MOLEC STRU DETER XRAY
MOLEC STRU DETER XRAY BCH 6744
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This 15 page Class Notes was uploaded by Kavon Huel on Friday September 18, 2015. The Class Notes belongs to BCH 6744 at University of Florida taught by Staff in Fall. Since its upload, it has received 32 views. For similar materials see /class/206967/bch-6744-university-of-florida in Biochemistry and Molecular Biology at University of Florida.
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Date Created: 09/18/15
L13 Map interpretation Model building and re nement cos 271 hxky lz am 1 PXayaz Z Z Z I Fwd thl REAL SPACE REPICROCAL SPACE 4 r navy Lecture 13 1 Initial chain trace First map Continuous density 2 Assign Cu positions 36A apart 3 Build polypeptide backbone 1r Note Keep model in asymmetric unit monomer Lecture 13 Be aware Phases maybe very approximate density smeared resolution maybe low 3A details maybe weak Goals Obtain the fold of the protein polypeptide chain tracing Obtain secondary structural elements maybe missing turns loops etc Lecture 13 Amino acid assignment of side chains require sequence Key to amino acids Ala Arg Asn Asp Cys Gln Glu Gly His Lecture 13 4 Lecture 13 Tyr V a l Thr Trpkkk M et Phe Pro Ile Leu Lys 5 lt a O N E 5 O N S Q m A O O 5 Sr 5 E D Q v Amino acid assignment of side chains Knowledge All What you learnt in biochemistry classes Aromatic rings Trp Phe Tyr His Sulfur containing Met Cys Hydrophobic Hydrophilic Find a characteristic sequence and locate it in the map Note When building use libraries of standard bond geometry and torsion angles for amino acids and main chain Lecture 13 Phase improvement Only parameter that can be re ned Fobs are the observed data and CAN NEVER be adjusted Phase can be improved TWO ways 1 Map modi cation real space A Solvent attening B electron density averaging C increase resolution phase extension Bulk solvent Averaging density Lecture 13 7 2 Model modi cation real space A Improving t of model to density Re nement B Add to model more building Amino acids ions ligands water molecules C Re ne knowledge of model occupancythermal parameters of atoms Lecture 13 Leastsquares model re nement The modelimproved electron density map can be used to calculate improved structure factors Goal to nd a set of atom positionsparameters that give Fcalc that minimize the difference to the observed Fobs minimize CD 2 WFobs 39 chalcl2 hkl Fobs measured structure factor F calculated structure factor calc W weighting reliability of Fobs lcshkl2 Lecture 13 9 Other considerations Model Geometry bond lengths bond angles 1 2 WFobs chalcl2 2 Wlideal lmodel2 2 Waideal amodel2 hkl 1 a torsion angles 2 Wtideal tmodel2 t Always check rms deviation of model geometry Additional re nement parameters Fcalc G 2 njfj e 21tihxjkyjlzj e BjsinGc2 1 nj occupancy Bj temperature factor of atom j G overall scale factor of Fcalc to lFobsl Lecture 13 10 RFactor Rfactor 2 W lFobsl 39 Kchalc hkl 2 lFobsl hkl RFree Rfactor 2 W lFobsl 39 Kchalc hleT 2 lFobsl hkl CT Where hkl CT means re ections belonging to a test set T of unique re ections not used during the model s re nement Lecture 13 11 Concept of minimization Rigid body Annealing local minimum Re nement x y z 75 u 3 g 0 39Fl g E a 3 H g 3 global rmmmum rebulldwaterlons a a c a 5 a 3 g m Re nement x y z b n a E 2 i Convergence changesin paxameters Lecture 13 12 Data Collection Iobdeobs lt3 Expression Puri cation Space group Crystallization 5 Phase assignment cecal D Fobs am Electron density pXyz D Rfactor 3 a model density improvement I Generate Fcal 0cm ecture l3 1 3 Ramachandran plot Check torsion angles 3 sheet k X20 3quot glycine a 1 1 psi 0 w a 339 gt60 vao Lecture 13 Final note Resolution Resolution Resolution Crystallography produces a model it can therefore only be a good as the observed data it was refined against G A data 392 5 A data L5 Adana us A data Lecture 13
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