96 INSTRUMENTAL METHODS
a major role in muscle contraction – relaxation cycles and are responsible for
transporting calcium into the lumen of the sarcoplasmic reticulum. The Ca 2+ -
ATPase from fast - twitch rabbit skeletal muscle sarcoplasmic reticulum
(SERCA1a) is a large membrane protein of 110 kDa. The enzyme actively
transports two Ca 2+ ions per ATP hydrolyzed across the membrane from the
cytoplasm to the lumen against a concentration gradient. At the same time,
two to three protons are transported in the opposite direction. Activity of
Ca2+ - ATPase is crucial to skeletal muscle function. In 2000, Japanese research-
ers determined the fi rst atomic - scale structural model for any P - type ATP -
dependent pump using crystals formed from the Ca 2+ - ATPase SERCA1a
protein.^19 The data are deposited in the Protein Data Bank (PDB) with the
accession number 1SU4. Since then, various X - ray crystallographic structures
corresponding to stages in the Ca 2+ - ATPases catalytic cycle (see Figures 6.28 –
6.30 ) have been determined. The text that follows here is adapted from the
Methods section of reference 19.
Ca 2+ - ATPase from rabbit hind leg muscle was purifi ed, and crystals were
grown by dialyzing the mixture of purifi ed protein and phosphatidylcholine (a
phospholipid) against a crystallization buffer. After one month, crystals of
dimensions 100 × 500 × 20 μ m were obtained. The crystals belonged to the
C 2 space group with unit cell dimensions of a = 166 Å , b = 64.4 Å , c = 147.1 Å.
β = 98.0 ° and contained one protein molecule in the asymmetric unit. The
crystals used for crystallography were fl ash - frozen in cold nitrogen gas. Heavy
atom derivatives were prepared by soaking native crystals in platinum, terbium,
or lanthanum compounds or salts for one hour. After crystal screening with a
laboratory X - ray source, the fi nal data sets were collected at SPring - 8 beam-
lines BL41XU ( λ = 0.800 Å ) or BL44B2 ( λ = 0.890 and 1.000 Å ) for native
crystals and BL44B2 ( λ = 0.890 Å ) for derivative crystals. Data were collected
on eight native crystals and crystal derivatives obtained by soaking other
native crystals in heavy metal compounds and salts. Table 1 of reference 19
contains a summary of the crystallographic analyses. Data were detected using
either Rigaku R - AXIS IV imaging plate or Mar CCD 165 detectors. Charge
coupled device (CCD) area detectors have faster readout times and better
resolution than imaging plate detectors. Data taken with the Rigaku R - AXIS
IV imaging plate detector were processed with Denzo and Scalepack (see
reference 17d ) software programs used to visualize and interpret X - ray crys-
tallographic data. Data taken with the Mar CCD 165 detector were processed
with MOSFLM and Scala, also software programs used to visualize and inter-
pret X - ray crystallographic data. Merging of different data sets was carried
out using Scalepack, subsequent processing using CCP4 program suites, avail-
able through the Collaborative Computational Project, Number 4. 1994 (The
CCP4 Suite: Programs for Protein Crystallography, Acta Crystallogr. D50 ,
760 – 763). References to information on all these software programs are found
in reference 19 and online. First, experimental phases were calculated using
Miphare (part of the CCP4 program suites) to 2.8 - Å resolution. The electron
density map refi ned by density modifi cation is shown in Figures 3a and 3b of