Technology development will dominate 2011 as we establish the tools necessary for attacking our protein targets
- Establish pipeline for protein production from bacteria, yeast and insect cells
- Establish standard conditions for screening 2D crystallization of novel targets
- Implement methods for detergent removal/crystallization
  - microfluidic 96-well dialysis block
  - robotic micropipettor for incremental addition of cyclodextrins
- Develop software for automated imaging and evaluation of crystallization trials by electron microscopy
- Demonstrate utility of phase extension methods for electron crystallography
- Implement iterative helical reconstruction methods

Targets
- P2A3 Zn transporter: Comparison of membrane bound form with X-ray structure of detergent-solubilized homolog (YiiP^?)

TEMIMPS Goals for next 5 years

Targets
- Existing crystals
  - AQP2: Comparison of constructs mimicking phosphorylated/dephosphorylated state
  - AQP0: Interaction of channel with native lipids
  - KvAP^? channel: Structure of membrane bound channel to address gating
- Novel targets
  - GPCRs: cannabinoid, seratonin
  - TRP channnels: TRPA1 pain receptor
  - Rhomboids
  - secondary transporters: Heme transporter FLVCR
- Oligomeric proteins
  - gamma-secretase
  - Kdp (ATP-dependent P-type potassium pump)
- Complexes
  - G-protein in complex with GIRK channel
  - AQP2 in complex with AKAP79 adaptor
  - Two-component system (PhoP^?/PhoQ)

Technologies
- establish general high-throughput pipeline for screening 2D crystallization within lipid bilayers
  - automation of crystallization
  - automation of imaging
  - integration with database
  - general enough and affordable to interest other EM groups
- develop software for automated data collection
  - low-dose imaging
  - electron diffraction
- improve software for structure determination
  - novel methods for image analysis of poorly ordered crystals
  - more robust/automated analysis of electron diffraction

Protocols
- define important parameters affecting 2D crystallization
  - define matrix of conditions for crystallization screen
- extend methods for structure determination by electron crystallography
  - molecular replacement
  - phase extension
  - density modification

Notable achievements

none to date

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-- DavidStokes - 14 Nov 2010

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