Proteins. Central Dogma : DNA RNA protein Amino acid polymers - defined composition & order. Perform nearly all cellular functions Drug Targets

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1 Proteins Central Dogma : DNA RNA protein Amino acid polymers - defined composition & order Perform nearly all cellular functions Drug Targets

2 Fold into discrete shapes. Proteins - cont. Specific shapes specific functions. >How do we determine the shape of a protein? >How does shape define function and influence drug action?

3 Revolutions in X-ray crystallography Getting faster - Hemoglobin: 30y! Your Favorite Protein: hours-weeks Not size limited - ribosome, viruses (> 2.5 MDa) Atomic resolution detail

4 The PDB surpasses >60,000 structures in 2009 Yearly Total...and is still growing!

5 Genome sequencing discoveries Genes Human >35,000 Fly ~13,600 Flat worm >19,000 Plant 25,498 Yeast 6,400 >50 microbes 500-5,000 Viruses < Genomic data is growing even faster!

6 X-ray crystallography can produce molecular images (Source) Data Protein Crystal Structure Electron Model building density

7 Crystal growth - general requirements Need: Pure protein (>98%) Chemically, conformationally homogeneous sample Add: Precipitating agents (mild organics such as PEG or salts) Buffers, inorganic or organic salts Cofactors, ligands, chemical additives Perturb: Hydration state, temperature, solubility response Get: Random aggregate Amorphous precipitate (common) Ordered phase transition Crystals (hard, rare)

8 Crystallization methods Direct mixing Free interface diffusion Precipitant Protein Precipitant Wait (Microbatch) Dehydrate - wait (Vapor diffusion) Protein Movie courtesy Fluidigm, Inc.

9 Crystals 101 Crystals are: Ordered arrays of ~10 14 molecules ~25-80% water - similar to cells Native protein structure/activity retained in crystalline state

$Diffracted X-rays$ Source Synchrotron

10 Data collection - experimental setup N2 stream (100 K) Diffracted X-rays Source Synchrotron Rotating anode Detector CCD Image plate Film Beam Optics (mirrors) Crystal (cryo-preserved)

11 Rotate the crystal to record all data - Oscillation 1º/frame Hexagonal lattice Each reflection (spot) arises from a set of Bragg planes Beam stop shadow Water ring

12 X-rays & crystals 101 Why use X-rays? X-rays are periodic waves (~1Å wavelength) Electrons (from protein atoms) scatter X-rays Scattering measurably perturbs incident X-ray properties Why use crystals? Crystals are periodic arrays of proteins Act as a micro-diffraction grating to constructively amplify scattering signal Scattered X-rays carry information about electron density distribution in a crystal

13 Crystal (Bravais) lattice types 14 lattice geometries can pack into repeating, 3D arrays

14 How do molecules pack? Unit cell - fundamental crystal repeat Asymmetric unit - minimal element within unit cell acted upon by symmetry operators Squiggles per: A.U U.C

15 Symmetry and lattice type define Space Groups 230 groups 65 accessible to biomolecules (no mirror planes!) Can also have symmetry within an AU! 3 orthogonal 2-fold axes 222 point group - four AUs AUs

16 X-rays are electromagnetic waves A simple wave: α=0 Can describe by: f(x)=fcos2π(hx+α) F Where: F=Amplitude λ=wavelength α=phase 1/λ=h

17 Fourier syntheses reconstruct electron density from diffraction data ρ(x) = Σ h F(h)cos2π(hx - α (h)) = sum of cosine terms Target function h F(h) α (h)/ / /5 0 Sum - approximates target function More terms better approximation Concept of RESOLUTION

$Diffraction data Onward to structure hkl = 18,17,0 hkl = 17,12,0 1) Collect data, index reflections (spots) - hkl terms ( addresses ) 2) Integrate spot intensities; calculate amplitudes ( F I) 3)$

18 Diffraction data Onward to structure hkl = 18,17,0 hkl = 17,12,0 1) Collect data, index reflections (spots) - hkl terms ( addresses ) 2) Integrate spot intensities; calculate amplitudes ( F I) 3) Calculate scattered wave phases: Experimentally (deliberately modulate spot intensities): Heavy atom substitution (Multiple isomorphous replacement, MIR) Multiwavelength anomolous dispersion (MAD) Computationally (use prior model): Molecular replacement (MR)

19 Waves can be represented as vectors α=0 F F α 1/λ=h

20 Atomic scattering is additive F PH = F P + F H F F PH - F H = F P - F H F PH Identify heavy atom positions (HA xyz ), can calculate F H But, two possibilities for F PH in solving for F P!

21 Get third derivative! F PH = F P + F H F PH - F H = F P F PH2 = F P + F H2 F PH2 - F H2 = F P F PH2 F - F H2 F PH - F H With HA1 xyz, calculate F H1 With HA2 xyz, calculate F H2 Leaves only one possibility for F P!

22 Multiwavelength Anomalous Dispersion -- MAD 1. Derivatize YFP with heavy metal(s) (commonly SeMet) 2. Change wavelength to change X-ray absorbtion by metals (anomalous dispersion), Synchrotron needed 3. When x-rays are absorbed, F(hkl) F(-h-k-l) 4. Use anomalous differences, F(hkl) - F(-h-k-l), to locate metals 5. Calculate amplitude and phase of scattering from metals 6. Calculate probability of α P (hkl) 7. Each wavelength limits protein phases to 2 most probable values 8. Resolve phase ambiguity with: multiple wavelengths (MAD) solvent flattening (SAD) noncrystallographic symmetry averaging (model)...

23 Onward to structure Diffraction data Electron density FT 4) Apply Fourier synthesis to reconstruct electron density: Structure factor equation

24 Structure factor equation ρ(xyz) = 1/V ΣΣΣ F(hkl)cos2π(hx + ky + lz - α (hkl)) h k l ρ = electron density x, y, z = positions in crystalline repeat (fractional coordinates) V = unit cell volume F(hkl) = amplitude for reflection hkl h, k, l = integers, coordinates of each spot hx + ky + lz = counter through the unit cell α (hkl) = phase angle ( ), α, of spot hkl divided by 360 hkl s: 0 h 0 k 0 l z 2,1,5. x y 4,4,1

25 Resolution affects electron density interpretability Higher scattering angles add more spots (Fourier terms) Resolution information content 6 Å resolution 3 Å resolution Side chains evident at > 3.5 Å resolution

From maps to model Electron density Interpretation/model building 6) Thread amino acid sequence through electron density (manually or automatically) 7) Use

26 From maps to model Electron density Interpretation/model building 6) Thread amino acid sequence through electron density (manually or automatically) 7) Use amino acid shape and sequence as a guide (3D jigsaw) 8) Refine model computationally to find best match to data (F calc vs. F obs ) and optimize stereochemistry

27 Refinement Model F calc, α calc F obs Data Manual rebuilding Iterate until convergence F obs - F calc ; α calc Covalent geometry (Molecular dynamics) Shifts -- Δ x,y,z and B B ( temperature ) factor = disorder relative to a point atom

28 R and Rfree values -- the gold standards R = Σ F obs - F calc = 0.59 for random model ΣF obs = for starting model > 0.25, good fit, errors still possible < 0.20, excellent fit R free = R value of a small, random subset never used in refinement. Ideally, Rfree< 0.30 & Rfree Rwork (this scales with resolution, however) Model is complete when: No interpretable difference electron density (F obs - F calc ) Geometry close to ideal No clashes, optimal rotamer stereochemistry

29 Interpreting the data - the structure table Data Collection Data set Remote Peak HgCl 2 derivative Space group P P P Unit cell a, b, c (Å) 52.8, 52.8, , 52.8, , 52.1, α, β, γ ( ) 90, 90, 90 90, 90, 90 90, 90, 90 Wavelength (Å) Resolution range (Å) Total reflections 206,536 72,214 41,453 Unique reflections 10,986 10,171 7,537 Redundancy 18.8 (15.5) a 7.1 (6.8) 5.5 (3.8) Completeness 99.9 (100) 99.5 (100) 98.6 (99.2) I/σ 43.3 (7.3) 41.7 (9.6) 34.9 (4.2) R sym (%) b 5.5 (30.8) 5.0 (20.0) 5.0 (37.4) a Values in parentheses are for highest-resolution shells. b I(h)j is the scaled observed intensity of the jth observation of reflection h, and <I(h)> is the mean value of corresponding symmetry-related reflections. (signal to noise) (data agreement) R sym = I(h) j < I(h) > / I(h) j j j

30 Interpreting the data - the structure table Refinement parameters Resolution (Å) No. of nonhydrogen atoms 29,845 Rmsd No. of waters 243 Bond lengths (Å) No. of ions 3 Bond angles ( ) 1.4 B factors Overall 30.1 Ramachandran Protein 29.2 Favored 90.4% (should be <0.02Å) (should be <2.0 ) (stereochemistry) Ligand/ion 39.5 Allowed 7.6% Water 34.6 Generous 2% R work /R e free 19.4/22.9 Disallowed 0 (model/data agreement) e where F obs and F calc are observed and model structure factors, respectively. R free was calculated by using a randomly selected set (5%) of reflections. R work = F obs F calc / F obs

31 Making sense of the PDB file - header info HEADER ISOMERASE 02-JUN-05 1ZVU TITLE STRUCTURE OF THE FULL-LENGTH E. COLI PARC SUBUNIT COMPND 2 MOLECULE: TOPOISOMERASE IV SUBUNIT A; SOURCE 2 ORGANISM_SCIENTIFIC: ESCHERICHIA COLI; KEYWDS BETA-PINWHEEL, ATPASE, SUPERCOILING, DECATENATION, DNA EXPDTA X-RAY DIFFRACTION AUTHOR K.D.CORBETT,A.J.SCHOEFFLER,N.D.THOMSEN,J.M.BERGER JRNL TITL THE STRUCTURAL BASIS FOR SUBSTRATE SPECIFICITY IN JRNL TITL 2 DNA TOPOISOMERASE IV. JRNL REF J.MOL.BIOL. V REMARK 1 REMARK 2 REMARK 2 RESOLUTION ANGSTROMS. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : REFMAC REMARK 3 AUTHORS : MURSHUDOV,VAGIN,DODSON REMARK 3 REMARK 3 REFINEMENT TARGET : MAXIMUM LIKELIHOOD REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 3.00 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : REMARK 3 DATA CUTOFF (SIGMA(F)) : REMARK 3 COMPLETENESS FOR RANGE (%) : 89.6 REMARK 3 NUMBER OF REFLECTIONS : 18167

32 Making sense of the PDB file - the guts SEQRES 1 A 716 MET ASP ARG ALA LEU PRO PHE ILE GLY ASP GLY LEU LYS SEQRES 2 A 716 PRO VAL GLN ARG ARG ILE VAL TYR ALA MET SER GLU LEU SEQRES 3 A 716 GLY LEU ASN ALA SER ALA LYS PHE LYS LYS SER ALA ARG HELIX 1 1 LYS A 39 SER A HELIX 2 2 THR A 66 GLY A 72 1 Atom number 7 HELIX 3 3 ASP A 79 ALA A Amino acid type SHEET 1 A 2 VAL A 100 GLY A SHEET 2 A 2 SER A 123 LEU A O ARG A 124 N ASP A 101 CRYST P ORIGX ORIGX ORIGX SCALE Occupancy SCALE SCALE ATOM 1 N ASP A N ATOM 2 CA ASP A C ATOM 3 C ASP A C ATOM 4 O ASP A O ATOM 5 CB ASP A C ATOM 6 CG ASP A C ATOM 7 OD1 ASP A O ATOM 8 OD2 ASP A O ATOM 9 N ARG A N TER END Atom identifier Protein chain ID Residue number Cell dimensions and space group B-factor Atomic position

33 Some things to keep in mind PDB file oddities: Occ<1 - partial occupancy, see for ligands sometimes B>>B avg - disordered region, interpret with caution Missing side chain or sequence gap - region not modeled, likely disordered Two copies of same amino acid - multiple conformations modeled Waters/ligands often at end of file The model is still a model: Best fit to data, doesn t mean everything is perfect or right Higher resolution models typically more accurate - use for homology modeling, molecular replacement, analysis of active site geometry, etc.

34 Representations of protein structure Ribbon representation: traces path of protein chain through space Surface representation: shows solid features of protein exterior Spheres and sticks: show atomic connections Remember - a model is still a model!

35 Where is crystallography headed? Dissect mechanism and catalysis Structure/function studies Time resolved reactions Harder problems Dynamic, metastable complexes and assemblies Membrane proteins Rational ligand/inhibitor design Define cellular proteome

36 Where do we need physics? Detectors Increase sensitivity, dynamic range, speed Sources Benchtop synchrotrons Overcome radiation damage Crystallization Develop rational guidelines & novel approaches Use of non-diffracting/poorly-diffracting crystals Functional prediction Extracting/simulating dynamics from models and data Docking/modeling interactions Single protein diffraction Free electron lasers Data analyses

X-ray Crystallography. Kalyan Das

X-ray Crystallography. Kalyan Das X-ray Crystallography Kalyan Das Electromagnetic Spectrum NMR 10 um - 10 mm 700 to 10 4 nm 400 to 700 nm 10 to 400 nm 10-1 to 10 nm 10-4 to 10-1 nm X-ray radiation was discovered by Roentgen in 1895. X-rays