CO CRYSTALLIZATION OF MULTIPROTEIN-DNA COMPLEXES FOR STRUCTURAL ANALYSIS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。MAT α 2/MCM 1/DNA复合物的结构先前已被解析为2.25埃（Tan S.，里士满·T Nature（1998）391，p660-666）。初步结构揭示了MAT α 2的臂结构域的不寻常构象（残基112-130，其将MAT α 2同源结构域连接到MCM 1结合口袋）。在一个拷贝（称为MAT α 2-顺式）中，该区域形成两个抗β-细胞β折叠构象，而在另一个（MAT α 2-反式）拷贝中，该区域形成α螺旋（从残基121-128）。这是两种不同构象的原因，即（1）MCM 1结合口袋和MAT α 2同源结构域之间的间距不同和（2）MAT α 2反式与MCM 1的对称相关拷贝接触，其中弯曲角改变了90度。我们用新的底物共结晶了MATalpha 2和MCM 1，所述底物在MATalpha 2同源结构域和MCM 1结合口袋之间具有改变的间隔。我们的晶体在我们的母源X射线中的作用很弱，并且通常在一维上很薄（通常为-0.2x.15x.02）。我们正在寻找衍射的改进，使结构可以解决在同步辐射源。我们的计划是收集更高分辨率的衍射数据。如果获得足够高的分辨率，我们应该能够利用这些数据通过分子置换技术来解决结构问题。我们还想测试MATalpha 1/MCM 1/DNA和Dot 1/核小体的晶体，这些晶体以前在我们的家庭源X射线上不会产生衍射。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structure of the MATalpha2/MCM1/DNA complex has been solved to 2.25 angstroms previously (Tan S., Richmond T., Nature (1998) 391, p660-666). The preliminary structure has revealed an unusual conformation of the arm domain of MATalpha2 (residues 112-130 which links the MATalpha2 homeodomain to the MCM1 binding pocket). In one copy (called MATalpha2-cis)this region forms two anti-paralell beta sheets conformation while in the other (MATalpha2-trans) copy, the region forms a alpha helix (from residues 121-128). Their are two reasons for the different conformation which are (1) the spaciing between the MCM1-binding pocket and the MATalpha2 homeodomain are different and (2) the MATalpha2-trans makes a contact with a symmetry related copy of MCM1 in which the bending angle is altered by 90 degrees. We have cocrystallized MATalpha2 and MCM1 with new substrates that have an altered spacing between the MATalpha2 homeodomain and the MCM1 binding pocket. Our crystals diffract weakly at our home source xrays and are often thin in one dimension (typically-.2x.15x.02). We are looking for diffraction improvement so that the structure could be solved at a synchotron source. Our plan is to collect diffraction data at higher resolution. If high enough resolution is obtained, we should be able to use the data to solve the structure by molecular replacement techniques. We would also like to test crystals of MATalpha1/MCM1/DNA and Dot1/nucleosomes which previously do not yeild diffraction on our home source xrays.