Download Use of Solvent Iodide Ions as an Effective In

Use of Solvent Iodide Ions
as an Effective In-house Tool
for Crystallographic Phasing
Michael R. Sawaya & Duilio Cascio
January 17, 2002
Phasing by solvent halide ions promises to be a
revolutionary new way to phase protein structures
Acta Cryst (2000). D56, 232-237
Quick soak in halide (30 seconds)
Easy to perform
Applicable to any protein
Non-toxic, no heavy atom waste
High quality phasing
Iodide is the choice of halide for
in-house phasing
Anomalous scattering
correction factors
at CuKa wavelength
Atom
f”
I-
6.9
Pt
7.0
Br-
1.3
C
0.01
N
0.02
O
0.03
S
0.56
At CuKa, f” is comparable to Pt, Hg, Au
54 electrons
F-RD generator high flux, fine focus
How successful is phasing by
iodide at UCLA?
Original study performed with only 4 proteins using a synchrotron source.
Will this method prove to be generally applicable in a real academic
research lab with real proteins with real problems?
What are the limitations? (e.g. resolution limits, lack of isomorphism,
data quality, soaking conditions, number of iodide sites required for good
phases)
Will all proteins bind a sufficient number
of iodides to generate good phases?
Dauter et al., suggest that the number
of iodides bound is simply proportional
to the surface area of the protein. Is this
always true?
Students, Post docs, and Staff from MBI generously
donated their crystals to test the effectiveness of
iodide soaks in phasing protein structure
Thank You for the crystals!
protein
contributor
Rv1926c
Celia Goulding
Rv3697c
Celia Goulding
Rv2878c
Celia Goulding
DsbD-N-term
Celia Goulding
P51
Chongwoo Kim
SmAP
Cameron Mura
NarLc complex
Ann Maris
RNase ds-trimer
Yanshun Liu
Protein isoaspartyl
methyl transferase
Scott Griffith
Daniel Boutz
Myoglobin
Maria Grzeskowiak
Proteinase K
Helty Adisetiyo
Thaumatin
Helty Adisetiyo
Xylanase
Helty Adisetiyo
lysozyme
Students of M230B (2001)
Experimental methods flow chart
SOAKING PROCEDURES
•Weigh 0.008g KI (one medium sized grain)
•Dissolve KI in 100 uL of reservoir solution
•Add appropriate % of glycerol (for cryoprotection)
•Soak crystal 30 seconds
•Mount on cold stream
DATA COLLECTION
PROCEDURES
•Collect data on F-RD when possible
•Collect 360 degrees of data
•Process with Denzo/Scalepack
DETERMINE IODIDE SUBSTRUCTURE
Import data with xprepx (Bruker)
•Calculate difference Patterson coefficients using SAS, SIRAS, SIR data
•Locate Iodide sites with ShelxD
•Verify quality of sites by overlapping predicted Patterson peaks on Patterson map
PHASING
•CCP4 suite: scalepack2mtz, truncate, cad, scaleit, mlphare, dm
MODEL BUILDING
•Arp/wArp
12/14 crystals soaked showed clear
evidence of iodide binding
8/14 Iodide soaks led to complete structure determination
1 structure had not been previously determined
Example of electron density generated by
SIRAS phasing based on 11 iodide sites
DsbD N-term
Summary: Iodide is my first choice for derivatization.
No other heavy atom as successful with so many proteins
under so many different conditions
Eight
structures
solved
using
phases
based on
iodide
Clear
iodide
sites but
poor
phasing
NonIsomorph
ous
1.8A
3
13%
Tips for successful phasing with iodide
Poor peak heights in difference Patterson map? High
redundancy of intensity measurements is crucial
to locating heavy atom sites and phasing. Collect 360 degrees of
data. Not just iodide, but any derivative would benefit.
Iodide soak is non isomorphous with native? Nonisomorphorism can be reduced by a quick back-soak in cryoconditions lacking iodide. (eg. Rv2878c)
Iodide sites not convincing? ShelxD often succeeds at finding
iodide sites based on anomalous differences alone. But, If the
solution is not clear, try using isomorphous differences (SIR) or a
combination of isomorphous and anomalous differences (SIRAS)
output by xprepx.
Data collected using FR-D generator can produce
better quality maps than RU200 generator
I/s (2.0 A)
Rsym (2.0 A)
7.6
37.3%
26.4
7.1%
Poor phasing is a direct consequence of too few
iodides/surface area
Need 1 iodide bound per 10-20 residues
Why do some proteins bind
disproportionately fewer
iodides/surface area?
Two possibilities
1) Soaking conditions (e.g. pH, salt, buffer) disfavor or
compete with iodide binding. If true then we could search for
conditions that favor iodide binding.
or
2) Residue composition of the protein surface disfavors
iodide binding. Make predictions about iodide binding based on
amino acid composition.
Iodide binding appears insensitive to
the composition of the cryo-solvent
Experiment to test effects of cryo-solvent on iodide substitution
Thaumatin
1.3M Na,K tartrate
35% glycerol
Bis-Tris pH 6.5
0.5M KI
Rv1926c
0.1M (NH4)2SO4
30% PEG 4000
Tris pH 7.0
0.5M KI
1 iodide/14 residues
1 iodide/47 residues
Soaking a Rv1926c crystal in thaumatin’s cryo-conditions did
not increase the number of iodides bound.
But, why expect conditions that are optimal for iodide binding to one protein to also be optimal for another protein?
Thaumatin is a more basic protein (pI=8.5) than Rv1926c (pI=6.1). Perhaps if I tried a more substantial change in pH to
change the electrostatic potential of the surface…
Higher pH appears to weaken
iodide binding
Experiment to test effects of pH on iodide substitution
Proteinase K
0.1M (NH4)2SO4
30% glycerol
Cacodylate pH 6.5
0.5M KI
Proteinase K
20% PEG 8000
20% glycerol
CHES pH 9.5
0.5M KI
Top 3 negative peaks in Fobs(pH9.5) –Fobs(pH6.5) difference
Fourier map correspond to iodide sites.
Tally of side chains in contact with
102 iodide sites
Note: Arginine and lysine are the two residues most frequently found in iodide
binding sites.
The amino acid composition favored by iodide is significantly
depleted in negatively charged side chains compared to the average
amino acid composition on the surface of most proteins
CC=0.77
Red data points taken from
The Atomic Structure of
Protein-Protein Recognition
Sites by Lo Conte, Chothia &
Janin, J. Mol. Biol.,
285,2177-2198
Most successful iodide experiments were conducted at a pH
below the pI with the exception of Rv2878c
>
>
>
>
>
<
>
>
<
<
<
<
1.8A
3
13%
A protein may still bind iodide
even if pH > pI since iodide
binding sites are often nonpolar
No consensus
coordination
geometry
Polar & nonpolar
Hydrogens at a radius
3.5-4 Angstroms
Peptide planes
Could involve any of the
20 amino acids
Conclusions
SIRAS phasing from iodide soaks in-house is effective,
quick, easy, and non-toxic. 8/14 structures could be
determined at UCLA
Even in cases where there are too few iodide sites to
produce a good map, iodide sites could be used in
combination with other derivatives (e.g. CsCl).
High redundancy, high resolution, and a bright, focused
x-ray source (F-RD) are important factors for success.
Soaking at pH < pI improves chances of success
Future: Lower the pH of cryo-conditions of Rv1926c or
xylanase to increase iodide binding and solve another
structure.
Acknowledgements
Duilio Cascio- partner in experiments, advice, inspiration
CRYSTALS
Celia Goulding
Chongwoo Kim
Cam Mura
Ann Maris
Yanshun Liu
Scott Griffith
Daniel Boutz
Maria Grzeskowiak
Helty Adisetiyo
STATISTICS
Gary Kleiger
Todd Norcross
SUPPORT
David Eisenberg
Todd Yeates
Richard Dickerson
James Bowie
Zbigniew Dauter-advice on
back-soaking, shelxD, xprepx.
Peter Muller-xprep connections
Kim Ma –X-ray maintenance