New methods
Download
1 / 21

“New” methods - PowerPoint PPT Presentation


  • 67 Views
  • Uploaded on

“New” methods. By Paul Ellis. Derivatizing with quick soaks. Quick soaks can be much less time consuming than traditional long soaks or cocrystallising High concentrations can be destructive of crystal order Ions used include: Br - , I - Cs + , Rb + Gd 3+ , Ho 3+ , Sm 3+ , Eu 3+. PPLO.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about ' “New” methods' - kalei


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
New methods

“New” methods

By Paul Ellis


Derivatizing with quick soaks
Derivatizing with quick soaks

  • Quick soaks can be much less time consuming than traditional long soaks or cocrystallising

  • High concentrations can be destructive of crystal order

  • Ions used include:

    • Br-, I-

    • Cs+, Rb+

    • Gd3+, Ho3+, Sm3+, Eu3+


PPLO

  • Pichia pastoris lysyl oxidase – an analogue for mammalian lysyl oxidase:

    • 70 kDa glycoprotein

    • 1 intrinsic Cu

    • 4 molecules in the asymmetric unit

  • Tried long soaks and cocrystallising with:

    • Hg(II), Yb(III), Sm(III), PIP, EMTS, WO42-, IrCl42-, Os(III), Kr…

    • Poor resolution

    • No peaks in anomalous Patterson

  • Tried short soaks in KBr

    • ≥ 1.0 M, ≥ 120 s destroyed crystal

    • Good resolution

    • Good peaks in anomalous Patterson with 90 s, 0.75 M




Krypton xenon

Experimental

Experimental

Energies for MAD were chosen using a fluorescence scan from each crystal

Energies for MAD were chosen using a fluorescence scan from each crystal

Mb

rotation angle /image: 0.5°

exposure time: 15 s

total rotation range: 180°

resolution range: 17-1.7 Å

measured reflections: 230000

unique reflections: 23000

completeness: 99.6%

anom. completeness: 99.6%

multiplicity: 10.0

I/(I) (overall): 26.6

I/(I) (1.71-1.70 Å): 11.4

Rmerge: 0.065

Mb

rotation angle /image: 0.5°

exposure time: 15 s

total rotation range: 180°

resolution range: 17-1.7 Å

measured reflections: 230000

unique reflections: 23000

completeness: 99.6%

anom. completeness: 99.6%

multiplicity: 10.0

I/(I) (overall): 26.6

I/(I) (1.71-1.70 Å): 11.4

Rmerge: 0.065

SP18

rotation angle /image: 0.5°

exposure time: 15 s

total rotation range: 180°

resolution range: 24-2.0 Å

measured reflections: 730000

unique reflections: 18800

completeness: 99.6%

anom. completeness: 99.9%

multiplicity: 38.0

I/(I) (overall): 30.0

I/(I) (2.02-2.00 Å): 1.41

Rmerge: 0.097

SP18

rotation angle /image: 0.5°

exposure time: 15 s

total rotation range: 180°

resolution range: 24-2.0 Å

measured reflections: 730000

unique reflections: 18800

completeness: 99.6%

anom. completeness: 99.9%

multiplicity: 38.0

I/(I) (overall): 30.0

I/(I) (2.02-2.00 Å): 1.41

Rmerge: 0.097

Krypton & Xenon

  • Underutilized

  • More isomorphous than traditional derivatives

  • Must be stable in cryoprotectant

  • Good chance of useful derivative

  • Quillin: large-to-small mutation to create binding site


Kr k edge
Kr K edge


Sp18 by kr mad
SP18 by Kr MAD

RAW

AFTER wARP


Sad v mad
SAD v. MAD

  • SAD will be the method of choice for high throughput

  • Programs designed for SAD data are becoming available

  • Robots will give experimenters more freedom to try SAD


Hibadh a sad example
HIBADH – a SAD example

  • 3-hydroxyisobutyrate dehydrogenase

    • a ubiquitous enzyme involved in valine catabolism

  • 1 Crystal

    • P43212, 103 × 103 × 108 Å

    • 2 × 295 residues in asu ≈ 70 kDa

    • Grown in 5 mM Pb2+

  • Data collection

    • Δφ = 94°

    • dmin = 2.2 Å

    • R = 9.7%

    • Multiplicity = 7.6

    • <I/σ(I)> = 15

    • λ = 0.79 Å (Pb f " ≈ 10e-)



Structure solution
Structure solution

SHARP

DM

wARP

<ΔF±>/<F> ≈ 2.5%





Missing elements
Missing elements

Incorrect

Correct





Summary
Summary

  • Quick soaks

    • Dauter, Z., Li., M., & Wlodawer, A. (2001). Practical experience with the use of halides for phasing macromolecular structures: a powerful tool for structural genomics. Acta Cryst. D57, 239-249.

    • Nagem, R.A.P., Dauter, Z., & Polikarpov, I. (2001). Protein crystal structure solution by fast incorporation of negatively and positively charged anomalous scatterers. Acta Cryst. D57, 996-1002.

  • Kr and Xe

    • Cohen, A.E., Ellis, P.J., Kresge, N. & Soltis, S.M. (2001). MAD phasing with krypton. Acta Cryst. D57, 233-238.

    • Quillin, M.L., & Matthews, B.W. (2002). Generation of noble-gas binding sites for crystallographic phasing using site-directed mutagenesis. Acta Cryst. D58, 97-103.

  • SAD

    • Dauter, Z., Dauter, M., & Dodson, E. (2002). Jolly SAD. Acta Cryst. D58, 494-506.

  • Low energy


Acknowledgements
Acknowledgements

  • School of Molecular & Microbial Biosciences, University of Sydney:

    • Hans Freeman

    • Mitchell Guss

    • Anthony Duff

  • Department of Chemistry & Biochemistry, Montana State University:

    • David M. Dooley

  • Department of Molecular Biochemistry, Ohio State University:

    • Russ Hille

    • Thomas Conrads

  • Structural Molecular Biology, SSRL:

    • Peter Kuhn

    • Mike Soltis

    • Aina Cohen

    • Nancy Fathali

  • Department of Energy:

    • Office of Basic Energy Sciences

    • Office of Biological and Environmental Research

  • National Institutes of Health, National Center for Research Resources, Biomedical Technology Program


ad