(2):171-93
references.pmid: 7568675
references.citation: J Struct Biol. 2006 Sep;155(3):470-81
references.pmid: 16762565
references.citation: J Struct Biol. 2008 Oct;164(1):166-9
references.pmid: 18621546
references.citation: Structure. 2008 Mar;16(3):441-8
references.pmid: 18334219
references.citation: J Struct Biol. 2006 Jun;154(3):303-11
references.pmid: 16632377
references.citation: J Struct Biol. 2003 Aug;143(2):135-44
references.pmid: 12972350
references.citation: J Struct Biol. 2005 Apr;150(1):69-80
references.pmid: 15797731
references.citation: J Struct Biol. 2009 Jan;165(1):1-9
references.pmid: 18926912
references.citation: Science. 2009 Dec 4;326(5958):1369-73
references.pmid: 19933108
references.citation: J Struct Biol. 2001 Sep;135(3):251-61
references.pmid: 11722165
references.citation: Nature. 2000 Jun 15;405(6788):804-7
references.pmid: 10866203
references.citation: Cell. 2009 Feb 6;136(3):485-95
references.pmid: 19203583
references.citation: J Microsc. 2007 Dec;228(Pt 3):384-9
references.pmid: 18045333
references.citation: Nature. 1994 Sep 15;371(6494):261-4
references.pmid: 7915827
references.citation: J Struct Biol. 2005 Jul;151(1):41-60
references.pmid: 15890530
references.citation: J Struct Biol. 2000 Oct;132(1):33-45
references.pmid: 11121305
references.citation: Nature. 2003 Feb 13;421(6924):715-8
references.pmid: 12610617
references.citation: Science. 2009 Dec 4;326(5958):1412-5
references.pmid: 19933110
references.citation: Nature. 2006 Jul 13;442(7099):212-5
references.pmid: 16838021