Supplementary Information v410, 235
Supplementary information item 1: MAD phasing
 PEAK
 INFLEX
 REM1
 REM2
Wavelength (Å)
0.97923
0.97939
0.99988
0.92873
Resolution (Å)
3.0
3.0
3.0
3.0
# images
300
150
150
150
Total data
253477
125641
125303
132478
Unique data
86086
43136
43258
43177
Completeness(%)
99.8
99.7
99.7
99.8
Mean I/ (I)
16.5
17.7
19.1
12.9
Rmerge (%)
Phasing Power
(Iso/Anom)
6.6
6.7
5.1
7.6
2.0/2.3
2.5/1.9
0.7/1.5
An inverse beam experiment was performed at the peak wavelength, followed by standard data collection
procedures for the remaining wavelengths. Images were collected with an oscillation of =0.5o and an
exposure time of 5 seconds. Data were processed using the HKL2000 and CCP4 suite of programs 1-3. A
slightly unconventional route was used to estimate Se structure factors. Two Patterson maps were
calculated, the first based on anomalous differences (F+peak - F-peak)2, and the second on isomorphous
differences (Finflex- Frem1)2. Each was optimised by careful removal of outlying data. Scaling, averaging
and back-transformation produced a set of calculated FSe to a resolution of 3.5 Å. Using these data
Shake’n’Bake4,5 located 45 of the expected 50 selenium atoms. These were elaborated to 49 atoms and
refined using SHARP6. A 3.0Å electron density map revealed 2 molecules in the asymmetric unit and was
improved by cyclical 2-fold averaging and solvent flattening (GAP, DIS and JMG unpublished program).
An automated model building procedure using BONES7, CA_TRACE (DIS, JMG, unpublished program),
CALPHA8 and MUTATE8 generated a model that was rebuilt9 and refined10 against a wavelength-merged
data set.
1.
Otwinowski, Z. & Minor, W. in Macromolecular Crystallography (eds. Carter Jr., C. W. & Sweet,
R. M.) 307-326 (Academic Press, San Diego, 1997).
2.
Collaborative Computational Project, N. The CCP4 suite: Programs for Protein Crystallography.
Acta Crystallogr. D50, 760-763 (1994).
3.
Howell, P. L. & Smith, G. D. Identification of heavy-atom derivatives by normal probability
methods. J. Appl. Crystallogr. 25, 81-86 (1992).
4.
Weeks, C. M. & Miller, R. The design and implementation of SnB v2.0. J. Appl. Crystallogr. 32,
120-124 (1999).
5.
Miller, R., Gallo, S. M., Khalak, H. G. & Weeks, C. M. SnB: crystal structure determination via
Shake-and-Bake. J. Appl. Crystallogr. 27, 613-621 (1994).
6.
La Fortelle, E. d. & Bricogne, G. in Macromolecular crystallography (eds. Carter Jr., C. W. &
Sweet, R. M.) 472-494 (Academic Press, San Diego, 1997).
7.
Greer, J. Three-dimensional pattern recognition: an approach to automated interpretation of
electron density maps of proteins. J. Mol. Biol. 82, 279-301 (1974).
8.
Esnouf, R. M. Polyalanine reconstruction from C positions using the program CALPHA can aid
initial phasing of data by molecular replacement procedures. Acta Crystallogr. D53, 665-672
(1997).
9.
10.
Jones, T. A., Zou, Y. J., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein
models in electron density and the location of errors in these models. Acta Crystallogr. A47, 110119 (1991).
Brunger, A. T. et al. Crystallography and NMR System: A New Software Suite for
Macromolecular Structure Determination. Acta Crystallogr. D54, 905-921 (1998).
Supplementary information item 2: experimental electron density
Sigmaa weighted 2¦Fo¦-¦Fc¦ 2Å resolution electron density for the active site residues D453 and D454, and
the octahedrally coordinated Mn++ ion.
Supplementary information item 3: 6–HCV sequence alignment
Sequence alignment based on structural alignment from SHP 1,2 (see Fig 1b). Strictly conserved residues
are in red blocks, and similar residues in blue boxes. Semi-transparent grey bars above the 6 P2 sequence
indicate structurally equivalent amino-acids. Secondary structural elements defined by DSSP 3 are shown.
The secondary structural elements are coloured by domain with fingers drawn in red, palm in green, thumb
in blue, C-terminal domain in yellow and connections between the fingers and thumb in pink. Sequence
motifs for RNA polymerases4 are marked by bars below the sequences. Motif A (324-334) is coloured red,
motif B (394-413) yellow, motif C (445-461) green, motif D (475-488) blue, motif E (493-505) pink and
motif F (268-272) orange. Conserved catalytic aspartates are marked by black ellipses. To avoid breaking
up the 6 P2 sequence, residues for HCV polymerase that are not matched by 6 are omitted, and the
position and number of residues removed is indicated under the HCV sequence.
1.
2.
3.
4.
Stuart, D. I., Levine, M., Muirhead, H. & Stammers, D. K. Crystal structure of cat muscle
pyruvate kinase at resolution of 2.6Å. J. Mol. Biol. 134, 109-142 (1979).
Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence
alignments in PostScript. Bioinformatics 15, 305-8 (1999).
Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of
hydrogen-bonded and geometrical features. Biopolymers 22, 2577-2637 (1983).
Lesburg, C. A. et al. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C
virus reveals a fully encircled active site. Nat. Struct. Biol. 6, 937-43 (1999).