Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

5A,6A/2004G356

Structure of ATP synthaseYasuo1 SHIRAKIHARA* 1, Aya SIRATORI 1, Satoshi Murakami 2, Hiroshi ITO 1
National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
2
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan
Introduction
ATP synthase is responsible for ATP production in living
cells, and is a membrane protein located in the energy
conversion membrane. ATP synthase consists of achannel Fo portion (about 100,000 dalton, subunit
composition of ab2c8-12) and a large soluble catalytic F1portion (380,000 dalton, α3β3γδε).
The unique rotational catalysis mechanism of F1 includes
rotation of the rod-like γ subunit, which is thought tocontrol the conformations of the three catalytic β-subunits
in a cyclic manner by its rotation.

Among various columns examined, useful ones were Qsepharose high performance, Superdex prep grade 200columns. An array of Q-sepharose high performance,
Superdex prep grade 200 and the second Q-sepharosehigh performance columns yielded a preparation ofcrystallization grade, but a smaller array of Superdex prep
grade 200 and Q-sepharose columns only wasindistinguishable from the first array.

Starting from elucidation of the α3β3 sub-assembly
structure of the thermophilic F1, we have been moving up
to the higher sub-assembly. The α3β3γ sub-assembly wasdifficult to crystallize, but the α3β3γε sub-assembly gave
crystals that allowed to see a novel conformation of F1.
We are now dealing with the holo-enzyme, ATP synthase.

Initial tiny crystals are now replaced by crystals withtypical dimensions of 0.2mm, 0.2mm, 0.01mm after anextensive and systematic crystallization condition search.
Among the detergents mentioned above, only decymaltoside and dodecyl-maltoside gave diffracting crystals,
though all of the four detergents gave similar-looking
crystals.

The membrane protein ATP synthase is still a challenging
target for a structural study, in view of relatively few
solved structures of the membrane proteins so far.

ADP is the most favourite ligand, as others like AMPPNP,an ATP analogue, gave crystals that diffracted poorly.
Other factors such as temperature in crystallization, kindand concentration of monovalent salts and those ofdivalent salts have been optimized.

In an initial crystallization trial using ATP synthase
extracted with dodecylmaltoside from the PS3 membrane,
and then purified with an array of Q-sepharose highperformance column, Superdex prep grade 200 column
and the second Q-sepharose high performance column,
we found that the preparation gave small crystals inpolyethylelene glycol solution containing MgADP as an
ATP synthase specific additive. The preparation
procedure was a conventional one.
Encouraged by this initial success, we proposed a further
crystallization experiment that included improvement ofthe preparations, improvement in crystallization
conditions and examination of resultant crystals usingsynchrotron beams.
Results
First a number of detergents have been examined forgood extraction capability in the preparation procedure.
In addition to dodecylmaltoside previously identified,decylglucoside and decylmaltoside have been shown tobe promising candidates. Using those promising
candidates, we examined preparation procedure andcrystallizability of each of the obtained preparations
carefully.
Detergents such as decylmaltoside, undecyl-maltoside,dodecyl-maltoside, tridecyl-malltoside were good inpreparation. But others like alkyl glucosides were not.

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For example, dectylglucoside showed good extraction
capability but gave an unusual elution profile in the Qsepharose high performance column chromatography.

Users' Report

ATP synthase in our crystals contains all the 8 subunits,
in contrast to yeast ATP synthase that lacks a and b
subunits in crystals.
Crystals of dodecylmaltoside-extracted ATP synthase
formed in presence of MgADP allowed us to record
diffraction patterns to a resolution of about 7 A with abeam of BL5A. Crystals of decylmaltoside-extracted ATPsynthase formed in similar conditions gave diffraction
patterns also to a resolution of about 7 A with a beam ofBL6A. Crystals from undecyl-maltoside or tridecylmalltoside extracted ATP synthase diffracted significantly
worse than the two. In the PF experiments, we had an
impression that crystals formed in presence of nucleotide
other than ADP were worse than those formed inpresence of ADP, irrespective of detergents. Furtherefforts are being made to get better diffracting crystals.
* yshiraki@lab.nig.ac.jp