WEBVTT 00:00:00.500 --> 00:00:03.270 The following content is provided under a Creative 00:00:03.270 --> 00:00:04.630 Commons license. 00:00:04.630 --> 00:00:07.140 Your support will help MIT OpenCourseWare 00:00:07.140 --> 00:00:11.470 continue to offer high quality educational resources for free. 00:00:11.470 --> 00:00:14.100 To make a donation or view additional materials 00:00:14.100 --> 00:00:18.050 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:18.050 --> 00:00:19.000 at ocw.mit.edu. 00:00:24.317 --> 00:00:27.770 SHIVA MANDALA: And so just as an overview, today 00:00:27.770 --> 00:00:30.340 we're going to be talking about different techniques that 00:00:30.340 --> 00:00:32.949 are used to determine protein structure. 00:00:32.949 --> 00:00:35.240 We're talking a little bit about the protein data bank, 00:00:35.240 --> 00:00:36.650 or the PDB. 00:00:36.650 --> 00:00:40.700 And then the latter half of the recitation will be on-- 00:00:40.700 --> 00:00:43.220 we'll be doing a worksheet to look 00:00:43.220 --> 00:00:46.740 at the structure of ubiquitin and di-ubiqiotin using PyMOL. 00:00:46.740 --> 00:00:50.670 So that's just to get you familiar with using PyMOL. 00:00:50.670 --> 00:00:53.840 And so for the first question I'd like to pose to you 00:00:53.840 --> 00:00:56.690 is why should we determine protein structure, what 00:00:56.690 --> 00:00:59.420 we can learn from determining protein structure? 00:00:59.420 --> 00:01:03.120 And so I look to you for answers, a lot of answers. 00:01:03.120 --> 00:01:04.760 But does anybody have any ideas? 00:01:08.232 --> 00:01:09.720 Yeah? 00:01:09.720 --> 00:01:10.712 AUDIENCE: [INAUDIBLE] 00:01:10.712 --> 00:01:13.324 SHIVA MANDALA: Yeah, absolutely. 00:01:13.324 --> 00:01:14.990 Knowing putting structures does help you 00:01:14.990 --> 00:01:16.700 determine enzyme mechanisms. 00:01:16.700 --> 00:01:18.947 Anything else? 00:01:18.947 --> 00:01:20.863 AUDIENCE: Structure can indicate function. 00:01:20.863 --> 00:01:21.821 SHIVA MANDALA: Structure what? 00:01:21.821 --> 00:01:22.385 AUDIENCE: Can indicate function. 00:01:22.385 --> 00:01:24.051 SHIVA MANDALA: Yes, absolutely structure 00:01:24.051 --> 00:01:25.220 does indicate function. 00:01:25.220 --> 00:01:28.878 Can you be a bit more specific with respect to that? 00:01:28.878 --> 00:01:33.189 AUDIENCE: [INAUDIBLE] 00:01:33.189 --> 00:01:34.600 SHIVA MANDALA: Yeah, absolutely. 00:01:34.600 --> 00:01:38.060 Yeah you can determine active site of enzymes. 00:01:38.060 --> 00:01:40.000 Any other ideas? 00:01:40.000 --> 00:01:41.300 Still a lot more to go. 00:01:44.300 --> 00:01:46.990 What about can you learn interactions 00:01:46.990 --> 00:01:48.280 with other macro molecules? 00:01:50.810 --> 00:01:51.670 OK. 00:01:51.670 --> 00:01:53.380 Well, let's just go through some of them. 00:01:53.380 --> 00:01:55.529 So yes, structure does, indeed, determine function. 00:01:55.529 --> 00:01:57.820 And the idea is if you know the structure of a protein, 00:01:57.820 --> 00:02:00.980 you can learn a lot about its function in vivo. 00:02:00.980 --> 00:02:03.650 And so some of the things that you can study 00:02:03.650 --> 00:02:05.570 is you can study enzyme mechanisms. 00:02:05.570 --> 00:02:07.840 It's relatively hard to do in x-ray crystallography, 00:02:07.840 --> 00:02:10.449 because every time you solve a crystal structure, 00:02:10.449 --> 00:02:13.640 it's only one snapshot of the enzyme. 00:02:13.640 --> 00:02:15.490 But you can definitely do it. 00:02:15.490 --> 00:02:18.372 You can design drugs or substrate that bind 00:02:18.372 --> 00:02:20.830 to the protein if you know what the active site looks like. 00:02:20.830 --> 00:02:23.650 You can design a high affinity inhibitor, 00:02:23.650 --> 00:02:27.160 and this is used in the pharmaceutical industry a lot. 00:02:27.160 --> 00:02:29.560 You can study translation and transcription, which 00:02:29.560 --> 00:02:32.260 is what we'll be learning about in class this week, 00:02:32.260 --> 00:02:34.550 and we'll be learning next week, as well. 00:02:34.550 --> 00:02:39.100 You can also make co-crystals of proteins with other proteins 00:02:39.100 --> 00:02:42.490 and nucleic acids and study interactions between macro 00:02:42.490 --> 00:02:43.580 molecules. 00:02:43.580 --> 00:02:46.170 And this is something that has been emerging. 00:02:46.170 --> 00:02:48.340 It makes it harder to solve a crystal structure, 00:02:48.340 --> 00:02:54.139 but x-ray crystallography is a very powerful tool for this. 00:02:54.139 --> 00:02:55.930 You can also study immune system functions. 00:02:55.930 --> 00:02:57.971 So this is more on the biological side of things. 00:02:57.971 --> 00:03:00.250 You can study host-pathogen receptors 00:03:00.250 --> 00:03:01.420 and their interactions. 00:03:01.420 --> 00:03:02.320 And many, many more. 00:03:02.320 --> 00:03:04.750 Really there's no reason why you shouldn't 00:03:04.750 --> 00:03:07.770 have a structure for whatever protein you're studying. 00:03:07.770 --> 00:03:12.310 And so the key idea is that if you know the protein structure, 00:03:12.310 --> 00:03:14.950 that allows you to carry out biochemical studies. 00:03:14.950 --> 00:03:17.590 But then also if you determine the structure, 00:03:17.590 --> 00:03:19.157 that can help rationalize results 00:03:19.157 --> 00:03:20.740 that you get from biochemical studies. 00:03:20.740 --> 00:03:22.810 So it goes both ways. 00:03:22.810 --> 00:03:24.760 And the final point that is not perhaps not 00:03:24.760 --> 00:03:26.343 emphasized that much is that structure 00:03:26.343 --> 00:03:27.710 is a result of sequence. 00:03:27.710 --> 00:03:29.590 And ideally, what we would like to know 00:03:29.590 --> 00:03:33.640 is if we know the primary amino acid sequence of a polypeptide, 00:03:33.640 --> 00:03:36.250 we'd like to be able to predict its complete three 00:03:36.250 --> 00:03:38.420 dimensional fold. 00:03:38.420 --> 00:03:40.825 And that's sort of the idea behind protein folding. 00:03:40.825 --> 00:03:43.927 But we're not quite at that stage yet computationally. 00:03:43.927 --> 00:03:45.760 And so that's why we need experimental data. 00:03:45.760 --> 00:03:48.040 But that's something we're moving forward, 00:03:48.040 --> 00:03:51.270 moving towards as a science. 00:03:51.270 --> 00:03:52.840 And so just to go over, I'm going 00:03:52.840 --> 00:03:55.180 to cover the three main techniques in protein structure 00:03:55.180 --> 00:03:56.510 determination. 00:03:56.510 --> 00:03:59.420 And so the most common one is X-ray diffraction. 00:03:59.420 --> 00:04:02.710 And the I'll go over some of the details of X-ray later. 00:04:02.710 --> 00:04:05.975 I'll be focusing on X-ray diffraction in this talk. 00:04:05.975 --> 00:04:09.490 Some of the selling points of X-ray diffraction are that you 00:04:09.490 --> 00:04:11.500 can study a protein of any size-- 00:04:11.500 --> 00:04:12.940 small proteins, large proteins. 00:04:12.940 --> 00:04:15.520 You can study complexes of proteins. 00:04:15.520 --> 00:04:18.010 There is a need to crystallize your sample, which 00:04:18.010 --> 00:04:18.950 makes it challenging. 00:04:18.950 --> 00:04:20.740 It's very hard to crystallize proteins. 00:04:20.740 --> 00:04:23.717 In your body, no protein is crystallized. 00:04:23.717 --> 00:04:26.050 So you're trying to make proteins do something that they 00:04:26.050 --> 00:04:27.840 don't really like doing. 00:04:27.840 --> 00:04:31.990 You can obtain a high resolution structure. 00:04:31.990 --> 00:04:34.910 So a resolution of two Angstroms or even less 00:04:34.910 --> 00:04:38.530 is pretty common for good protein X-ray structures. 00:04:38.530 --> 00:04:41.340 But then also it's difficult to observe dynamics. 00:04:41.340 --> 00:04:43.510 So an X-ray structure is just a snapshot 00:04:43.510 --> 00:04:46.220 of the protein at a specific time. 00:04:46.220 --> 00:04:48.700 And so you need often a series of X-ray structures 00:04:48.700 --> 00:04:50.830 to really learn something about the mechanism 00:04:50.830 --> 00:04:52.690 or the dynamics of these proteins. 00:04:55.125 --> 00:04:56.500 The second most popular technique 00:04:56.500 --> 00:04:59.700 is nuclear magnetic resonance, or NMR. 00:04:59.700 --> 00:05:02.630 NMR is typically used to study small proteins. 00:05:02.630 --> 00:05:05.380 And the reason for this is that if you look at-- 00:05:05.380 --> 00:05:07.720 so this is a 2D NMR spectra. 00:05:07.720 --> 00:05:09.610 If you look at the 2D NMR spectra, 00:05:09.610 --> 00:05:11.230 there's a lot of peaks, right? 00:05:11.230 --> 00:05:14.260 And the more residues you have, the more amino acids you have, 00:05:14.260 --> 00:05:16.770 the more peaks you have and it gets very crowded. 00:05:16.770 --> 00:05:19.210 And so that's the limiting factor 00:05:19.210 --> 00:05:22.290 with using NMR to study large proteins 00:05:22.290 --> 00:05:26.225 is that you can't resolve all the chemical shifts. 00:05:26.225 --> 00:05:27.850 The plus point is that there is no need 00:05:27.850 --> 00:05:29.260 to crystallize your protein. 00:05:29.260 --> 00:05:31.940 You can study it in solution state or solid state. 00:05:31.940 --> 00:05:36.730 Solid state NMR is typically used for memory in proteins. 00:05:36.730 --> 00:05:40.330 You can use solution NMR to study any soluble proteins. 00:05:40.330 --> 00:05:42.460 You do need isotopically labeled samples. 00:05:42.460 --> 00:05:46.160 So these are 13c or 15n enriched samples, 00:05:46.160 --> 00:05:47.980 which is very hard and expensive to do. 00:05:47.980 --> 00:05:49.990 So that's one of the drawbacks with NMR 00:05:49.990 --> 00:05:52.660 is that it's a relatively expensive technique while X-ray 00:05:52.660 --> 00:05:55.280 is more accessible. 00:05:55.280 --> 00:05:58.780 You can obtain a high resolution picture with NMR, as well, 00:05:58.780 --> 00:06:02.180 but it often requires more work than X-ray crystallography 00:06:02.180 --> 00:06:05.800 in that you need to do about five NMR experiments. 00:06:05.800 --> 00:06:09.190 That can sometimes take months to determine high resolution 00:06:09.190 --> 00:06:10.000 structure. 00:06:10.000 --> 00:06:12.070 X-ray is more accessible. 00:06:12.070 --> 00:06:14.590 But really, the big upshot of NMR 00:06:14.590 --> 00:06:17.030 is that you can observe dynamics within proteins. 00:06:17.030 --> 00:06:18.960 So you can really see-- 00:06:18.960 --> 00:06:20.930 proteins are living, breathing machines. 00:06:20.930 --> 00:06:23.410 And you can see that with NMR better than you 00:06:23.410 --> 00:06:26.220 can with any other technique. 00:06:26.220 --> 00:06:29.170 Another quick point which I don't have written up here 00:06:29.170 --> 00:06:31.900 is that NMR is sensitive to protons. 00:06:31.900 --> 00:06:34.120 And you can study hydrogens with NMR. 00:06:34.120 --> 00:06:37.270 You cannot study hydrogens with X-ray diffraction, 00:06:37.270 --> 00:06:40.825 for reasons that I'll come back to later in the talk. 00:06:40.825 --> 00:06:46.160 AUDIENCE: Can you explain how exactly NMR observes dynamics? 00:06:46.160 --> 00:06:49.720 SHIVA MANDALA: Yeah, so there is a series of different-- 00:06:49.720 --> 00:06:52.480 I mean, I don't know how much detail you guys know about NMR. 00:06:52.480 --> 00:06:55.140 But basically, you can study the relaxation of nuclei. 00:06:55.140 --> 00:06:56.790 That's often one that's used. 00:06:56.790 --> 00:06:59.830 So you study T1 and T2 relaxation of nuclei. 00:06:59.830 --> 00:07:04.440 And more mobile residues and more mobile atoms relax faster. 00:07:04.440 --> 00:07:07.320 But really, the idea is you can use-- 00:07:07.320 --> 00:07:10.110 there are a whole bunch of different experiments in NMR. 00:07:10.110 --> 00:07:13.890 And you can access timescales from the nanosecond 00:07:13.890 --> 00:07:15.080 up to till millisecond. 00:07:15.080 --> 00:07:17.100 So 10 to the negative 9 to about 10 00:07:17.100 --> 00:07:19.860 to the negative 3 seconds of motion. 00:07:19.860 --> 00:07:22.620 So it's quite-- and they use different experiments 00:07:22.620 --> 00:07:25.340 for different parts of that timescale. 00:07:25.340 --> 00:07:29.360 And we can talk more about that in detail. 00:07:29.360 --> 00:07:32.250 The third technique is electron microscopy. 00:07:32.250 --> 00:07:36.300 So this is restricted so far to large proteins. 00:07:36.300 --> 00:07:39.575 The reason for this is that resolution is not so good. 00:07:39.575 --> 00:07:41.700 Again, you don't need to crystallize your proteins. 00:07:41.700 --> 00:07:43.930 So that's an option. 00:07:43.930 --> 00:07:46.050 You don't need to use labeled samples, either. 00:07:46.050 --> 00:07:48.180 So the sample preparation is probably the easiest 00:07:48.180 --> 00:07:50.780 for electron microscopy. 00:07:50.780 --> 00:07:56.370 The picture that you get is sometimes lower resolution, 00:07:56.370 --> 00:07:59.010 but the technology is moving forward to the point 00:07:59.010 --> 00:08:03.580 where we can get a resolution as good as X-ray structures. 00:08:03.580 --> 00:08:06.990 And I know that there's a 2.2 Angstrom resolution 00:08:06.990 --> 00:08:09.060 structure out there definitely, and there are 00:08:09.060 --> 00:08:10.790 others that are 3.2 Angstroms. 00:08:10.790 --> 00:08:13.040 But maybe there's something better than that out there 00:08:13.040 --> 00:08:13.960 in the literature. 00:08:13.960 --> 00:08:15.710 Again, it's difficult to observe dynamics. 00:08:15.710 --> 00:08:19.824 So similar to X-ray, it's just a snapshot of your enzyme. 00:08:19.824 --> 00:08:21.045 AUDIENCE: Is that picture-- 00:08:25.100 --> 00:08:30.245 is the concept similar to a normal microscope? 00:08:30.245 --> 00:08:32.370 SHIVA MANDALA: Yeah, absolutely it is very similar. 00:08:32.370 --> 00:08:34.940 And the only thing is you're looking 00:08:34.940 --> 00:08:38.960 at how electrons interact with with your sample as 00:08:38.960 --> 00:08:40.130 compared to light, right? 00:08:40.130 --> 00:08:41.150 Visible light, I guess. 00:08:45.000 --> 00:08:49.190 So each of these particles here is your protein, 00:08:49.190 --> 00:08:51.030 is a protein molecule. 00:08:51.030 --> 00:08:54.270 And then these are three dimensional reconstructions. 00:08:54.270 --> 00:08:56.250 So there's computer software that does this. 00:08:56.250 --> 00:08:57.770 So to go from this, you basically 00:08:57.770 --> 00:09:00.590 signal average over all of these different molecules. 00:09:00.590 --> 00:09:02.630 And then you signal average over all 00:09:02.630 --> 00:09:04.700 of your different orientations of the protein 00:09:04.700 --> 00:09:08.540 that are trapped in your static electron microscope image. 00:09:08.540 --> 00:09:10.350 And then using some image processing, 00:09:10.350 --> 00:09:13.310 you generate a three dimensional image of your protein 00:09:13.310 --> 00:09:15.620 that has a higher resolution than what you can see just 00:09:15.620 --> 00:09:17.750 with one single photo, I guess. 00:09:17.750 --> 00:09:19.549 So there's lot of computer processing 00:09:19.549 --> 00:09:20.840 that happens behind the scenes. 00:09:23.440 --> 00:09:25.530 AUDIENCE: So is the electron-- 00:09:25.530 --> 00:09:27.910 the interactions with electrons, is that similar 00:09:27.910 --> 00:09:30.106 to fluorescence microscopy? 00:09:30.106 --> 00:09:33.071 Because that's where you're seeing where your proteins are 00:09:33.071 --> 00:09:33.960 located, right? 00:09:33.960 --> 00:09:36.310 SHIVA MANDALA: So the difference with fluorescence-- 00:09:36.310 --> 00:09:39.670 I mean, here electrons can interact with any atoms, right? 00:09:39.670 --> 00:09:40.470 Any material. 00:09:40.470 --> 00:09:42.760 AUDIENCE: Oh, so you can distinguish 00:09:42.760 --> 00:09:45.809 what different atoms the electrons are interacting with? 00:09:45.809 --> 00:09:47.600 SHIVA MANDALA: Yes, because different atoms 00:09:47.600 --> 00:09:50.370 interact with-- different nuclei interact, 00:09:50.370 --> 00:09:53.330 and electron densities interact with electrons differently. 00:09:53.330 --> 00:09:54.860 But with fluorescence microscopy, 00:09:54.860 --> 00:09:57.220 you're usually looking at just a single molecule 00:09:57.220 --> 00:10:00.350 a fluorophore that's reporting on where your protein is. 00:10:00.350 --> 00:10:03.020 But electron microscopy is a much higher resolution picture. 00:10:03.020 --> 00:10:04.940 It's actually an atomic level-- 00:10:04.940 --> 00:10:07.190 well, maybe a few atoms level-- 00:10:07.190 --> 00:10:07.874 picture. 00:10:07.874 --> 00:10:09.290 Fluorescence microscopy is usually 00:10:09.290 --> 00:10:12.290 just used to study where your protein is 00:10:12.290 --> 00:10:13.590 if proteins are interacting. 00:10:13.590 --> 00:10:18.500 So that's more macromolecular interactions. 00:10:18.500 --> 00:10:20.950 But you can get single molecule resolution 00:10:20.950 --> 00:10:26.000 with fluorescence microscopy if you use the correct techniques. 00:10:26.000 --> 00:10:29.560 And so just as an introduction to the protein data bank, so 00:10:29.560 --> 00:10:33.370 the first graph tells you the number of structures in the PDB 00:10:33.370 --> 00:10:37.510 as a function of a year going from 1975 all the way to 2015. 00:10:37.510 --> 00:10:41.900 So you'll see today there are about 110,000 structures, 00:10:41.900 --> 00:10:49.150 of which 100,000 were determined using X-ray crystallography, 00:10:49.150 --> 00:10:52.630 and about 10,000 using NMR, and about 1,000 00:10:52.630 --> 00:10:54.700 using electron microscopy. 00:10:54.700 --> 00:10:56.890 So really quite a nice ratio there. 00:10:56.890 --> 00:10:58.570 And if you see the yearly increase 00:10:58.570 --> 00:11:00.670 in the number of PDB structures, you'll 00:11:00.670 --> 00:11:04.180 see that X-ray is, of course, really big. 00:11:04.180 --> 00:11:06.257 NMR has been fairly consistent over time. 00:11:06.257 --> 00:11:08.590 I think that has to do with the fact that it's expensive 00:11:08.590 --> 00:11:10.660 and it takes time to prepare your samples. 00:11:10.660 --> 00:11:14.140 But you also see a huge spike in electron microscopy of late. 00:11:14.140 --> 00:11:19.060 And so with the advent of cryo EM, a lot more 00:11:19.060 --> 00:11:21.890 people start using cry EM to determine protein structure. 00:11:21.890 --> 00:11:24.496 AUDIENCE: Doesn't [INAUDIBLE] produce [INAUDIBLE] or you 00:11:24.496 --> 00:11:26.950 just put it in [INAUDIBLE] 00:11:26.950 --> 00:11:29.910 SHIVA MANDALA: Yeah, it can be. 00:11:29.910 --> 00:11:34.500 But the problem with that is when 00:11:34.500 --> 00:11:36.300 you put your sample in the [INAUDIBLE],, 00:11:36.300 --> 00:11:38.067 you can get chemical shift information. 00:11:38.067 --> 00:11:39.900 But chemical shift doesn't tell you anything 00:11:39.900 --> 00:11:41.220 about protein structure. 00:11:41.220 --> 00:11:42.810 I mean, it tells you a little bit. 00:11:42.810 --> 00:11:44.310 It tells you about what the electron 00:11:44.310 --> 00:11:46.320 density is at the atoms. 00:11:46.320 --> 00:11:48.810 But what you really need to get from NMR experiments 00:11:48.810 --> 00:11:50.160 are distance of strains. 00:11:50.160 --> 00:11:52.330 And so this is through space experiments. 00:11:52.330 --> 00:11:55.140 So you can say that, oh, this one carbon 00:11:55.140 --> 00:11:57.090 nuclei is at a distance of 6 Angstroms 00:11:57.090 --> 00:11:59.780 away from this other carbon nuclei. 00:11:59.780 --> 00:12:01.350 And you typically want to accumulate 00:12:01.350 --> 00:12:04.200 about five strains per atom. 00:12:04.200 --> 00:12:06.150 And so to collect five times how many ever 00:12:06.150 --> 00:12:08.380 atoms you have in your sample, it can take time. 00:12:08.380 --> 00:12:09.140 It's hard to do. 00:12:09.140 --> 00:12:10.980 So chemical shift by itself doesn't tell you 00:12:10.980 --> 00:12:15.180 much about protein structure. 00:12:15.180 --> 00:12:17.810 Any other questions so far? 00:12:17.810 --> 00:12:21.220 All right. 00:12:21.220 --> 00:12:23.230 So now we will focus the rest of the talk 00:12:23.230 --> 00:12:26.770 on-- well, another part of the talk on X-ray crystallography. 00:12:26.770 --> 00:12:28.630 And so crystallography is the science 00:12:28.630 --> 00:12:33.520 of determining the t dimensional position of atoms in a crystal. 00:12:33.520 --> 00:12:35.410 And so what crystal is, a crystal 00:12:35.410 --> 00:12:37.270 is a solid material whose constituents 00:12:37.270 --> 00:12:40.840 are arranged in an ordered pattern expanding and extending 00:12:40.840 --> 00:12:42.640 in all three spatial dimensions. 00:12:42.640 --> 00:12:46.090 And so the key idea is that this translational symmetry-- 00:12:46.090 --> 00:12:48.540 so if you go in any of the three directions 00:12:48.540 --> 00:12:50.970 for a certain amount of time, a certain amount of length, 00:12:50.970 --> 00:12:52.469 you'll come back to the same pattern 00:12:52.469 --> 00:12:53.770 that you start off with. 00:12:53.770 --> 00:12:57.796 And so this is a crystal of your protein of interest. 00:12:57.796 --> 00:12:59.170 What you want to know is you want 00:12:59.170 --> 00:13:01.330 to know how the proteins are packed or arranged 00:13:01.330 --> 00:13:02.830 within this crystal structure. 00:13:02.830 --> 00:13:04.420 And also as a result, how the atoms 00:13:04.420 --> 00:13:06.520 are arranged within the crystal structure. 00:13:06.520 --> 00:13:09.580 And the way this works is by diffracting X-rays 00:13:09.580 --> 00:13:11.590 through your sample of interest. 00:13:11.590 --> 00:13:13.240 And with this slide, I just wanted 00:13:13.240 --> 00:13:15.790 to point out that it's not restricted to proteins. 00:13:15.790 --> 00:13:17.470 You can study salts, you can study 00:13:17.470 --> 00:13:19.060 your favorite small organic molecule. 00:13:19.060 --> 00:13:21.790 Whatever you want, really. 00:13:21.790 --> 00:13:24.580 And so the general workflow is that you have a source 00:13:24.580 --> 00:13:28.210 of x-rays that can be a singleton-- or local source, 00:13:28.210 --> 00:13:30.760 singletons are much brighter than local sources-- 00:13:30.760 --> 00:13:32.170 that you shine in your crystal. 00:13:32.170 --> 00:13:35.250 And you obtain what is known as a diffraction pattern. 00:13:35.250 --> 00:13:37.900 And so this tells you how the X-rays are in track-- 00:13:37.900 --> 00:13:40.450 this tells you something about how the X-rays are interacting 00:13:40.450 --> 00:13:43.400 with the atoms in the crystal. 00:13:43.400 --> 00:13:46.300 And so this used to be collected on a photographic plate. 00:13:46.300 --> 00:13:49.510 This particular image is on a photographic plate, 00:13:49.510 --> 00:13:51.430 but now people use CCD sensors. 00:13:51.430 --> 00:13:52.870 It's a lot easier. 00:13:52.870 --> 00:13:57.100 And knowing the-- sorry, one more thing. 00:13:57.100 --> 00:13:59.330 Each of these dots, light and dark, 00:13:59.330 --> 00:14:01.650 on the diffraction pattern is called the reflection. 00:14:01.650 --> 00:14:03.250 And that contains some information 00:14:03.250 --> 00:14:06.250 about the electron density and the crystal structure. 00:14:06.250 --> 00:14:07.960 And from your diffraction pattern, 00:14:07.960 --> 00:14:11.110 you can then back calculate the electron density 00:14:11.110 --> 00:14:12.970 in your crystal structure that gave rise 00:14:12.970 --> 00:14:14.680 to this diffraction pattern. 00:14:14.680 --> 00:14:16.360 And the way you do that is by looking 00:14:16.360 --> 00:14:18.890 at the intensity of these reflections. 00:14:18.890 --> 00:14:21.390 And you also need-- there's also something called phase, 00:14:21.390 --> 00:14:22.870 so you need to determine phase. 00:14:22.870 --> 00:14:26.110 And sometimes you'll see in the literature 00:14:26.110 --> 00:14:27.870 you'll see heavy atoms being introduced, 00:14:27.870 --> 00:14:29.414 or mercury being introduced. 00:14:29.414 --> 00:14:31.330 And that's often to determine the phase, which 00:14:31.330 --> 00:14:34.900 is essential for calculating the electron density. 00:14:34.900 --> 00:14:36.920 Once you determine the electron density, 00:14:36.920 --> 00:14:39.070 you know what protein you started off with. 00:14:39.070 --> 00:14:41.350 And so you know what your protein 00:14:41.350 --> 00:14:43.600 looks-- you know the sequence of your protein. 00:14:43.600 --> 00:14:46.180 And so then you just take your electron density 00:14:46.180 --> 00:14:49.320 and fit whatever polypeptide change you have to that. 00:14:49.320 --> 00:14:52.300 And then usually this is all automated nowadays. 00:14:52.300 --> 00:14:56.050 So you press a few buttons and it goes through, 00:14:56.050 --> 00:14:57.850 software does everything for you. 00:14:57.850 --> 00:15:01.090 But it was much more challenging early on. 00:15:01.090 --> 00:15:04.850 And even now, the computers will get you up to a certain point. 00:15:04.850 --> 00:15:07.780 And then in the last, last stages of refinement, 00:15:07.780 --> 00:15:08.950 you always want to-- 00:15:08.950 --> 00:15:12.480 usually people do that by hand. 00:15:12.480 --> 00:15:16.356 Any questions about X-ray crystallography? 00:15:16.356 --> 00:15:20.670 AUDIENCE: [INAUDIBLE] how strongly or complex, 00:15:20.670 --> 00:15:23.390 but how do you get that electron density from diffraction 00:15:23.390 --> 00:15:24.760 pattern. 00:15:24.760 --> 00:15:30.500 Like in organic chemistry, in basic [INAUDIBLE] I thought 00:15:30.500 --> 00:15:31.750 that you can-- 00:15:31.750 --> 00:15:34.350 from diffraction pattern, you can 00:15:34.350 --> 00:15:41.970 learn the distance between atoms in the lattice points. 00:15:41.970 --> 00:15:47.640 But here with proteins, every point is itself a protein, 00:15:47.640 --> 00:15:49.120 right? 00:15:49.120 --> 00:15:50.130 In the lattice? 00:15:50.130 --> 00:15:50.720 No? 00:15:50.720 --> 00:15:51.970 SHIVA MANDALA: No, no, no, no. 00:15:51.970 --> 00:15:54.386 Because you're still looking at every point in the lattice 00:15:54.386 --> 00:15:56.740 is still an atom if you're doing proteins. 00:15:56.740 --> 00:15:58.540 It's just that there are a lot more atoms, 00:15:58.540 --> 00:16:00.510 and the lattice is a lot bigger, which 00:16:00.510 --> 00:16:02.260 is what makes protein crystallography hard 00:16:02.260 --> 00:16:04.670 compared to small molecule crystallography. 00:16:04.670 --> 00:16:06.820 So it's harder to solve a protein crystal structure 00:16:06.820 --> 00:16:08.694 than it is a small molecule crystal structure 00:16:08.694 --> 00:16:11.420 just because there's so many more atoms in your lattice. 00:16:11.420 --> 00:16:14.020 But the idea is exactly the same as a small molecule. 00:16:14.020 --> 00:16:15.890 It's just a lot harder to do. 00:16:15.890 --> 00:16:17.530 And for more information, I actually 00:16:17.530 --> 00:16:19.930 have a resource at the end that goes in-depth 00:16:19.930 --> 00:16:22.570 into the math of the process. 00:16:22.570 --> 00:16:24.880 But just briefly, this diffraction pattern 00:16:24.880 --> 00:16:27.610 is collected into what's called reciprocal space. 00:16:27.610 --> 00:16:29.914 And to go from this to electron density, 00:16:29.914 --> 00:16:31.330 you need to do a Fourier transform 00:16:31.330 --> 00:16:33.890 into Hilbert space, which is what electron density is spaced 00:16:33.890 --> 00:16:34.390 in. 00:16:34.390 --> 00:16:40.670 But I will provide a reference for more information on that. 00:16:40.670 --> 00:16:43.610 And so the next part of the discussion part 00:16:43.610 --> 00:16:46.300 of this recitation will be thinking about some 00:16:46.300 --> 00:16:48.400 of the limitations of X-ray crystallography. 00:16:48.400 --> 00:16:52.330 So there's a lot of them, but I'll turn to all of you 00:16:52.330 --> 00:16:54.490 for your inputs. 00:16:54.490 --> 00:16:56.614 AUDIENCE: Is it difficult to develop crystals 00:16:56.614 --> 00:16:58.012 of certain types of proteins? 00:16:58.012 --> 00:16:59.470 SHIVA MANDALA: Yes, absolutely yes. 00:16:59.470 --> 00:17:02.910 First point, it's really hard to purify and crystallize 00:17:02.910 --> 00:17:03.729 proteins. 00:17:03.729 --> 00:17:05.020 It's really not a trivial task. 00:17:05.020 --> 00:17:07.359 It can take months or even years to do so. 00:17:07.359 --> 00:17:10.979 And nowadays you have robots that can set up reactions 00:17:10.979 --> 00:17:13.270 under hundreds of different crystallization conditions. 00:17:13.270 --> 00:17:16.677 It's sort of a black magic sort of hard. 00:17:16.677 --> 00:17:19.010 It's hard to predict what crystallization conditions are 00:17:19.010 --> 00:17:22.369 going to give you a high quality crystal. 00:17:22.369 --> 00:17:23.190 Anything else? 00:17:23.190 --> 00:17:24.047 Yes? 00:17:24.047 --> 00:17:26.130 AUDIENCE: Like the crystals you get may or may not 00:17:26.130 --> 00:17:28.369 be physiologically relevant? 00:17:28.369 --> 00:17:29.821 SHIVA MANDALA: Yes, absolutely. 00:17:29.821 --> 00:17:32.180 Just on point with questions. 00:17:32.180 --> 00:17:34.910 But yeah, it's hard to tell whether the crystal 00:17:34.910 --> 00:17:37.970 structure that you get is depicting what's happening 00:17:37.970 --> 00:17:39.230 with the protein in vivo. 00:17:39.230 --> 00:17:42.742 And I mean, this is a problem that's inherent to X-ray 00:17:42.742 --> 00:17:43.700 crystallography, right? 00:17:43.700 --> 00:17:45.408 Every time you solve a crystal structure, 00:17:45.408 --> 00:17:47.210 you don't know whether it's relevant. 00:17:47.210 --> 00:17:50.810 But usually I think it turns out that it's 00:17:50.810 --> 00:17:54.500 pretty close, if not completely accurate in solution. 00:17:54.500 --> 00:17:56.919 But sometimes you do have to be careful about this. 00:17:56.919 --> 00:17:58.460 It's especially challenging for stuff 00:17:58.460 --> 00:18:01.730 like membrane proteins, where you don't really know. 00:18:01.730 --> 00:18:02.570 Any other ideas? 00:18:05.720 --> 00:18:08.690 So when proteins are translated, do they usually-- 00:18:08.690 --> 00:18:10.595 are they usually used just like that, 00:18:10.595 --> 00:18:13.178 or does something else happened to the proteins in most cells? 00:18:13.178 --> 00:18:14.402 AUDIENCE: [INAUDIBLE] 00:18:14.402 --> 00:18:15.770 SHIVA MANDALA: Yes, absolutely. 00:18:15.770 --> 00:18:17.150 Post translation modifications. 00:18:17.150 --> 00:18:20.180 So a lot of proteins are post-translationally modified. 00:18:20.180 --> 00:18:25.119 And so when you're growing a crystal of your protein, 00:18:25.119 --> 00:18:27.410 you usually just use a purified version of your protein 00:18:27.410 --> 00:18:28.850 so you can't really calculate. 00:18:28.850 --> 00:18:32.106 And sometimes these PTMs are essential for the function 00:18:32.106 --> 00:18:32.730 of the protein. 00:18:32.730 --> 00:18:35.456 So you're missing some part of the picture. 00:18:35.456 --> 00:18:38.480 Anything else? 00:18:38.480 --> 00:18:39.900 What about movement? 00:18:39.900 --> 00:18:44.710 Can you tell what proteins are flexible, what parts of the-- 00:18:44.710 --> 00:18:45.630 sorry. 00:18:45.630 --> 00:18:49.126 AUDIENCE: Well it's like if part of protein is mobile, 00:18:49.126 --> 00:18:53.960 then you won't have the density for it. 00:18:53.960 --> 00:18:55.640 SHIVA MANDALA: Yes, that is true. 00:18:55.640 --> 00:18:58.120 You can discern anything about dynamics and flexibility. 00:18:58.120 --> 00:19:01.321 And the answer is you can tell something. 00:19:01.321 --> 00:19:03.320 You can tell something about the relative motion 00:19:03.320 --> 00:19:05.236 of different parts of the protein with respect 00:19:05.236 --> 00:19:07.280 to each other, but it's hard to tell something 00:19:07.280 --> 00:19:09.600 about the absolute motion of these proteins. 00:19:09.600 --> 00:19:12.146 So you can't see, say, larger scale motions, right? 00:19:12.146 --> 00:19:14.020 Most proteins are living, breathing machines, 00:19:14.020 --> 00:19:18.650 and it's hard to capture that in an X-ray structure. 00:19:18.650 --> 00:19:21.000 And one more thing. 00:19:21.000 --> 00:19:24.650 So is there any element that you cannot detect in X-ray 00:19:24.650 --> 00:19:26.120 crystallography very well? 00:19:26.120 --> 00:19:28.500 This has to do with the way-- 00:19:28.500 --> 00:19:30.020 so in X-ray crystallography, you're 00:19:30.020 --> 00:19:33.770 setting interactions of X-rays with electrons, right? 00:19:33.770 --> 00:19:35.520 So does anybody know? 00:19:38.150 --> 00:19:39.493 Yeah, I heard somewhere. 00:19:39.493 --> 00:19:39.856 AUDIENCE: Protons. 00:19:39.856 --> 00:19:40.460 SHIVA MANDALA: Protons, yeah. 00:19:40.460 --> 00:19:42.160 So protons have one electron. 00:19:42.160 --> 00:19:46.640 And so their X-ray signal, so-called, is really weak. 00:19:46.640 --> 00:19:47.480 Really, really weak. 00:19:47.480 --> 00:19:49.430 And so you can't really see protons 00:19:49.430 --> 00:19:50.900 with X-ray crystallography. 00:19:50.900 --> 00:19:53.980 And so you can't really study hydrogens or hydrogen bonds. 00:19:53.980 --> 00:19:55.850 And if you look at the structure of proteins 00:19:55.850 --> 00:19:58.130 that has hydrogens in it, those hydrogens 00:19:58.130 --> 00:20:01.160 were put there as a result of an average bond length, 00:20:01.160 --> 00:20:02.820 the typical bond calculation. 00:20:02.820 --> 00:20:04.940 So it's not actually experimentally determined. 00:20:04.940 --> 00:20:07.680 You can use neutron diffraction to get around this, 00:20:07.680 --> 00:20:09.260 but neutron diffraction is hard to do 00:20:09.260 --> 00:20:12.540 because you need to grow very large crystals to study. 00:20:12.540 --> 00:20:15.350 And I think there are about 80, I think, neutron-- 00:20:15.350 --> 00:20:18.490 around 100 neutron structures in the PDB so far. 00:20:18.490 --> 00:20:21.470 But for small molecules, neutron is much more accessible. 00:20:21.470 --> 00:20:23.096 Neutron diffraction? 00:20:23.096 --> 00:20:26.270 And so the idea is the same as X-ray crystallography, 00:20:26.270 --> 00:20:27.990 except for you're using neutrons. 00:20:31.730 --> 00:20:34.014 And the final point is that one structure only 00:20:34.014 --> 00:20:35.180 tells you part of the story. 00:20:35.180 --> 00:20:37.580 Again, this is emphasizing the fact 00:20:37.580 --> 00:20:41.120 that one structure is just a snapshot of the protein 00:20:41.120 --> 00:20:42.320 at a certain time. 00:20:42.320 --> 00:20:44.600 And you want to correctly interpret your data 00:20:44.600 --> 00:20:46.880 and learn something more about the protein, 00:20:46.880 --> 00:20:50.420 you often have to use complementary biochemical 00:20:50.420 --> 00:20:52.820 techniques. 00:20:52.820 --> 00:20:55.410 Are there any questions at this point? 00:20:55.410 --> 00:20:58.130 So the last part of the talk is on how 00:20:58.130 --> 00:21:00.960 to assess the quality of structures in the PDB. 00:21:00.960 --> 00:21:02.530 They're large structures, and you 00:21:02.530 --> 00:21:05.120 want to be able to know whether the model that's 00:21:05.120 --> 00:21:07.564 presented to you is actually accurate, 00:21:07.564 --> 00:21:10.500 actually reflects the data that was collected. 00:21:10.500 --> 00:21:13.640 And so the first point is what is the resolution 00:21:13.640 --> 00:21:14.830 of the structure? 00:21:14.830 --> 00:21:18.650 And so the take home message is that a lower number 00:21:18.650 --> 00:21:20.150 means a greater resolution. 00:21:20.150 --> 00:21:21.800 And the resolution actually here is 00:21:21.800 --> 00:21:24.806 referring to the distances between the atoms in the plane. 00:21:24.806 --> 00:21:26.430 And so that's where that's coming from. 00:21:26.430 --> 00:21:28.190 So that's why if you have a lower resolution, 00:21:28.190 --> 00:21:29.614 that means you can resolve atoms. 00:21:29.614 --> 00:21:31.280 A one Angstrom resolution means that you 00:21:31.280 --> 00:21:33.970 can resolve atoms that are one Angstrom apart 00:21:33.970 --> 00:21:36.200 on parallel planes. 00:21:36.200 --> 00:21:38.930 But the take home message at a one Angstrom resolution, 00:21:38.930 --> 00:21:41.090 you can see individual atoms and you 00:21:41.090 --> 00:21:42.890 can discern the identities of those atoms 00:21:42.890 --> 00:21:45.180 by looking at their electron density. 00:21:45.180 --> 00:21:48.131 But if you come bound to a four Angstrom structure, 00:21:48.131 --> 00:21:49.880 you'll see the benzene ring doesn't really 00:21:49.880 --> 00:21:51.650 have a clearly defined electron density, 00:21:51.650 --> 00:21:53.560 and there's no hole in the center. 00:21:53.560 --> 00:21:55.476 But if you look at the one Angstrom structure, 00:21:55.476 --> 00:21:58.227 you can see that there's even a hole in the benzene ring 00:21:58.227 --> 00:21:59.810 to confirm the electron density there. 00:22:02.410 --> 00:22:05.610 And then if you look at-- so these are the data statistics. 00:22:05.610 --> 00:22:09.330 So this is just pulled from PDB off 2JF5. 00:22:09.330 --> 00:22:12.129 So this is the PDB ID for di-ubiquitin, 00:22:12.129 --> 00:22:13.920 and we'll be looking at the structure later 00:22:13.920 --> 00:22:15.470 in the worksheet. 00:22:15.470 --> 00:22:17.850 The resolution tells you, of course, 00:22:17.850 --> 00:22:20.560 about the resolution of the crystal structure. 00:22:20.560 --> 00:22:23.580 And so in this case, it's at 1.95 Angstroms 00:22:23.580 --> 00:22:26.340 or two Angstroms, and so that's pretty high resolution. 00:22:29.659 --> 00:22:31.950 Reflections are each of those points in the diffraction 00:22:31.950 --> 00:22:34.380 pattern that you collect, and a unique reflection 00:22:34.380 --> 00:22:37.290 refers to the fact that you've only collected it once. 00:22:37.290 --> 00:22:41.370 So usually when you collect diffraction patterns, 00:22:41.370 --> 00:22:44.730 you put your protein a certain orientation with respect 00:22:44.730 --> 00:22:47.070 to the X-ray beam, and then you collect the diffraction 00:22:47.070 --> 00:22:49.500 pattern, and then you rotate your crystal a whole bunch 00:22:49.500 --> 00:22:50.700 of times, and you collect a whole bunch 00:22:50.700 --> 00:22:52.080 of different diffraction patterns. 00:22:52.080 --> 00:22:53.640 And then you superimpose all of those 00:22:53.640 --> 00:22:57.540 together to get the master diffraction pattern. 00:22:57.540 --> 00:23:01.530 Redundancy refers to the fact of how often each reflection was 00:23:01.530 --> 00:23:02.250 observed. 00:23:02.250 --> 00:23:03.750 And so this is a signal averaging. 00:23:03.750 --> 00:23:08.040 The more redundancy you have, the greater number of times 00:23:08.040 --> 00:23:10.710 you observed that particular reflection. 00:23:10.710 --> 00:23:13.080 Completedness refers to how many of the data 00:23:13.080 --> 00:23:15.480 points were actually measured. 00:23:15.480 --> 00:23:18.330 And so this is when you created your model, 00:23:18.330 --> 00:23:21.160 you can back calculate your diffraction pattern. 00:23:21.160 --> 00:23:24.150 And then you see how many of those 00:23:24.150 --> 00:23:28.660 reflections were experimentally observed in our data set. 00:23:28.660 --> 00:23:31.190 And so for this usually you want as close to 100%, 00:23:31.190 --> 00:23:35.298 and anything above 95% is considered fairly good. 00:23:35.298 --> 00:23:41.350 R merge is an indicator of how consistent measurements are. 00:23:41.350 --> 00:23:45.220 So this is a measure of what the difference 00:23:45.220 --> 00:23:50.050 between different measurements for the same reflection are. 00:23:50.050 --> 00:23:54.400 So if you look at the intensity for the same reflection 00:23:54.400 --> 00:23:55.870 a different number of times, you're 00:23:55.870 --> 00:23:58.300 seeing the standard deviation of that. 00:23:58.300 --> 00:24:00.280 So you want as low a number as possible. 00:24:00.280 --> 00:24:05.140 So usually you want it to be about 1/10 00:24:05.140 --> 00:24:07.120 of the resolution of your crystal structure, 00:24:07.120 --> 00:24:10.570 which is 0.2 Angstroms in this case. 00:24:10.570 --> 00:24:13.030 I forgot to mention this, but the values in parentheses 00:24:13.030 --> 00:24:14.830 are for a high resolution bin. 00:24:14.830 --> 00:24:18.760 So this is just a certain subset of this data set 00:24:18.760 --> 00:24:21.322 that's considered to be higher quality than the original data 00:24:21.322 --> 00:24:21.750 set. 00:24:21.750 --> 00:24:23.830 And that's actually coming from this value, which 00:24:23.830 --> 00:24:28.840 is signal intensity over sigma of signal intensity, 00:24:28.840 --> 00:24:31.310 and that's a measure of the signal to noise ratio. 00:24:31.310 --> 00:24:34.010 So you want a higher signal to noise ratio is better. 00:24:34.010 --> 00:24:36.310 And this is fairly good. 00:24:36.310 --> 00:24:37.880 And the higher the better. 00:24:37.880 --> 00:24:40.850 And cutoff is at 2 for the high resolution 00:24:40.850 --> 00:24:44.890 bin of your data points. 00:24:44.890 --> 00:24:47.040 All, right so this is just the raw data. 00:24:47.040 --> 00:24:49.410 And then the refinement statistics 00:24:49.410 --> 00:24:51.900 tell you something about your refinement process that 00:24:51.900 --> 00:24:54.240 gave you the crystal structure that you calculated. 00:24:54.240 --> 00:24:57.300 So our crystallization, which is R cryst, which is also 00:24:57.300 --> 00:25:00.900 called R work, and also called the R factor. 00:25:00.900 --> 00:25:05.070 That tells you how well your model and your data match. 00:25:05.070 --> 00:25:06.990 And so this is where you calculate 00:25:06.990 --> 00:25:09.030 the difference in the diffraction 00:25:09.030 --> 00:25:13.710 patterns between what you experimentally observed 00:25:13.710 --> 00:25:19.035 and what you calculated using the model of electron density. 00:25:19.035 --> 00:25:23.220 R free tells you how well your model and data match 00:25:23.220 --> 00:25:24.930 when corrected for overfitting. 00:25:24.930 --> 00:25:29.430 So the idea behind this is that when you collect your data set, 00:25:29.430 --> 00:25:32.200 you put aside about 5% of the reflections 00:25:32.200 --> 00:25:37.080 that you observe in the data set to prevent overfitting. 00:25:37.080 --> 00:25:38.940 And then when you've created your model, 00:25:38.940 --> 00:25:41.820 you go back and see how well those 5% of data points 00:25:41.820 --> 00:25:44.530 fit the model that you've come up with. 00:25:44.530 --> 00:25:47.130 And if they fit well, that means you've accurately 00:25:47.130 --> 00:25:49.985 predicted your crystal structure using the data that you have. 00:25:49.985 --> 00:25:51.360 If you don't fit well, that means 00:25:51.360 --> 00:25:54.774 you've just fit whatever data points you have to some model. 00:25:54.774 --> 00:25:56.190 You've used a bunch of data points 00:25:56.190 --> 00:25:59.670 and you fit it to some model, but that's not actually 00:25:59.670 --> 00:26:02.670 accurate to the protein structure that you determined. 00:26:02.670 --> 00:26:06.780 And so for this you want it to be about 1/10 of a resolution. 00:26:06.780 --> 00:26:08.640 That's also true for R cryst. 00:26:08.640 --> 00:26:10.590 But then the other key point is that it 00:26:10.590 --> 00:26:12.895 should be very close to the R cryst, 00:26:12.895 --> 00:26:16.950 because it's telling you that it's random error, 00:26:16.950 --> 00:26:18.450 or it's the quality of your data set 00:26:18.450 --> 00:26:21.900 that's causing this and not sum overrefinement 00:26:21.900 --> 00:26:25.340 that you've done during the refinement process. 00:26:25.340 --> 00:26:28.145 B factor tells you about how mobile atoms 00:26:28.145 --> 00:26:29.720 are in the crystal lattice. 00:26:29.720 --> 00:26:31.470 And this is something that's it's 00:26:31.470 --> 00:26:35.430 not particularly useful if you look at the bulk statistic. 00:26:35.430 --> 00:26:38.910 But if you need to, it's important to evaluate this 00:26:38.910 --> 00:26:39.940 by residue. 00:26:39.940 --> 00:26:42.870 And so if you look at amino acids in the loops of proteins, 00:26:42.870 --> 00:26:45.300 you'll find that they have a higher B factor usually, 00:26:45.300 --> 00:26:48.120 meaning that they're more mobile. 00:26:48.120 --> 00:26:50.140 And so you can often tell what residues 00:26:50.140 --> 00:26:54.830 are important for function by looking at the B factor. 00:26:54.830 --> 00:26:58.477 B factor is kind of-- there's inherent vibration motions 00:26:58.477 --> 00:26:59.310 in all atoms, right? 00:26:59.310 --> 00:27:03.270 So there will be a B factor at any temperature greater than 0. 00:27:03.270 --> 00:27:06.000 But it also does tell you a little bit about the disorder 00:27:06.000 --> 00:27:08.070 in protein structures. 00:27:08.070 --> 00:27:09.750 And the B factor of water is often 00:27:09.750 --> 00:27:12.710 included just so that people who are looking at the crystal 00:27:12.710 --> 00:27:15.210 structure afterwards can decide whether that water is really 00:27:15.210 --> 00:27:18.786 there, or really was part of the electron density, 00:27:18.786 --> 00:27:20.910 or whether it's just an artifact of ore refinement, 00:27:20.910 --> 00:27:23.230 or something like that. 00:27:23.230 --> 00:27:25.500 And then the final statistic that you can look at 00:27:25.500 --> 00:27:27.510 is the RMSD from ideal geometry. 00:27:27.510 --> 00:27:30.450 So the geometries of these bonds and angles 00:27:30.450 --> 00:27:31.690 are usually well known. 00:27:31.690 --> 00:27:36.060 And so if you compare the results from your structure 00:27:36.060 --> 00:27:39.030 to the known stereochemistry, you'll 00:27:39.030 --> 00:27:42.124 find that this is actually just at the threshold of the cutoff 00:27:42.124 --> 00:27:43.290 for what is considered good. 00:27:43.290 --> 00:27:46.890 So 0.015 Angstroms, this is standard deviation 00:27:46.890 --> 00:27:51.070 of 1 lens of your model versus what is already known. 00:27:51.070 --> 00:27:55.230 And so 0.015 Angstroms is just about the cutoff 00:27:55.230 --> 00:27:58.080 that's considered good for X-ray structures. 00:27:58.080 --> 00:27:59.490 And same for bond angles. 00:27:59.490 --> 00:28:02.700 1.5 degrees is considered the threshold of what 00:28:02.700 --> 00:28:04.640 is considered acceptable. 00:28:04.640 --> 00:28:07.510 And you can also look at Ramachandran's statistics-- 00:28:07.510 --> 00:28:10.760 so this is looking at five sided back one angles-- 00:28:10.760 --> 00:28:16.290 to tell you if there are any static clashes, say, 00:28:16.290 --> 00:28:19.290 for side chains that really shouldn't be there. 00:28:19.290 --> 00:28:21.690 And you can if you do have static clash nowadays, 00:28:21.690 --> 00:28:23.700 you have to report it to PDB. 00:28:23.700 --> 00:28:25.410 The only exception is for glycine, which 00:28:25.410 --> 00:28:26.493 doesn't have a side chain. 00:28:26.493 --> 00:28:30.840 And so it can adopt a strange phi psi angle that's 00:28:30.840 --> 00:28:33.240 outside the Ramachandran plot. 00:28:33.240 --> 00:28:36.520 Any questions on this? 00:28:36.520 --> 00:28:38.372 Anything else about X-ray crystallography? 00:28:38.372 --> 00:28:40.080 So this brings us to the end of the talk, 00:28:40.080 --> 00:28:43.830 and this is a resource that I found 00:28:43.830 --> 00:28:46.860 that has more information about X-ray crystallography, 00:28:46.860 --> 00:28:48.610 and the math behind X-ray crystallography, 00:28:48.610 --> 00:28:49.985 and more of the theory behind it. 00:28:49.985 --> 00:28:52.130 But again, I want to emphasize that today it's 00:28:52.130 --> 00:28:54.720 a very automated process is that you click a few buttons 00:28:54.720 --> 00:28:57.020 and anybody can do it.