Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering By Rambo and Tainer

Introduction Importance of development of techniques that probe nucleic acid or protein-nucleic acid complex Three Predominant Techniques Used in Structural Biology –Macromolecular X-ray Crystallography (MX) –Nuclear Magnetic Resonance (NMR) –Electron Microscopy (EM) These techniques have limitations for macromolecules with functional flexibility and intrinsic disorder 1

Instrumentation 2

Sample Preparation Stresses high purity, high homogeneity similar to crystallography Amount needed is 15 μ L with protein concentration ranging from mg/ml. Typically 2-5 mg/ml is best higher concentration yields better signal but can lead to aggregation 3

SAXS Theory Three things to examine –SAXS profile in reciprocal and real space –Gunier Plot (not shown) –Kratky Plot 4

SAXS Profile Transformation of the scattering data I(q) yield P(r) a histogram of interatomic vectors Calculate a structure based on a atomic resolution macromolecular structure 5

Idealized Data Measurements at different range of concentration X-ray sensitivity can be detected by changes in scattering by repeat exposures 6

Real Data Raw shattering curves for all samples. 1 st exposure. See that with increasing concentration, sample is increased. Better signal at high concentration. 7 6/23/2015

Guinier Plot Non linear dependence of log(I(q)) indicates presence of aggregation Presence of aggregation means no data 8

Gunier Real Data 6/23/20159

Radius of Gyration Radius of gyration is calculated by taking I(0) at q= 0. Needs to be compared against a set of standards 10

Interparticle Interference Increasing concentration can reveal concentration dependence Visible in decrease in intensity at small q. 11

P(r) Distribution in Real Space From this distribution you can tell two things –Dmax –Some general information about shape 12

Real Data 13 Dmax = 110 Dmax = 115

Kratky Plot Kratky plot also is an indication of protein folding/ unfolding Globular proteins macromolecules follow Porod’s law and are bell shaped 14

Kratky Plot Real Data 6/23/201515

No Atomic Structure Without previously known structure can still make shape prediction Programs such as GASBOR and DAMMIF allow for low resolution structure 16

Atomic Structure Solved Calculate curve from known data and compare to experimental data Disagreement –Investigate alternate states –Investigate mixture of states –Investigate flexibility 17

Gasbor Ub-PCNA

Conformation Assembly Use of a variety of software to find best fit X 2 vs Rg gives good idea about entire ensemble 19

SAM-I: Comparison of Crystal Against SAXS Crystal structure was determined in presence of ligand and poorly fit SAXS data SAXS guided hypothesis about conformational switching as mechanism 20

Abscisic Acid Hormone Receptor PYR1 Crystalized with open- lid and closed-lid conformations. Crystal contacts show three possible dimers α-α, β-β, α-β SAXS profile distinguishes between three conformations. 21

VS Ribozyme Solution Structure Ab initio modeling which lead to identificaiton of helical regions based on helical secondary structure Resulting model was converted to residue specific model 22

Erp72 Solution Structure Parts previously solved by NMR and MX but solution structure unknown Ab initio modeling allowed for putting together of parts into correct orientation in solution 23

p53-Taz2-DNA complex Parts had been solved previously –Core and tetramerization solved by MX –Taz2 by NMR Used in rigid body analysis and protein with and without DNA to model 24

Future of SAXS Data analysis are contuining to be developed computational tools Synchotron-based facilities can extend SAXS into high throughput region Can answer fundamental questions in DNA repair, modeling of large multidomain macromolecular machines and suggests flexibility are criticical for biological funcitions. 25

Questions? Froliche Weinachten! (Merry Christmas in German) 26